Sine SM


Full name : Sine Steven M

First name : Steven M

Mail : Mayo Clinic 200, 1st Street SW - Rm. 1-135 MSB, Rochester, MN 55905

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Country : USA

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References (125)

Title : Stoichiometry for activation of neuronal alpha7 nicotinic receptors - Andersen_2013_Proc.Natl.Acad.Sci.U.S.A_110_20819
Author(s) : Andersen N , Corradi J , Sine SM , Bouzat C
Ref : Proc Natl Acad Sci U S A , 110 :20819 , 2013
Abstract : Neuronal alpha7 nicotinic receptors elicit rapid cation influx in response to acetylcholine (ACh) or its hydrolysis product choline. They contribute to cognition, synaptic plasticity, and neuroprotection and have been implicated in neurodegenerative and neuropsychiatric disorders. alpha7, however, often localizes distal to sites of nerve-released ACh and binds ACh with low affinity, and thus elicits its biological response with low agonist occupancy. To assess the function of alpha7 when ACh occupies fewer than five of its identical binding sites, we measured the open-channel lifetime of individual receptors in which four of the five ACh binding sites were disabled. To improve the time resolution of the inherently brief alpha7 channel openings, background mutations or a potentiator was used to increase open duration. We find that, in receptors with only one intact binding site, the open-channel lifetime is indistinguishable from receptors with five intact binding sites, counter to expectations from prototypical neurotransmitter-gated ion channels where the open-channel lifetime increases with the number of binding sites occupied by agonist. Replacing the membrane-embedded domain of alpha7 by that of the related 5-HT3A receptor increases the number of sites that need to be occupied to achieve the maximal open-channel lifetime, thus revealing a unique interdependence between the detector and actuator domains of these receptors. The distinctive ability of a single occupancy to elicit a full biological response adapts alpha7 to volume transmission, a prevalent mechanism of ACh-mediated signaling in the nervous system and nonneuronal cells.
ESTHER : Andersen_2013_Proc.Natl.Acad.Sci.U.S.A_110_20819
PubMedSearch : Andersen_2013_Proc.Natl.Acad.Sci.U.S.A_110_20819
PubMedID: 24297903

Title : Inter-residue coupling contributes to high-affinity subtype-selective binding of alpha-bungarotoxin to nicotinic receptors - Sine_2013_Biochem.J_454_311
Author(s) : Sine SM , Huang S , Li SX , daCosta CJ , Chen L
Ref : Biochemical Journal , 454 :311 , 2013
Abstract : The crystal structure of a pentameric alpha7 ligand-binding domain chimaera with bound alpha-btx (alpha-bungarotoxin) showed that of the five conserved aromatic residues in alpha7, only Tyr184 in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr184 depends on local residues, we generated mutations in an alpha7/5HT3A (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured 125I-labelled alpha-btx binding. The results show that mutations of individual residues near Tyr184 do not affect alpha-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurements show that the affinity decreases arise through increases in the alpha-btx dissociation rate with little change in the association rate. Replacing loop C in alpha7 with loop C from the alpha-btx-insensitive alpha2 or alpha3 subunits abolishes high-affinity alpha-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the alpha2 and alpha3 construct, mutating either residue that flanks Tyr184 to its alpha7 counterpart restores high-affinity alpha-btx binding. Analogously, in alpha7, mutating both residues that flank Tyr184 to the alpha2 or alpha3 counterparts abolishes high-affinity alpha-btx binding. Thus interaction between Tyr184 and local residues contributes to high-affinity subtype-selective alpha-btx binding.
ESTHER : Sine_2013_Biochem.J_454_311
PubMedSearch : Sine_2013_Biochem.J_454_311
PubMedID: 23802200

Title : Stoichiometry for drug potentiation of a pentameric ion channel - daCosta_2013_Proc.Natl.Acad.Sci.U.S.A_110_6595
Author(s) : daCosta CJ , Sine SM
Ref : Proc Natl Acad Sci U S A , 110 :6595 , 2013
Abstract : Drug modulation of ion channels is a powerful means to alter physiological responses for therapeutic benefit, yet the structural bases of modulation remain poorly understood. Here we study potentiation of nicotinic alpha7 acetylcholine receptors, which are emerging drug targets in several neurological disorders. alpha7 receptors are ligand-gated ion channels composed of five identical subunits, each bearing a site for the potentiating drug PNU-120596 (PNU). How the individual subunits contribute to PNU potentiation is not known. Taking advantage of a PNU-resistant mutant, we generated receptors composed of normal and PNU-resistant subunits and tagged one of the subunits with conductance mutations to report subunit stoichiometry. We then used patch clamp recording to monitor PNU potentiation of single alpha7 receptors with defined stoichiometry in real time. We find that potentiation depends steeply on the number of PNU-resistant subunits and that four, and possibly five, subunits must be sensitive to PNU for potentiation to occur. Thus, by monitoring the activity of every possible subunit combination, our findings predict that at the macroscopic level, PNU potentiation is highly cooperative.
ESTHER : daCosta_2013_Proc.Natl.Acad.Sci.U.S.A_110_6595
PubMedSearch : daCosta_2013_Proc.Natl.Acad.Sci.U.S.A_110_6595
PubMedID: 23576748

Title : Complex between alpha-bungarotoxin and an alpha7 nicotinic receptor ligand-binding domain chimaera - Huang_2013_Biochem.J_454_303
Author(s) : Huang S , Li SX , Bren N , Cheng K , Gomoto R , Chen L , Sine SM
Ref : Biochemical Journal , 454 :303 , 2013
Abstract : To identify high-affinity interactions between long-chain alpha-neurotoxins and nicotinic receptors, we determined the crystal structure of the complex between alpha-btx (alpha-bungarotoxin) and a pentameric ligand-binding domain constructed from the human alpha7 AChR (acetylcholine receptor) and AChBP (acetylcholine-binding protein). The complex buries ~2000 A2 (1 A=0.1 nm) of surface area, within which Arg36 and Phe32 from finger II of alpha-btx form a pi-cation stack that aligns edge-to-face with the conserved Tyr184 from loop-C of alpha7, while Asp30 of alpha-btx forms a hydrogen bond with the hydroxy group of Tyr184. These inter-residue interactions diverge from those in a 4.2 A structure of alpha-ctx (alpha-cobratoxin) bound to AChBP, but are similar to those in a 1.94 A structure of alpha-btx bound to the monomeric alpha1 extracellular domain, although compared with the monomer-bound complex, the alpha-btx backbone exhibits a large shift relative to the protein surface. Mutational analyses show that replacing Tyr184 with a threonine residue abolishes high-affinity alpha-btx binding, whereas replacing with a phenylalanine residue maintains high affinity. Comparison of the alpha-btx complex with that coupled to the agonist epibatidine reveals structural rearrangements within the binding pocket and throughout each subunit. The overall findings highlight structural principles by which alpha-neurotoxins interact with nicotinic receptors.
ESTHER : Huang_2013_Biochem.J_454_303
PubMedSearch : Huang_2013_Biochem.J_454_303
PubMedID: 23800261

Title : Nicotinic receptor transduction zone: invariant arginine couples to multiple electron-rich residues - Mukhtasimova_2013_Biophys.J_104_355
Author(s) : Mukhtasimova N , Sine SM
Ref : Biophysical Journal , 104 :355 , 2013
Abstract : Gating of the muscle-type acetylcholine receptor (AChR) channel depends on communication between the ACh-binding site and the remote ion channel. A key region for this communication is located within the structural transition zone between the ligand-binding and pore domains. Here, stemming from beta-strand 10 of the binding domain, the invariant alphaArg209 lodges within the hydrophobic interior of the subunit and is essential for rapid and efficient channel gating. Previous charge-reversal experiments showed that the contribution of alphaArg209 to channel gating depends strongly on alphaGlu45, also within this region. Here we determine whether the contribution of alphaArg209 to channel gating depends on additional anionic or electron-rich residues in this region. Also, to reconcile diverging findings in the literature, we compare the dependence of alphaArg209 on alphaGlu45 in AChRs from different species, and compare the full agonist ACh with the weak agonist choline. Our findings reveal that the contribution of alphaArg209 to channel gating depends on additional nearby electron-rich residues, consistent with both electrostatic and steric contributions. Furthermore, alphaArg209 and alphaGlu45 show a strong interdependence in both human and mouse AChRs, whereas the functional consequences of the mutation alphaE45R depend on the agonist. The emerging picture shows a multifaceted network of interdependent residues that are required for communication between the ligand-binding and pore domains.
ESTHER : Mukhtasimova_2013_Biophys.J_104_355
PubMedSearch : Mukhtasimova_2013_Biophys.J_104_355
PubMedID: 23442857

Title : Highly fatal fast-channel syndrome caused by AChR epsilon subunit mutation at the agonist binding site - Shen_2012_Neurology_79_449
Author(s) : Shen XM , Brengman JM , Edvardson S , Sine SM , Engel AG
Ref : Neurology , 79 :449 , 2012
Abstract : OBJECTIVE: To characterize the molecular basis of a novel fast-channel congenital myasthenic syndrome.
METHODS: We used the candidate gene approach to identify the pathogenic mutation in the acetylcholine receptor (AChR) epsilon subunit, genetically engineered the mutant AChR into HEK cells, and evaluated the level of expression and kinetic properties of the mutant receptor.
RESULTS: An 8-year-old boy born to consanguineous parents had severe myasthenic symptoms since birth. He is wheelchair bound and pyridostigmine therapy enables him to take only a few steps. Three similarly affected siblings died in infancy. He carries a homozygous p.W55R mutation at the alpha/epsilon subunit interface of the AChR agonist binding site. The mutant protein expresses well in HEK cells. Patch-clamp analysis of the mutant receptor expressed in HEK cells reveals 30-fold reduced apparent agonist affinity, 75-fold reduced apparent gating efficiency, and strikingly attenuated channel opening probability (P(open)) over a range agonist concentrations. CONCLUSION: Introduction of a cationic Arg into the anionic environment of alpha/epsilon AChR binding site hinders stabilization of cationic ACh by aromatic residues and accounts for the markedly perturbed kinetic properties of the receptor. The very low P(open) explains the poor response to pyridostigmine and the high fatality of the disease.
ESTHER : Shen_2012_Neurology_79_449
PubMedSearch : Shen_2012_Neurology_79_449
PubMedID: 22592360

Title : Myasthenic syndrome AChRalpha C-loop mutant disrupts initiation of channel gating - Shen_2012_J.Clin.Invest_122_2613
Author(s) : Shen XM , Brengman JM , Sine SM , Engel AG
Ref : J Clinical Investigation , 122 :2613 , 2012
Abstract : Congenital myasthenic syndromes (CMSs) are neuromuscular disorders that can be caused by defects in ace-tylcholine receptor (AChR) function. Disease-associated point mutants can reveal the unsuspected functional significance of mutated residues. We identified two pathogenic mutations in the extracellular domain of the AChR alpha subunit (AChRalpha) in a patient with myasthenic symptoms since birth: a V188M mutation in the C-loop and a heteroallelic G74C mutation in the main immunogenic region. The G74C mutation markedly reduced surface AChR expression in cultured cells, whereas the V188M mutant was expressed robustly but had severely impaired kinetics. Single-channel patch-clamp analysis indicated that V188M markedly decreased the apparent AChR channel opening rate and gating efficiency. Mutant cycle analysis of energetic coupling among conserved residues within or dispersed around the AChRalpha C-loop revealed that V188 is functionally linked to Y190 in the C-loop and to D200 in beta-strand 10, which connects to the M1 transmembrane domain. Furthermore, V188M weakens inter-residue coupling of K145 in beta-strand 7 with Y190 and with D200. Cumulatively, these results indicate that V188 of AChRalpha is part of an interdependent tetrad that contributes to rearrangement of the C-loop during the initial coupling of agonist binding to channel gating.
ESTHER : Shen_2012_J.Clin.Invest_122_2613
PubMedSearch : Shen_2012_J.Clin.Invest_122_2613
PubMedID: 22728938

Title : End-plate acetylcholine receptor: structure, mechanism, pharmacology, and disease - Sine_2012_Physiol.Rev_92_1189
Author(s) : Sine SM
Ref : Physiol Rev , 92 :1189 , 2012
Abstract : The synapse is a localized neurohumoral contact between a neuron and an effector cell and may be considered the quantum of fast intercellular communication. Analogously, the postsynaptic neurotransmitter receptor may be considered the quantum of fast chemical to electrical transduction. Our understanding of postsynaptic receptors began to develop about a hundred years ago with the demonstration that electrical stimulation of the vagus nerve released acetylcholine and slowed the heart beat. During the past 50 years, advances in understanding postsynaptic receptors increased at a rapid pace, owing largely to studies of the acetylcholine receptor (AChR) at the motor endplate. The endplate AChR belongs to a large superfamily of neurotransmitter receptors, called Cys-loop receptors, and has served as an exemplar receptor for probing fundamental structures and mechanisms that underlie fast synaptic transmission in the central and peripheral nervous systems. Recent studies provide an increasingly detailed picture of the structure of the AChR and the symphony of molecular motions that underpin its remarkably fast and efficient chemoelectrical transduction.
ESTHER : Sine_2012_Physiol.Rev_92_1189
PubMedSearch : Sine_2012_Physiol.Rev_92_1189
PubMedID: 22811427

Title : Intramembrane proton binding site linked to activation of bacterial pentameric ion channel - Wang_2012_J.Biol.Chem_287_6482
Author(s) : Wang HL , Cheng X , Sine SM
Ref : Journal of Biological Chemistry , 287 :6482 , 2012
Abstract : Prokaryotic orthologs of eukaryotic Cys-loop receptor channels recently emerged as structural and mechanistic surrogates to investigate this superfamily of intercellular signaling proteins. Here, we examine proton activation of the prokaryotic ortholog GLIC using patch clamp electrophysiology, mutagenesis, and molecular dynamics (MD) simulations. Whole-cell current recordings from human embryonic kidney (HEK) 293 cells expressing GLIC show half-maximal activation at pH 6, close to the pK(a) of histidine, implicating the three native His residues in proton sensing linked to activation. The mutation H235F abolishes proton activation, H277Y is without effect, and all nine mutations of His-127 prevent expression on the cell surface. In the GLIC crystal structure, His-235 on transmembrane (TM) alpha-helix 2, hydrogen bonds to the main chain carbonyl oxygen of Ile-259 on TM alpha-helix 3. MD simulations show that when His-235 is protonated, the hydrogen bond persists, and the channel remains in the open conformation, whereas when His-235 is deprotonated, the hydrogen bond dissociates, and the channel closes. Mutations of the proximal Tyr-263, which also links TM alpha-helices 2 and 3 via a hydrogen bond, alter proton sensitivity over a 1.5 pH unit range. MD simulations show that mutations of Tyr-263 alter the hydrogen bonding capacity of His-235. The overall findings show that His-235 in the TM region of GLIC is a novel proton binding site linked to channel activation.
ESTHER : Wang_2012_J.Biol.Chem_287_6482
PubMedSearch : Wang_2012_J.Biol.Chem_287_6482
PubMedID: 22084238

Title : Ligand-binding domain of an alpha7-nicotinic receptor chimera and its complex with agonist - Li_2011_Nat.Neurosci_14_1253
Author(s) : Li SX , Huang S , Bren N , Noridomi K , Dellisanti CD , Sine SM , Chen L
Ref : Nat Neurosci , 14 :1253 , 2011
Abstract : The alpha(7) acetylcholine receptor (AChR) mediates pre- and postsynaptic neurotransmission in the central nervous system and is a potential therapeutic target in neurodegenerative, neuropsychiatric and inflammatory disorders. We determined the crystal structure of the extracellular domain of a receptor chimera constructed from the human alpha(7) AChR and Lymnaea stagnalis acetylcholine binding protein (AChBP), which shares 64% sequence identity and 71% similarity with native alpha(7). We also determined the structure with bound epibatidine, a potent AChR agonist. Comparison of the structures revealed molecular rearrangements and interactions that mediate agonist recognition and early steps in signal transduction in alpha(7) AChRs. The structures further revealed a ring of negative charge within the central vestibule, poised to contribute to cation selectivity. Structure-guided mutational studies disclosed distinctive contributions to agonist recognition and signal transduction in alpha(7) AChRs. The structures provide a realistic template for structure-aided drug design and for defining structure-function relationships of alpha(7) AChRs.
ESTHER : Li_2011_Nat.Neurosci_14_1253
PubMedSearch : Li_2011_Nat.Neurosci_14_1253
PubMedID: 21909087

Title : Single-channel and structural foundations of neuronal alpha7 acetylcholine receptor potentiation - daCosta_2011_J.Neurosci_31_13870
Author(s) : daCosta CJ , Free CR , Corradi J , Bouzat C , Sine SM
Ref : Journal of Neuroscience , 31 :13870 , 2011
Abstract : Potentiation of neuronal nicotinic acetylcholine receptors by exogenous ligands is a promising strategy for treatment of neurological disorders including Alzheimer's disease and schizophrenia. To gain insight into molecular mechanisms underlying potentiation, we examined ACh-induced single-channel currents through the human neuronal alpha7 acetylcholine receptor in the presence of the alpha7-specific potentiator PNU-120596 (PNU). Compared to the unusually brief single-channel opening episodes elicited by agonist alone, channel opening episodes in the presence of agonist and PNU are dramatically prolonged. Dwell time analysis reveals that PNU introduces two novel components into open time histograms, indicating at least two degrees of PNU-induced potentiation. Openings of the longest potentiated class coalesce into clusters whose frequency and duration change over a narrow range of PNU concentration. At PNU concentrations approaching saturation, these clusters last up to several minutes, prolonging the submillisecond alpha7 opening episodes by several orders of magnitude. Mutations known to reduce PNU potentiation at the whole-cell level still give rise to multisecond-long single-channel clusters. However mutation of five residues lining a cavity within each subunit's transmembrane domain abolishes PNU potentiation, defining minimal structural determinants of PNU potentiation.
ESTHER : daCosta_2011_J.Neurosci_31_13870
PubMedSearch : daCosta_2011_J.Neurosci_31_13870
PubMedID: 21957249

Title : Functional relationships between agonist binding sites and coupling regions of homomeric Cys-loop receptors - Andersen_2011_J.Neurosci_31_3662
Author(s) : Andersen N , Corradi J , Bartos M , Sine SM , Bouzat C
Ref : Journal of Neuroscience , 31 :3662 , 2011
Abstract : Each subunit in a homopentameric Cys-loop receptor contains a specialized coupling region positioned between the agonist binding domain and the ion conductive channel. To determine the contribution of each coupling region to the stability of the open channel, we constructed a receptor subunit (alpha7-5-HT(3A)) with both a disabled coupling region and a reporter mutation that alters unitary conductance, and coexpressed normal and mutant subunits. The resulting receptors show single-channel current amplitudes that are quantized according to the number of reporter mutations per receptor, allowing correlation of the number of intact coupling regions with mean open time. We find that each coupling region contributes an equal increment to the stability of the open channel. However, by altering the numbers and locations of active coupling regions and binding sites, we find that a coupling region in a subunit flanked by inactive binding sites can still stabilize the open channel. We also determine minimal requirements for channel opening regardless of stability and find that channel opening can occur in a receptor with one active coupling region flanked by functional binding sites or with one active binding site flanked by functional coupling regions. The overall findings show that, whereas the agonist binding sites contribute interdependently and asymmetrically to open-channel stability, the coupling regions contribute independently and symmetrically.
ESTHER : Andersen_2011_J.Neurosci_31_3662
PubMedSearch : Andersen_2011_J.Neurosci_31_3662
PubMedID: 21389221

Title : On the origin of ion selectivity in the Cys-loop receptor family - Sine_2010_J.Mol.Neurosci_40_70
Author(s) : Sine SM , Wang HL , Hansen S , Taylor P
Ref : Journal of Molecular Neuroscience , 40 :70 , 2010
Abstract : Agonist binding to Cys-loop receptors promotes a large transmembrane ion flux of several million cations or anions per second. To investigate structural bases for the dynamics (MD) simulations, X-ray crystallography, and single channel recording. MD simulations of the muscle nicotinic receptor, imbedded in a lipid bilayer with an applied transmembrane potential, reveal single cation translocation events during transient periods of channel hydration. During the simulation trajectory, cations paused for prolonged periods near several rings of anionic residues projecting from the lumen of the extracellular domain of the receptor, but subsequently the cation moved rapidly through the hydrophobic transmembrane region as the constituent alpha-helices exhibited back and forth rocking motions. Cocrystallization of acetylcholine binding protein with sulfate ions revealed coordination of five sulfates with residues from one of these charged rings; in cation-selective Cys-loop receptors this ring contains negatively charged residues, whereas in anion-selective receptors it contains positively charged residues. In the muscle nicotinic receptor, charge reversal of residues of this ring decreases unitary conductance by up to 80%. Thus in Cys-loop receptors, a series of charged rings along the ion translocation pathway concentrates hydrated ions relative to bulk solution, giving rise to charge selectivity, and then subtle motions of the hydrophobic transmembrane, coupled with transient periods of water filling, enable rapid ion flux.
ESTHER : Sine_2010_J.Mol.Neurosci_40_70
PubMedSearch : Sine_2010_J.Mol.Neurosci_40_70
PubMedID: 19728176

Title : What have we learned from the congenital myasthenic syndromes - Engel_2010_J.Mol.Neurosci_40_143
Author(s) : Engel AG , Shen XM , Selcen D , Sine SM
Ref : Journal of Molecular Neuroscience , 40 :143 , 2010
Abstract : The congenital myasthenic syndromes have now been traced to an array of molecular targets at the neuromuscular junction encoded by no fewer than 11 disease genes. The disease genes were identified by the candidate gene approach, using clues derived from clinical, electrophysiological, cytochemical, and ultrastructural features. For example, electrophysiologic studies in patients suffering from sudden episodes of apnea pointed to a defect in acetylcholine resynthesis and CHAT as the candidate gene (Ohno et al., Proc Natl Acad Sci USA 98:2017-2022, 2001); refractoriness to anticholinesterase medications and partial or complete absence of acetylcholinesterase (AChE) from the endplates (EPs) has pointed to one of the two genes (COLQ and ACHE ( T )) encoding AChE, though mutations were observed only in COLQ. After a series of patients carrying mutations in a disease gene have been identified, the emerging genotype-phenotype correlations provided clues for targeted mutation analysis in other patients. Mutations in EP-specific proteins also prompted expression studies that proved pathogenicity, highlighted important functional domains of the abnormal proteins, and pointed to rational therapy.
ESTHER : Engel_2010_J.Mol.Neurosci_40_143
PubMedSearch : Engel_2010_J.Mol.Neurosci_40_143
PubMedID: 19688192

Title : Molecular-dynamics simulations of ELIC-a prokaryotic homologue of the nicotinic acetylcholine receptor - Cheng_2009_Biophys.J_96_4502
Author(s) : Cheng X , Ivanov I , Wang H , Sine SM , McCammon JA
Ref : Biophysical Journal , 96 :4502 , 2009
Abstract : The ligand-gated ion channel from Erwinia chrysanthemi (ELIC) is a prokaryotic homolog of the eukaryotic nicotinic acetylcholine receptor (nAChR) that responds to the binding of neurotransmitter acetylcholine and mediates fast signal transmission. ELIC is similar to the nAChR in its primary sequence and overall subunit organization, but despite their structural similarity, it is not clear whether these two ligand-gated ion channels operate in a similar manner. Further, it is not known to what extent mechanistic insights gleaned from the ELIC structure translate to eukaryotic counterparts such as the nAChR. Here we use molecular-dynamics simulations to probe the conformational dynamics and hydration of the transmembrane pore of ELIC. The results are compared with those from our previous simulation of the human alpha7 nAChR. Overall, ELIC displays increased stability compared to the nAChR, whereas the two proteins exhibit remarkable similarity in their global motion and flexibility patterns. The majority of the increased stability of ELIC does not stem from the deficiency of the models used in the simulations, and but rather seems to have a structural basis. Slightly altered dynamical correlation features are also observed among several loops within the membrane region. In sharp contrast to the nAChR, ELIC is completely dehydrated from the pore center to the extracellular end throughout the simulation. Finally, the simulation of an ELIC mutant substantiates the important role of F246 on the stability, hydration and possibly function of the ELIC channel.
ESTHER : Cheng_2009_Biophys.J_96_4502
PubMedSearch : Cheng_2009_Biophys.J_96_4502
PubMedID: 19486673

Title : Single-channel current through nicotinic receptor produced by closure of binding site C-loop - Wang_2009_Biophys.J_96_3582
Author(s) : Wang HL , Toghraee R , Papke D , Cheng XL , McCammon JA , Ravaioli U , Sine SM
Ref : Biophysical Journal , 96 :3582 , 2009
Abstract : We investigated the initial coupling of agonist binding to channel gating of the nicotinic acetylcholine receptor using targeted molecular-dynamics (TMD) simulation. After TMD simulation to accelerate closure of the C-loops at the agonist binding sites, the region of the pore that passes through the cell membrane expands. To determine whether the structural changes in the pore result in ion conduction, we used a coarse-grained ion conduction simulator, Biology Boltzmann transport Monte Carlo, and applied it to two structural frames taken before and after TMD simulation. The structural model before TMD simulation represents the channel in the proposed "resting" state, whereas the model after TMD simulation represents the channel in the proposed "active" state. Under external voltage biases, the channel in the "active" state was permeable to cations. Our simulated ion conductance approaches that obtained experimentally and recapitulates several functional properties characteristic of the nicotinic acetylcholine receptor. Thus, closure of the C-loop triggers a structural change in the channel sufficient to account for the open channel current. This approach of applying Biology Boltzmann transport Monte Carlo simulation can be used to further investigate the binding to gating transduction mechanism and the structural bases for ion selection and translocation.
ESTHER : Wang_2009_Biophys.J_96_3582
PubMedSearch : Wang_2009_Biophys.J_96_3582
PubMedID: 19413963

Title : Number and locations of agonist binding sites required to activate homomeric Cys-loop receptors - Rayes_2009_J.Neurosci_29_6022
Author(s) : Rayes D , De Rosa MJ , Sine SM , Bouzat C
Ref : Journal of Neuroscience , 29 :6022 , 2009
Abstract : Homo-pentameric Cys-loop receptors contain five identical agonist binding sites, each formed at a subunit interface. To determine the number and locations of binding sites required to generate a stable active state, we constructed a receptor subunit with a mutation that disables the agonist binding site and a reporter mutation that alters unitary conductance and coexpressed mutant and nonmutant subunits. Although receptors with a range of different subunit compositions are produced, patch-clamp recordings reveal that the amplitude of each single-channel opening event reports the number and, for certain subunit combinations, the locations of subunits with intact binding sites. We find that receptors with three binding sites at nonconsecutive subunit interfaces exhibit maximal mean channel open time, receptors with binding sites at three consecutive or two nonconsecutive interfaces exhibit intermediate open time, and receptors with binding sites at two consecutive or one interface exhibit brief open time. Macroscopic recordings after rapid application of agonist reveal that channel activation slows and the extent of desensitization decreases as the number of binding sites per receptor decreases. The overall results provide a framework for defining mechanisms of activation and drug modulation for homo-pentameric Cys-loop receptors.
ESTHER : Rayes_2009_J.Neurosci_29_6022
PubMedSearch : Rayes_2009_J.Neurosci_29_6022
PubMedID: 19420269

Title : Detection and trapping of intermediate states priming nicotinic receptor channel opening - Mukhtasimova_2009_Nature_459_451
Author(s) : Mukhtasimova N , Lee WY , Wang HL , Sine SM
Ref : Nature , 459 :451 , 2009
Abstract : In the course of synaptic transmission in the brain and periphery, acetylcholine receptors (AChRs) rapidly transduce a chemical signal into an electrical impulse. The speed of transduction is facilitated by rapid ACh association and dissociation, suggesting a binding site relatively non-selective for small cations. Selective transduction has been thought to originate from the ability of ACh, over that of other organic cations, to trigger the subsequent channel-opening step. However, transitions to and from the open state were shown to be similar for agonists with widely different efficacies. By studying mutant AChRs, we show here that the ultimate closed-to-open transition is agonist-independent and preceded by two primed closed states; the first primed state elicits brief openings, whereas the second elicits long-lived openings. Long-lived openings and the associated primed state are detected in the absence and presence of an agonist, and exhibit the same kinetic signatures under both conditions. By covalently locking the agonist-binding sites in the bound conformation, we find that each site initiates a priming step. Thus, a change in binding-site conformation primes the AChR for channel opening in a process that enables selective activation by ACh while maximizing the speed and efficiency of the biological response.
ESTHER : Mukhtasimova_2009_Nature_459_451
PubMedSearch : Mukhtasimova_2009_Nature_459_451
PubMedID: 19339970

Title : Binding to gating transduction in nicotinic receptors: Cys-loop energetically couples to pre-M1 and M2-M3 regions - Lee_2009_J.Neurosci_29_3189
Author(s) : Lee WY , Free CR , Sine SM
Ref : Journal of Neuroscience , 29 :3189 , 2009
Abstract : The nicotinic acetylcholine receptor (AChR) transduces binding of nerve-released ACh into opening of an intrinsic ion channel, yet the intraprotein interactions behind transduction remain to be fully elucidated. Attention has focused on the region of the AChR in which the beta1-beta2 and Cys-loops from the extracellular domain project into a cavity framed by residues preceding the first transmembrane domain (pre-M1) and the linker spanning transmembrane domains M2 and M3. Previous studies identified a principal transduction pathway in which the pre-M1 domain is coupled to the M2-M3 linker through the beta1-beta2 loop. Here we identify a parallel pathway in which the pre-M1 domain is coupled to the M2-M3 linker through the Cys-loop. Mutagenesis, single-channel kinetic analyses and thermodynamic mutant cycle analyses reveal energetic coupling among alphaLeu 210 from the pre-M1 domain, alphaPhe 135 and alphaPhe 137 from the Cys-loop, and alphaLeu 273 from the M2-M3 linker. Residues at equivalent positions of non-alpha-subunits show negligible coupling, indicating these interresidue couplings are specific to residues in the alpha-subunit. Thus, the extracellular beta1-beta2 and Cys-loops bridge the pre-M1 domain and M2-M3 linker to transduce agonist binding into channel gating.
ESTHER : Lee_2009_J.Neurosci_29_3189
PubMedSearch : Lee_2009_J.Neurosci_29_3189
PubMedID: 19279256

Title : Congenital myasthenia-related AChR delta subunit mutation interferes with intersubunit communication essential for channel gating - Shen_2008_J.Clin.Invest_118_1867
Author(s) : Shen XM , Fukuda T , Ohno K , Sine SM , Engel AG
Ref : J Clinical Investigation , 118 :1867 , 2008
Abstract : Congenital myasthenias (CMs) arise from defects in neuromuscular junction-associated proteins. Deciphering the molecular bases of the CMs is required for therapy and illuminates structure-function relationships in these proteins. Here, we analyze the effects of a mutation in 1 of 4 homologous subunits in the AChR from a CM patient, a Leu to Pro mutation at position 42 of the delta subunit. The mutation is located in a region of contact between subunits required for rapid opening of the AChR channel and impedes the rate of channel opening. Substitutions of Gly, Lys, or Asp for deltaL42, or substitutions of Pro along the local protein chain, also slowed channel opening. Substitution of Pro for Leu in the epsilon subunit slowed opening, whereas this substitution had no effect in the beta subunit and actually sped opening in the alpha subunit. Analyses of energetic coupling between residues at the subunit interface showed that deltaL42 is functionally linked to alphaT127, a key residue in the adjacent alpha subunit required for rapid channel opening. Thus, deltaL42 is part of an intersubunit network that enables ACh binding to rapidly open the AChR channel, which may be compromised in patients with CM.
ESTHER : Shen_2008_J.Clin.Invest_118_1867
PubMedSearch : Shen_2008_J.Clin.Invest_118_1867
PubMedID: 18398509

Title : Recent structural and mechanistic insights into endplate acetylcholine receptors - Sine_2008_Ann.N.Y.Acad.Sci_1132_53
Author(s) : Sine SM , Gao F , Lee WY , Mukhtasimova N , Wang HL , Engel AG
Ref : Annals of the New York Academy of Sciences , 1132 :53 , 2008
Abstract : Voluntary movement mediated by skeletal muscle relies on endplate acetylcholine receptors (AChR) to detect nerve-released ACh and depolarize the muscle fiber. Recent structural and mechanistic studies of the endplate AChR have catalyzed a leap in our understanding of the molecular steps in this chemical-to-electrical transduction process. Studies of acetylcholine binding protein (AChBP) give insight into ACh recognition, the first step in activation of the AChR. An atomic structural model of the Torpedo AChR at a resolution of 0.4 nm, together with single-ion channel recording methods, allow tracing of the link between the agonist binding event and gating of the ion channel, as well as determination of how the channel moves when it opens to allow flow of cations. Structural models of the human AChR enable precise mapping of disease-causing mutations, while studies of the speed with which single AChR channels open and close cast light on pathogenic mechanisms.
ESTHER : Sine_2008_Ann.N.Y.Acad.Sci_1132_53
PubMedSearch : Sine_2008_Ann.N.Y.Acad.Sci_1132_53
PubMedID: 18567853

Title : Control of cation permeation through the nicotinic receptor channel - Wang_2008_PLoS.Comput.Biol_4_e41
Author(s) : Wang HL , Cheng X , Taylor P , McCammon JA , Sine SM
Ref : PLoS Comput Biol , 4 :e41 , 2008
Abstract : We used molecular dynamics (MD) simulations to explore the transport of single cations through the channel of the muscle nicotinic acetylcholine receptor (nAChR). Four MD simulations of 16 ns were performed at physiological and hyperpolarized membrane potentials, with and without restraints of the structure, but all without bound agonist. With the structure unrestrained and a potential of -100 mV, one cation traversed the channel during a transient period of channel hydration; at -200 mV, the channel was continuously hydrated and two cations traversed the channel. With the structure restrained, however, cations did not traverse the channel at either membrane potential, even though the channel was continuously hydrated. The overall results show that cation selective transport through the nAChR channel is governed by electrostatic interactions to achieve charge selectivity, but ion translocation relies on channel hydration, facilitated by a trans-membrane field, coupled with dynamic fluctuations of the channel structure.
ESTHER : Wang_2008_PLoS.Comput.Biol_4_e41
PubMedSearch : Wang_2008_PLoS.Comput.Biol_4_e41
PubMedID: 18282090

Title : Morantel allosterically enhances channel gating of neuronal nicotinic acetylcholine alpha 3 beta 2 receptors - Wu_2008_Mol.Pharmacol_74_466
Author(s) : Wu TY , Smith CM , Sine SM , Levandoski MM
Ref : Molecular Pharmacology , 74 :466 , 2008
Abstract : We studied allosteric potentiation of rat alpha3beta2 neuronal nicotinic acetylcholine receptors (nAChRs) by the anthelmintic compound morantel. Macroscopic currents evoked by acetylcholine (ACh) from nAChRs expressed in Xenopus laevis oocytes increase up to 8-fold in the presence of low concentrations of morantel (< or =10 microM); the magnitude of the potentiation depends on both agonist and modulator concentrations. It is noteworthy that the potentiated currents exceed the maximum currents achieved by saturating (millimolar) concentrations of agonist. Studies of macroscopic currents elicited by prolonged drug applications (100-300 s) indicate that morantel does not increase alpha3beta2 receptor activity by reducing slow (> or =1 s) desensitization. Instead, using outside-out patch-clamp recordings, we demonstrate that morantel increases the frequency of single-channel openings and alters the bursting characteristics of the openings in a manner consistent with enhanced channel gating; these results quantitatively explain the macroscopic current potentiation. Morantel is a very weak agonist alone, but we show that the classic competitive antagonist dihydro-beta-erythroidine inhibits morantel-evoked currents noncompetitively, indicating that morantel does not bind to the canonical ACh binding sites.
ESTHER : Wu_2008_Mol.Pharmacol_74_466
PubMedSearch : Wu_2008_Mol.Pharmacol_74_466
PubMedID: 18458055

Title : The interface between extracellular and transmembrane domains of homomeric Cys-loop receptors governs open-channel lifetime and rate of desensitization - Bouzat_2008_J.Neurosci_28_7808
Author(s) : Bouzat C , Bartos M , Corradi J , Sine SM
Ref : Journal of Neuroscience , 28 :7808 , 2008
Abstract : The lifetimes of activated postsynaptic receptor channels contribute to the efficiency of synaptic transmission. Here we show that structural differences within the interface dividing extracellular and transmembrane domains of homomeric alpha7 and 5-HT(3A) receptors account for the large differences in open-channel lifetime and time of desensitization onset between these contrasting members of the Cys-loop receptor superfamily. For alpha7 receptors, agonist-evoked single-channel currents appear mainly as isolated brief openings (tau(o) = 0.35 ms), whereas macroscopic currents after a step pulse of agonist desensitize rapidly (tau(d) = 0.4 ms). In contrast for 5-HT(3A) receptors, agonist-evoked single-channel currents appear as clusters of many long openings in quick succession (tau(cluster) = 1.2 s), whereas macroscopic currents desensitize slowly (tau(d) = 1.1 s). A chimeric alpha7-5HT(3A) receptor exhibits functional properties intermediate between those of the parent receptors, but the functional signatures of each parent are reconstituted after substituting the major loops within the interface of the extracellular and transmembrane domains from the corresponding parent receptor. Furthermore, these structural loops contribute to open-channel lifetime and time of desensitization onset in a nonadditive manner. The results suggest that desensitization is the major determinant of the lifetimes of activated alpha7 and 5-HT(3A) receptors and that functional differences between the two receptors arise primarily through structural differences at the interface between extracellular and transmembrane domains.
ESTHER : Bouzat_2008_J.Neurosci_28_7808
PubMedSearch : Bouzat_2008_J.Neurosci_28_7808
PubMedID: 18667613

Title : Further observations in congenital myasthenic syndromes - Engel_2008_Ann.N.Y.Acad.Sci_1132_104
Author(s) : Engel AG , Shen XM , Selcen D , Sine SM
Ref : Annals of the New York Academy of Sciences , 1132 :104 , 2008
Abstract : During the past five years many patients suffering from congenital myasthenic syndromes (CMS) have been identified worldwide and novel causative genes and mutations have been discovered. The disease genes now include those encoding each subunit of the acetylcholine receptor (AChR), the ColQ part of acetylcholinesterase (AChE), choline acetyltransferase, Na(v)1.4, MuSK, and Dok-7. Moreover, emerging genotype-phenotype correlations are providing clues for targeted mutation analysis. This review focuses on the recent observations in selected CMS.
ESTHER : Engel_2008_Ann.N.Y.Acad.Sci_1132_104
PubMedSearch : Engel_2008_Ann.N.Y.Acad.Sci_1132_104
PubMedID: 18567859

Title : Nicotinic receptor interloop proline anchors beta1-beta2 and Cys loops in coupling agonist binding to channel gating - Lee_2008_J.Gen.Physiol_132_265
Author(s) : Lee WY , Free CR , Sine SM
Ref : Journal of General Physiology , 132 :265 , 2008
Abstract : Nicotinic acetylcholine receptors (AChRs) mediate rapid excitatory synaptic transmission throughout the peripheral and central nervous systems. They transduce binding of nerve-released ACh into opening of an intrinsic channel, yet the structural basis underlying transduction is not fully understood. Previous studies revealed a principal transduction pathway in which alphaArg 209 of the pre-M1 domain and alphaGlu 45 of the beta1-beta2 loop functionally link the two regions, positioning alphaVal 46 of the beta1-beta2 loop in a cavity formed by alphaPro 272 through alphaSer 269 of the M2-M3 loop. Here we investigate contributions of residues within and proximal to this pathway using single-channel kinetic analysis, site-directed mutagenesis, and thermodynamic mutant cycle analysis. We find that in contributing to channel gating, alphaVal 46 and alphaVal 132 of the signature Cys loop couple energetically to alphaPro 272. Furthermore, these residues are optimized in both their size and hydrophobicity to mediate rapid and efficient channel gating, suggesting naturally occurring substitutions at these positions enable a diverse range of gating rate constants among the Cys-loop receptor superfamily. The overall results indicate that alphaPro 272 functionally couples to flanking Val residues extending from the beta1-beta2 and Cys loops within the ACh binding to channel opening transduction pathway.
ESTHER : Lee_2008_J.Gen.Physiol_132_265
PubMedSearch : Lee_2008_J.Gen.Physiol_132_265
PubMedID: 18663134

Title : An ion selectivity filter in the extracellular domain of Cys-loop receptors reveals determinants for ion conductance - Hansen_2008_J.Biol.Chem_283_36066
Author(s) : Hansen SB , Wang HL , Taylor P , Sine SM
Ref : Journal of Biological Chemistry , 283 :36066 , 2008
Abstract : Neurotransmitter binding to Cys-loop receptors promotes a prodigious transmembrane flux of several million ions/s, but to date, structural determinants of ion flux have been identified flanking the membrane-spanning region. Using x-ray crystallography, sequence analysis, and single-channel recording, we identified a novel determinant of ion conductance near the point of entry of permeant ions. Co-crystallization of acetylcholine-binding protein with sulfate anions revealed coordination of SO4(2-) with a ring of lysines at a position equivalent to 24 A above the lipid membrane in homologous Cys-loop receptors. Analysis of multiple sequence alignments revealed that residues equivalent to the ring of lysines are negatively charged in cation-selective receptors but are positively charged in anion-selective receptors. Charge reversal of side chains at homologous positions in the nicotinic receptor from the motor end plate decreases unitary conductance up to 80%. Selectivity filters stemming from transmembrane alpha-helices have similar pore diameters and compositions of amino acids. These findings establish that when the channel opens under a physiological electrochemical gradient, permeant ions are initially stabilized within the extracellular vestibule of Cys-loop receptors, and this stabilization is a major determinant of ion conductance.
ESTHER : Hansen_2008_J.Biol.Chem_283_36066
PubMedSearch : Hansen_2008_J.Biol.Chem_283_36066
PubMedID: 18940802

Title : Barriers to ion translocation in cationic and anionic receptors from the Cys-loop family - Ivanov_2007_J.Am.Chem.Soc_129_8217
Author(s) : Ivanov I , Cheng X , Sine SM , McCammon JA
Ref : J Am Chem Soc , 129 :8217 , 2007
Abstract : Understanding the mechanisms of gating and ion permeation in biological channels and receptors has been a long-standing challenge in biophysics. Recent advances in structural biology have revealed the architecture of a number of transmembrane channels and allowed detailed, molecular-level insight into these systems. Herein, we have examined the barriers to ion conductance and origins of ion selectivity in models of the cationic human alpha7 nicotinic acetylcholine receptor (nAChR) and the anionic alpha1 glycine receptor (GlyR), based on the structure of Torpedo nAChR. Molecular dynamics simulations were used to determine water density profiles along the channel length, and they established that both receptor pores were fully hydrated. The very low water density in the middle of the nAChR pore indicated the existence of a hydrophobic constriction. By contrast, the pore of GlyR was lined with hydrophilic residues and remained well-hydrated throughout. Adaptive biasing force simulations allowed us to reconstruct potentials of mean force (PMFs) for chloride and sodium ions in the two receptors. For the nicotinic receptor we observed barriers to ion translocation associated with rings of hydrophobic residues-Val13' and Leu9'-in the middle of the transmembrane domain. This finding further substantiates the hydrophobic gating hypothesis for nAChR. The PMF revealed no significant hydrophobic barrier for chloride translocation in GlyR. For both receptors nonpermeant ions displayed considerable barriers. Thus, the overall electrostatics and the presence of rings of charged residues at the entrance and exit of the channels were sufficient to explain the experimentally observed anion and cation selectivity.
ESTHER : Ivanov_2007_J.Am.Chem.Soc_129_8217
PubMedSearch : Ivanov_2007_J.Am.Chem.Soc_129_8217
PubMedID: 17552523

Title : An intersubunit trigger of channel gating in the muscle nicotinic receptor - Mukhtasimova_2007_J.Neurosci_27_4110
Author(s) : Mukhtasimova N , Sine SM
Ref : Journal of Neuroscience , 27 :4110 , 2007
Abstract : Binding of neurotransmitter triggers gating of synaptic receptor channels, but our understanding of the structures that link the binding site to the channel is just beginning to develop. Here, we identify an intersubunit triggering element required for rapid and efficient gating of muscle nicotinic receptors using a structural model of the Torpedo receptor at 4 A resolution, recordings of currents through single receptor channels, measurements of inter-residue energetic coupling, and functional consequences of disulfide trapping. Mutation of the conserved residues, alphaTyr 127, epsilonAsn 39, and deltaAsn 41, located at the two subunit interfaces that form the agonist binding sites, markedly attenuates acetylcholine-elicited channel gating; mutant cycle analyses based on changes in the channel gating equilibrium constant reveal strong energetic coupling among these residues. After each residue is substituted with Cys, oxidizing conditions that promote disulfide bond formation attenuate gating of mutant, but not wild-type receptors. Gating is similarly attenuated when the Cys substitutions are confined to either of the binding-site interfaces, but can be restored by reducing conditions that promote disulfide bond breakage. Thus, the Tyr-Asn pair is an intersubunit trigger of rapid and efficient gating of muscle nicotinic receptors.
ESTHER : Mukhtasimova_2007_J.Neurosci_27_4110
PubMedSearch : Mukhtasimova_2007_J.Neurosci_27_4110
PubMedID: 17428989

Title : Nanosecond-timescale conformational dynamics of the human alpha7 nicotinic acetylcholine receptor - Cheng_2007_Biophys.J_93_2622
Author(s) : Cheng X , Ivanov I , Wang H , Sine SM , McCammon JA
Ref : Biophysical Journal , 93 :2622 , 2007
Abstract : We explore the conformational dynamics of a homology model of the human alpha7 nicotinic acetylcholine receptor using molecular dynamics simulation and analyses of root mean-square fluctuations, block partitioning of segmental motion, and principal component analysis. The results reveal flexible regions and concerted global motions of the subunits encompassing extracellular and transmembrane domains of the subunits. The most relevant motions comprise a bending, hinged at the beta10-M1 region, accompanied by concerted tilting of the M2 helices that widens the intracellular end of the channel. Despite the nanosecond timescale, the observations suggest that tilting of the M2 helices may initiate opening of the pore. The results also reveal direct coupling between a twisting motion of the extracellular domain and dynamic changes of M2. Covariance analysis of interresidue motions shows that this coupling arises through a network of residues within the Cys and M2-M3 loops where Phe135 is stabilized within a hydrophobic pocket formed by Leu270 and Ile271. The resulting concerted motion causes a downward shift of the M2 helices that disrupts a hydrophobic girdle formed by 9' and 13' residues.
ESTHER : Cheng_2007_Biophys.J_93_2622
PubMedSearch : Cheng_2007_Biophys.J_93_2622
PubMedID: 17573436

Title : Targeted molecular dynamics study of C-loop closure and channel gating in nicotinic receptors - Cheng_2006_PLoS.Comput.Biol_2_e134
Author(s) : Cheng X , Wang H , Grant B , Sine SM , McCammon JA
Ref : PLoS Comput Biol , 2 :e134 , 2006
Abstract : The initial coupling between ligand binding and channel gating in the human alpha7 nicotinic acetylcholine receptor (nAChR) has been investigated with targeted molecular dynamics (TMD) simulation. During the simulation, eight residues at the tip of the C-loop in two alternating subunits were forced to move toward a ligand-bound conformation as captured in the crystallographic structure of acetylcholine binding protein (AChBP) in complex with carbamoylcholine. Comparison of apo- and ligand-bound AChBP structures shows only minor rearrangements distal from the ligand-binding site. In contrast, comparison of apo and TMD simulation structures of the nAChR reveals significant changes toward the bottom of the ligand-binding domain. These structural rearrangements are subsequently translated to the pore domain, leading to a partly open channel within 4 ns of TMD simulation. Furthermore, we confirmed that two highly conserved residue pairs, one located near the ligand-binding pocket (Lys145 and Tyr188), and the other located toward the bottom of the ligand-binding domain (Arg206 and Glu45), are likely to play important roles in coupling agonist binding to channel gating. Overall, our simulations suggest that gating movements of the alpha7 receptor may involve relatively small structural changes within the ligand-binding domain, implying that the gating transition is energy-efficient and can be easily modulated by agonist binding/unbinding.
ESTHER : Cheng_2006_PLoS.Comput.Biol_2_e134
PubMedSearch : Cheng_2006_PLoS.Comput.Biol_2_e134
PubMedID: 17009865

Title : Slow-channel mutation in acetylcholine receptor alphaM4 domain and its efficient knockdown - Shen_2006_Ann.Neurol_60_128
Author(s) : Shen XM , Deymeer F , Sine SM , Engel AG
Ref : Annals of Neurology , 60 :128 , 2006
Abstract : OBJECTIVE: To identify the genetic basis of a slow-channel myasthenic syndrome, characterize functional properties of the mutant receptor, and selectively silence the mutant allele.
METHODS: We performed nutation analysis, cloning, and patch-clamp analysis of the functional properties of the mutant receptor; screening for a small interfering RNA with check plasmid; and assessed of the efficacy of small interfering RNA at the messenger RNA, protein, and functional levels.
RESULTS: We traced the cause of a slow-channel myasthenic syndrome to a C418W mutation in the M4 domain of the acetylcholine receptor alpha subunit. The mutation is the first one to occur spontaneously in an M4 domain of the receptor, and it is positioned within a stripe of hydrophobic residues facing the lipid bilayer. Kinetic analysis shows that alphaC418W enhances the channel opening equilibrium constant 26-fold without altering agonist affinity. Using a check plasmid as a screening tool, we identified a small interfering RNA that markedly suppresses the mutant but not the wild-type allele at the messenger RNA, protein, and functional levels. INTERPRETATION: alphaC418W occurring in humans causes a slow-channel syndrome by enhancing the relative stability of the channel open state. Efficient and selective knockdown of the mutant allele holds promise of therapeutic gene silencing.
ESTHER : Shen_2006_Ann.Neurol_60_128
PubMedSearch : Shen_2006_Ann.Neurol_60_128
PubMedID: 16685696

Title : Recent advances in Cys-loop receptor structure and function - Sine_2006_Nature_440_448
Author(s) : Sine SM , Engel AG
Ref : Nature , 440 :448 , 2006
Abstract : Throughout the nervous system, moment-to-moment communication relies on postsynaptic receptors to detect neurotransmitters and change the membrane potential. For the Cys-loop superfamily of receptors, recent structural data have catalysed a leap in our understanding of the three steps of chemical-to-electrical transduction: neurotransmitter binding, communication between the binding site and the barrier to ions, and opening and closing of the barrier. The emerging insights might be expected to explain how mutations of receptors cause neurological disease, but the opposite is generally true. Namely, analyses of disease-causing mutations have clarified receptor structure-function relationships as well as mechanisms governing the postsynaptic response.
ESTHER : Sine_2006_Nature_440_448
PubMedSearch : Sine_2006_Nature_440_448
PubMedID: 16554804

Title : Solution NMR of acetylcholine binding protein reveals agonist-mediated conformational change of the C-loop - Gao_2006_Mol.Pharmacol_70_1230
Author(s) : Gao F , Mer G , Tonelli M , Hansen SB , Burghardt TP , Taylor P , Sine SM
Ref : Molecular Pharmacology , 70 :1230 , 2006
Abstract : Previous X-ray crystallography, molecular dynamics simulation, fluorescence spectroscopy, and deuterium-hydrogen exchange of acetylcholine binding protein (AChBP) suggest that after binding of the agonist, the C-loop at the periphery of the binding site draws inward to cap the site and envelop the agonist. In this study, we use high-resolution solution NMR to monitor changes in the chemical environment of the C-loop without and with acetylcholine (ACh) bound. Substitution of [15N]cysteine for the native cysteines 123, 136, 187, and 188 provided intrinsic monitors of the chemical environments of the Cys- and C-loops, respectively. Two-dimensional transverse relaxation-optimized spectroscopy 15N-1H HSQC spectroscopy of apo-AChBP revealed seven well resolved cross-peaks for the group of cysteines. The spectrum of AChBP with Ser substituted for Cys 187 and 188 shows only two main cross-peaks, corresponding to Cys 123 and 136 from the Cys-loop, enabling resonance assignments. After binding of ACh, the five cross-peaks associated with cysteines from the C-loop condense into two predominant cross-peaks not observed in the spectrum from the apo protein, indicating a restricted range of conformations and change in chemical environment of the C-loop. The results show that isotopic cysteine can be incorporated into specified positions of AChBP expressed from a eukaryotic source, that the C-loop assumes multiple conformations without ACh, but that its conformation becomes restricted with ACh bound. The collective findings suggest a structural mechanism for agonist recognition in AChBP and related Cys-loop receptors.
ESTHER : Gao_2006_Mol.Pharmacol_70_1230
PubMedSearch : Gao_2006_Mol.Pharmacol_70_1230
PubMedID: 16847142

Title : Principal pathway coupling agonist binding to channel gating in nicotinic receptors - Lee_2005_Nature_438_243
Author(s) : Lee WY , Sine SM
Ref : Nature , 438 :243 , 2005
Abstract : Synaptic receptors respond to neurotransmitters by opening an intrinsic ion channel in the final step in synaptic transmission. How binding of the neurotransmitter is conveyed over the long distance to the channel remains a central question in neurobiology. Here we delineate a principal pathway that links neurotransmitter binding to channel gating by using a structural model of the Torpedo acetylcholine receptor at 4-A resolution, recordings of currents through single receptor channels and determinations of energetic coupling between pairs of residues. We show that a pair of invariant arginine and glutamate residues in each receptor alpha-subunit electrostatically links peripheral and inner beta-sheets from the binding domain and positions them to engage with the channel. The key glutamate and flanking valine residues energetically couple to conserved proline and serine residues emerging from the top of the channel-forming alpha-helix, suggesting that this is the point at which the binding domain triggers opening of the channel. The series of interresidue couplings identified here constitutes a primary allosteric pathway that links neurotransmitter binding to channel gating.
ESTHER : Lee_2005_Nature_438_243
PubMedSearch : Lee_2005_Nature_438_243
PubMedID: 16281039

Title : Subunit-specific contribution to agonist binding and channel gating revealed by inherited mutation in muscle acetylcholine receptor M3-M4 linker - Shen_2005_Brain_128_345
Author(s) : Shen XM , Ohno K , Sine SM , Engel AG
Ref : Brain , 128 :345 , 2005
Abstract : We trace the cause of congenital myasthenic syndromes in two patients to mutations in the epsilon subunit of the muscle acetylcholine receptor (AChR). Both patients harbour deletion of an asparagine residue in the epsilon subunit (epsilonN436del) at the C-terminus of the cytoplasmic loop linking the third (M3) and fourth (M4) transmembrane domains. The presence of a null mutation in the second allele of the epsilon subunit shows that epsilonN346del determines the phenotype. Endplate studies show markedly reduced expression of the epsilonN346del-AChR and compensatory accumulation of fetal gamma-AChR. Expression studies in HEK cells reveal decreased expression of epsilonN436del-AChR and abnormally brief channel openings. Thus, neuromuscular transmission is compromised by AChR deficiency, fast channel kinetics of the epsilonN346del-AChR and incomplete phenotypic rescue by gamma-AChR. Single-channel kinetic analysis shows that the epsilonN436del shortens channel openings by reducing stability of the diliganded receptor: rates of channel closing and of ACh dissociation are increased and the rate of channel opening is decreased. In addition to shortening the M3-M4 loop, epsilonN436del shifts a negatively charged aspartic acid residue adjacent to M4; the effects of epsilonN436del are shown to result from shortening of the M3-M4 loop and not from juxtaposition of a negative charge to M4. To determine whether the consequences of epsilonN346del are subunit-specific, we deleted residues that align with epsilonN436 in beta, delta and alpha subunits. Each deletion mutant reduces AChR expression, but whereas the beta and delta mutants curtail channel open duration, the alpha mutant strikingly prolongs open duration. Kinetic analysis reveals that the alpha mutant increases the stability of the diliganded receptor: rates of channel closing and of ACh dissociation are decreased and the rate of channel opening is increased. The overall studies reveal subunit asymmetry in the contributions of the M3-M4 loops in optimizing AChR activation through allosteric links to the channel and the agonist binding site.
ESTHER : Shen_2005_Brain_128_345
PubMedSearch : Shen_2005_Brain_128_345
PubMedID: 15615813

Title : Ligand-induced conformational change in the alpha7 nicotinic receptor ligand binding domain - Henchman_2005_Biophys.J_88_2564
Author(s) : Henchman RH , Wang HL , Sine SM , Taylor P , McCammon JA
Ref : Biophysical Journal , 88 :2564 , 2005
Abstract : Molecular dynamics simulations of a homology model of the ligand binding domain of the alpha7 nicotinic receptor are conducted with a range of bound ligands to induce different conformational states. Four simulations of 15 ns each are run with no ligand, antagonist d-tubocurarine (dTC), agonist acetylcholine (ACh), and agonist ACh with potentiator Ca(2+), to give insight into the conformations of the active and inactive states of the receptor and suggest the mechanism for conformational change. The main structural factor distinguishing the active and inactive states is that a more open, symmetric arrangement of the five subunits arises for the two agonist simulations, whereas a more closed and asymmetric arrangement results for the apo and dTC cases. Most of the difference arises in the lower portion of the ligand binding domain near its connection to the adjacent transmembrane domain. The transfer of the more open state to the transmembrane domain could then promote ion flow through the channel. Variation in how subunits pack together with no ligand bound appears to give rise to asymmetry in the apo case. The presence of dTC expands the receptor but induces rotations in alternate directions in adjacent subunits that lead to an asymmetric arrangement as in the apo case. Ca(2+) appears to promote a slightly greater expansion in the subunits than ACh alone by stabilizing the C-loop and ACh positions. Although the simulations are unlikely to be long enough to view the full conformational changes between open and closed states, a collection of different motions at a range of length scales are observed that are likely to participate in the conformational change.
ESTHER : Henchman_2005_Biophys.J_88_2564
PubMedSearch : Henchman_2005_Biophys.J_88_2564
PubMedID: 15665135

Title : Structural basis for epibatidine selectivity at desensitized nicotinic receptors - Pennington_2005_Mol.Pharmacol_67_123
Author(s) : Pennington RA , Gao F , Sine SM , Prince RJ
Ref : Molecular Pharmacology , 67 :123 , 2005
Abstract : The agonist binding sites of the fetal muscle nicotinic acetylcholine receptor are formed at the interfaces of alpha-subunits and neighboring gamma- and delta-subunits. When the receptor is in the nonconducting desensitized state, the alpha-gamma site binds the agonist epibatidine 200-fold more tightly than does the alpha-delta site. To determine the structural basis for this selectivity, we constructed gamma/delta-subunit chimeras, coexpressed them with complementary wild-type subunits in HEK 293 cells, and determined epibatidine affinity of the resulting complexes. The results reveal three determinants of epibatidine selectivity: gamma104-117/delta106-delta119, gamma164-171/delta166-177, and gammaPro190/deltaAla196. Point mutations reveal that three sequence differences within the gamma104-117/delta106-delta119 region are determinants of epibatidine selectivity: gammaLys104/deltaTyr106, gammaSer111/deltaTyr113, and gammaTyr117/deltaTyr119. In the delta-subunit, simultaneous mutation of these residues to their gamma equivalent produces high affinity, gamma-like epibatidine binding. However, converting gamma to delta affinity requires replacement of the gamma104-117 segment with delta sequence, suggesting interplay of residues in this region. The structural basis for epibatidine selectivity is explained by computational docking of epibatidine to a homology model of the alpha-gamma binding site.
ESTHER : Pennington_2005_Mol.Pharmacol_67_123
PubMedSearch : Pennington_2005_Mol.Pharmacol_67_123
PubMedID: 15496507

Title : Agonist-mediated conformational changes in acetylcholine-binding protein revealed by simulation and intrinsic tryptophan fluorescence - Gao_2005_J.Biol.Chem_280_8443
Author(s) : Gao F , Bren N , Burghardt TP , Hansen S , Henchman RH , Taylor P , McCammon JA , Sine SM
Ref : Journal of Biological Chemistry , 280 :8443 , 2005
Abstract : We delineated acetylcholine (ACh)-dependent conformational changes in a prototype of the nicotinic receptor ligand binding domain by molecular dynamics simulation and changes in intrinsic tryptophan (Trp) fluorescence. Prolonged molecular dynamics simulation of ACh-binding protein showed that binding of ACh establishes close register of Trps from adjacent subunits, Trp(143) and Trp(53), and draws the peripheral C-loop inward to occlude the entrance to the binding cavity. Close register of Trp(143) and Trp(53) was demonstrated by ACh-mediated quenching of intrinsic Trp fluorescence, elimination of quenching by mutation of one or both Trps to Phe, and decreased lifetime of Trp fluorescence by bound ACh. Occlusion of the binding cavity by the C-loop was demonstrated by restricted access of an extrinsic quencher of binding site Trp fluorescence by ACh. The collective findings showed that ACh initially establishes close register of conserved Trps from adjacent subunits and then draws the C-loop inward to occlude the entrance to the binding cavity.
ESTHER : Gao_2005_J.Biol.Chem_280_8443
PubMedSearch : Gao_2005_J.Biol.Chem_280_8443
PubMedID: 15591050

Title : Single-channel kinetic analysis of chimeric alpha7-5HT3A receptors - Rayes_2005_Mol.Pharmacol_68_1475
Author(s) : Rayes D , Spitzmaul G , Sine SM , Bouzat C
Ref : Molecular Pharmacology , 68 :1475 , 2005
Abstract : The receptor chimera alpha7-5HT3A has served as a prototype for understanding the pharmacology of alpha7 neuronal nicotinic receptors, yet its low single channel conductance has prevented studies of the activation kinetics of single receptor channels. In this study, we show that introducing mutations in the M3-M4 cytoplasmic linker of the chimera alters neither the apparent affinity for the agonist nor the EC50 but increases the amplitude of agonist-evoked single channel currents to enable kinetic analysis. Channel events appear as single brief openings flanked by long closings or as bursts of several openings in quick succession. Both the open and closed time distributions are described as the sum of multiple exponential components, but these do not change over a wide range of acetylcholine (ACh), nicotine, or choline concentrations. Bursts elicited by a saturating concentration of ACh contain brief and long openings and closings, and a cyclic scheme containing two open and two closed states is found to adequately describe the data. The analysis indicates that once fully occupied, the receptor opens rapidly and efficiently, and closes and reopens several times before it desensitizes. Channel closing and desensitization occur at similar rates and account for the invariant open and closed time distributions.
ESTHER : Rayes_2005_Mol.Pharmacol_68_1475
PubMedSearch : Rayes_2005_Mol.Pharmacol_68_1475
PubMedID: 16118362

Title : Initial coupling of binding to gating mediated by conserved residues in the muscle nicotinic receptor - Mukhtasimova_2005_J.Gen.Physiol_126_23
Author(s) : Mukhtasimova N , Free C , Sine SM
Ref : Journal of General Physiology , 126 :23 , 2005
Abstract : We examined functional consequences of intrasubunit contacts in the nicotinic receptor alpha subunit using single channel kinetic analysis, site-directed mutagenesis, and structural modeling. At the periphery of the ACh binding site, our structural model shows that side chains of the conserved residues alphaK145, alphaD200, and alphaY190 converge to form putative electrostatic interactions. Structurally conservative mutations of each residue profoundly impair gating of the receptor channel, primarily by slowing the rate of channel opening. The combined mutations alphaD200N and alphaK145Q impair channel gating to the same extent as either single mutation, while alphaK145E counteracts the impaired gating due to alphaD200K, further suggesting electrostatic interaction between these residues. Interpreted in light of the crystal structure of acetylcholine binding protein (AChBP) with bound carbamylcholine (CCh), the results suggest in the absence of ACh, alphaK145 and alphaD200 form a salt bridge associated with the closed state of the channel. When ACh binds, alphaY190 moves toward the center of the binding cleft to stabilize the agonist, and its aromatic hydroxyl group approaches alphaK145, which in turn loosens its contact with alphaD200. The positional changes of alphaK145 and alphaD200 are proposed to initiate the cascade of perturbations that opens the receptor channel: the first perturbation is of beta-strand 7, which harbors alphaK145 and is part of the signature Cys-loop, and the second is of beta-strand 10, which harbors alphaD200 and connects to the M1 domain. Thus, interplay between these three conserved residues relays the initial conformational change from the ACh binding site toward the ion channel.
ESTHER : Mukhtasimova_2005_J.Gen.Physiol_126_23
PubMedSearch : Mukhtasimova_2005_J.Gen.Physiol_126_23
PubMedID: 15955875

Title : Current understanding of congenital myasthenic syndromes - Engel_2005_Curr.Opin.Pharmacol_5_308
Author(s) : Engel AG , Sine SM
Ref : Curr Opin Pharmacol , 5 :308 , 2005
Abstract : Investigation of congenital myasthenic syndromes (CMSs) disclosed a diverse array of molecular targets at the motor endplate. Clinical, electrophysiologic and morphologic studies paved the way for detecting CMS-related mutations in proteins such as the acetylcholine receptor, acetylcholinesterase, choline acetyltransferase, rapsyn, MuSK and Na(v)1.4. Analysis of electrophysiologic and biochemical properties of mutant proteins expressed in heterologous systems contributed crucially to defining the molecular consequences of the observed mutations and resulted in improved therapy of different CMSs. Recent crystallography studies of choline acetyltransferase and homology structural models of the acetylcholine receptor are providing further clues to how point mutations alter protein function.
ESTHER : Engel_2005_Curr.Opin.Pharmacol_5_308
PubMedSearch : Engel_2005_Curr.Opin.Pharmacol_5_308
PubMedID: 15907919

Title : Toward atomic-scale understanding of ligand recognition in the muscle nicotinic receptor - Sine_2004_Curr.Med.Chem_11_559
Author(s) : Sine SM , Wang HL , Gao F
Ref : Curr Med Chem , 11 :559 , 2004
Abstract : The nicotinic receptor at the motor endplate has served as a prototype for understanding structure, function and ligand recognition in the superfamily of pentameric ligand-gated ion channels. Yet despite this advanced state of knowledge, atomic-scale understanding of such elementary processes as ligand recognition has remained elusive owing to the lack of a high-resolution x-ray structure. However, the field has recently entered a state of rapid advancement following the discovery and atomic structural determination of the water-soluble acetylcholine binding protein (AChBP), a homolog of the receptor ligand binding domain. The AChBP structure provides the theoretical foundation for generating homology models of the corresponding receptor ligand binding domains within this structural family of receptors. Experimental assignment of residue equivalence between AChBP and receptor subunits subsequently yielded homology models ready for experimental testing. One such test is computational determination of ligand docking orientation in conjunction with mutagenesis of predicted contact residues and measurements of ligand binding affinity. Applied to different analogs of the competitive antagonist curare, docking computations that incorporate intrinsic protein flexibility reveal fundamentally distinct orientations of each analog bound to AChBP. The different contact residues predicted for each analog were tested and confirmed by mutagenesis of AChBP followed by measurements of ligand binding. By applying the same computational and experimental approaches to the adult human muscle AChR, we find that the two curare analogs also dock in distinctly different orientations. Thus subtle structural changes in the ligand, and by extension, structural differences in non-conserved residues among receptor subtypes and species, can dramatically alter the orientation of the bound ligand. The results have important implications for design of drugs targeting nicotinic receptors and members of the superfamily of pentameric ligand-gated ion channels.
ESTHER : Sine_2004_Curr.Med.Chem_11_559
PubMedSearch : Sine_2004_Curr.Med.Chem_11_559
PubMedID: 15032604

Title : Coupling of agonist binding to channel gating in an ACh-binding protein linked to an ion channel - Bouzat_2004_Nature_430_896
Author(s) : Bouzat C , Gumilar F , Spitzmaul G , Wang HL , Rayes D , Hansen SB , Taylor P , Sine SM
Ref : Nature , 430 :896 , 2004
Abstract : Neurotransmitter receptors from the Cys-loop superfamily couple the binding of agonist to the opening of an intrinsic ion pore in the final step in rapid synaptic transmission. Although atomic resolution structural data have recently emerged for individual binding and pore domains, how they are linked into a functional unit remains unknown. Here we identify structural requirements for functionally coupling the two domains by combining acetylcholine (ACh)-binding protein, whose structure was determined at atomic resolution, with the pore domain from the serotonin type-3A (5-HT3A) receptor. Only when amino-acid sequences of three loops in ACh-binding protein are changed to their 5-HT3A counterparts does ACh bind with low affinity characteristic of activatable receptors, and trigger opening of the ion pore. Thus functional coupling requires structural compatibility at the interface of the binding and pore domains. Structural modelling reveals a network of interacting loops between binding and pore domains that mediates this allosteric coupling process.
ESTHER : Bouzat_2004_Nature_430_896
PubMedSearch : Bouzat_2004_Nature_430_896
PubMedID: 15318223

Title : Alpha-conotoxins ImI and ImII target distinct regions of the human alpha7 nicotinic acetylcholine receptor and distinguish human nicotinic receptor subtypes - Ellison_2004_Biochemistry_43_16019
Author(s) : Ellison M , Gao F , Wang HL , Sine SM , McIntosh JM , Olivera BM
Ref : Biochemistry , 43 :16019 , 2004
Abstract : The Conus peptides alpha-conotoxin ImI (alpha-ImI) and ImII (alpha-ImII) differ by only three of 11 residues in their primary sequences and yet are shown to inhibit the human alpha7 nicotinic acetylcholine receptor (nAChR) by targeting different sites. Mutations at both faces of the classical ligand binding site of the alpha7 nAChR strongly affect antagonism by alpha-ImI but not alpha-ImII. The effects of the mutations on alpha-ImI binding and functional antagonism are explained by computational docking of the NMR structure of alpha-ImI to a homology model of the ligand binding domain of the alpha7 nAChR. A distinct binding site for alpha-ImII is further demonstrated by its weakened antagonism for a chimeric receptor in which the membrane-spanning domains and intervening linkers of the alpha7 nAChR are replaced with the corresponding sequence from the serotonin type-3 receptor (5HT(3)). The two toxins also discriminate between different subtypes of human nicotinic receptors; alpha-ImII most strongly blocks the human alpha7 and alpha1beta1deltaepsilon receptor subtypes, while alpha-ImI most potently blocks the human alpha3beta2 subtype. Collectively, the data show that while alpha-ImI targets the classical competitive ligand binding site in a subtype selective manner, alpha-ImII is a probe of a novel inhibitory site in homomeric alpha7 nAChRs.
ESTHER : Ellison_2004_Biochemistry_43_16019
PubMedSearch : Ellison_2004_Biochemistry_43_16019
PubMedID: 15609996

Title : Invariant aspartic Acid in muscle nicotinic receptor contributes selectively to the kinetics of agonist binding - Lee_2004_J.Gen.Physiol_124_555
Author(s) : Lee WY , Sine SM
Ref : Journal of General Physiology , 124 :555 , 2004
Abstract : We examined functional contributions of interdomain contacts within the nicotinic receptor ligand binding site using single channel kinetic analyses, site-directed mutagenesis, and a homology model of the major extracellular region. At the principal face of the binding site, the invariant alphaD89 forms a highly conserved interdomain contact near alphaT148, alphaW149, and alphaT150. Patch-clamp recordings show that the mutation alphaD89N markedly slows acetylcholine (ACh) binding to receptors in the resting closed state, but does not affect rates of channel opening and closing. Neither alphaT148L, alphaT150A, nor mutations at both positions substantially affects the kinetics of receptor activation, showing that hydroxyl side chains at these positions are not hydrogen bond donors for the strong acceptor alphaD89. However substituting a negative charge at alphaT148, but not at alphaT150, counteracts the effect of alphaD89N, demonstrating that a negative charge in the region of interdomain contact confers rapid association of ACh. Interpreted within the structural framework of ACh binding protein and a homology model of the receptor ligand binding site, these results implicate main chain amide groups in the domain harboring alphaW149 as principal hydrogen bond donors for alphaD89. The specific effect of alphaD89N on ACh association suggests that interdomain hydrogen bonding positions alphaW149 for optimal interaction with ACh.
ESTHER : Lee_2004_J.Gen.Physiol_124_555
PubMedSearch : Lee_2004_J.Gen.Physiol_124_555
PubMedID: 15504901

Title : Mutation causing severe myasthenia reveals functional asymmetry of AChR signature cystine loops in agonist binding and gating - Shen_2003_J.Clin.Invest_111_497
Author(s) : Shen XM , Ohno K , Tsujino A , Brengman JM , Gingold M , Sine SM , Engel AG
Ref : J Clinical Investigation , 111 :497 , 2003
Abstract : We describe a highly disabling congenital myasthenic syndrome (CMS) associated with rapidly decaying, low-amplitude synaptic currents, and trace its cause to a valine to leucine mutation in the signature cystine loop (cys-loop) of the AChR alpha subunit. The recently solved crystal structure of an ACh-binding protein places the cys-loop at the junction between the extracellular ligand-binding and transmembrane domains where it may couple agonist binding to channel gating. We therefore analyzed the kinetics of ACh-induced single-channel currents to identify elementary steps in the receptor activation mechanism altered by the alphaV132L mutation. The analysis reveals that alphaV132L markedly impairs ACh binding to receptors in the resting closed state, decreasing binding affinity for the second binding step 30-fold, but attenuates gating efficiency only about twofold. By contrast, mutation of the equivalent valine residue in the delta subunit impairs channel gating approximately fourfold with little effect on ACh binding, while corresponding mutations in the beta and epsilon subunits are without effect. The unique functional contribution of the alpha subunit cys-loop likely owes to its direct connection via a beta strand to alphaW149 at the center of the ligand-binding domain. The overall findings reveal functional asymmetry between cys-loops of the different AChR subunits in contributing to ACh binding and channel gating.
ESTHER : Shen_2003_J.Clin.Invest_111_497
PubMedSearch : Shen_2003_J.Clin.Invest_111_497
PubMedID: 12588888

Title : Asymmetric structural motions of the homomeric alpha7 nicotinic receptor ligand binding domain revealed by molecular dynamics simulation - Henchman_2003_Biophys.J_85_3007
Author(s) : Henchman RH , Wang HL , Sine SM , Taylor P , McCammon JA
Ref : Biophysical Journal , 85 :3007 , 2003
Abstract : A homology model of the ligand binding domain of the alpha7 nicotinic receptor is constructed based on the acetylcholine-binding protein crystal structure. This structure is refined in a 10 ns molecular dynamics simulation. The modeled structure proves fairly resilient, with no significant changes at the secondary or tertiary structural levels. The hypothesis that the acetylcholine-binding protein template is in the activated or desensitized state, and the absence of a bound agonist in the simulation suggests that the structure may also be relaxing from this state to the activatable state. Candidate motions that take place involve not only the side chains of residues lining the binding sites, but also the subunit positions that determine the overall shape of the receptor. In particular, two nonadjacent subunits move outward, whereas their partners counterclockwise to them move inward, leading to a marginally wider interface between themselves and an overall asymmetric structure. This in turn affects the binding sites, producing two that are more open and characterized by distinct side-chain conformations of W54 and L118, although motions of the side chains of all residues in every binding site still contribute to a reduction in binding site size, especially the outward motion of W148, which hinders acetylcholine binding. The Cys loop at the membrane interface also displays some flexibility. Although the short simulation timescale is unlikely to sample adequately all the conformational states, the pattern of observed motions suggests how ligand binding may correlate with larger-scale subunit motions that would connect with the transmembrane region that controls the passage of ions. Furthermore, the shape of the asymmetry with binding sites of differing affinity for acetylcholine, characteristic of other nicotinic receptors, may be a natural property of the relaxed, activatable state of alpha7.
ESTHER : Henchman_2003_Biophys.J_85_3007
PubMedSearch : Henchman_2003_Biophys.J_85_3007
PubMedID: 14581202

Title : Curariform antagonists bind in different orientations to the nicotinic receptor ligand binding domain - Wang_2003_J.Biol.Chem_278_32284
Author(s) : Wang HL , Gao F , Bren N , Sine SM
Ref : Journal of Biological Chemistry , 278 :32284 , 2003
Abstract : Curariform alkaloids competitively inhibit muscle acetylcholine receptors (AChR) by bridging the alpha and non-alpha subunits that form the ligand-binding site. Here we delineate bound orientations of d-tubocurarine (d-TC) and its methylated derivative metocurine using mutagenesis, ligand binding measurements, and computational methods. When tested against a series of lysine mutations in the epsilon subunit, the two antagonists show marked differences in the consequences of the mutations on binding affinity. The mutations epsilon L117K, epsilon Y111K, and epsilon L109K decrease affinity of metocurine by up to 3 orders of magnitude but only slightly alter affinity of d-TC. At the alpha subunit face of the binding site, the mutation alpha Y198T decreases affinity of both antagonists, but alpha Y198F preferentially enhances affinity of d-TC. Computation of antagonist docking orientations, based on our structural model of the alpha-epsilon site of the human AChR, indicates distinct orientations of each antagonist; the flatter metocurine fits into a pocket formed principally by the epsilon subunit, whereas the more compact d-TC spans the narrower crevasse between alpha and epsilon subunits. The side chains of epsilon Tyr-111 and epsilon Thr-117 juxtapose one of two quaternary nitrogens in metocurine but are remote from the equivalent quaternary nitrogen in d-TC, which instead closely approaches alpha Tyr-198. The different docked orientations arise through tilt of the curariform scaffold by approximately 60 degrees normal to the nitrogen-nitrogen axis, together with a 20 degrees rotation about the axis. The overall mutagenesis and computational results show that despite their similar structures, d-TC and metocurine bind in distinctly different orientations to the adult human AChR.
ESTHER : Wang_2003_J.Biol.Chem_278_32284
PubMedSearch : Wang_2003_J.Biol.Chem_278_32284
PubMedID: 12799358

Title : Mechanistic diversity underlying fast channel congenital myasthenic syndromes - Sine_2003_Ann.N.Y.Acad.Sci_998_128
Author(s) : Sine SM , Wang HL , Ohno K , Shen XM , Lee WY , Engel AG
Ref : Annals of the New York Academy of Sciences , 998 :128 , 2003
Abstract : A host of missense mutations in muscle nicotinic receptor subunits have been identified as the cause of congenital myasthenic syndromes (CMS). Two classes of CMS phenotypes have been identified: slow channel myasthenic syndromes (SCCMSs) and fast channel myasthenic syndromes (FCCMSs). Although both have similar phenotypic consequences, they are physiologic opposites. Expression of the FCCMS phenotype requires the missense mutation to be accompanied by a second mutation, either a null or a missense mutation, in the second allele encoding the same receptor subunit. This seemingly rare scenario has arisen with surprisingly high incidence over the past few years, and analyses of the syndromes have revealed a diverse array of mechanisms underlying the pathology. This review focuses on new mechanisms underlying the FCCMS.
ESTHER : Sine_2003_Ann.N.Y.Acad.Sci_998_128
PubMedSearch : Sine_2003_Ann.N.Y.Acad.Sci_998_128
PubMedID: 14592870

Title : Sleuthing molecular targets for neurological diseases at the neuromuscular junction -
Author(s) : Engel AG , Ohno K , Sine SM
Ref : Nat Rev Neurosci , 4 :339 , 2003
PubMedID: 12728262

Title : Curariform antagonists bind in different orientations to acetylcholine-binding protein - Gao_2003_J.Biol.Chem_278_23020
Author(s) : Gao F , Bern N , Little A , Wang HL , Hansen SB , Talley TT , Taylor P , Sine SM
Ref : Journal of Biological Chemistry , 278 :23020 , 2003
Abstract : Acetylcholine-binding protein (AChBP) recently emerged as a prototype for relating structure to function of the ligand binding domain of nicotinic acetylcholine receptors (AChRs). To understand interactions of competitive antagonists at the atomic structural level, we studied binding of the curare derivatives d-tubocurarine (d-TC) and metocurine to AChBP using computational methods, mutagenesis, and ligand binding measurements. To account for protein flexibility, we used a 2-ns molecular dynamics simulation of AChBP to generate multiple snapshots of the equilibrated dynamic structure to which optimal docking orientations were determined. Our results predict a predominant docking orientation for both d-TC and metocurine, but unexpectedly, the bound orientations differ fundamentally for each ligand. At one subunit interface of AChBP, the side chain of Tyr-89 closely approaches a positively charged nitrogen in d-TC but is farther away from the equivalent nitrogen in metocurine, whereas, at the opposing interface, side chains of Trp-53 and Gln-55 closely approach the metocurine scaffold but not that of d-TC. The different orientations correspond to approximately 170 degrees rotation and approximately 30 degrees degree tilt of the curare scaffold within the binding pocket. Mutagenesis of binding site residues in AChBP, combined with measurements of ligand binding, confirms the different docking orientations. Thus structurally similar ligands can adopt distinct orientations at receptor binding sites, posing challenges for interpreting structure-activity relationships for many drugs.
ESTHER : Gao_2003_J.Biol.Chem_278_23020
PubMedSearch : Gao_2003_J.Biol.Chem_278_23020
PubMedID: 12682067

Title : Congenital myasthenic syndromes: multiple molecular targets at the neuromuscular junction - Engel_2003_Ann.N.Y.Acad.Sci_998_138
Author(s) : Engel AG , Ohno K , Shen XM , Sine SM
Ref : Annals of the New York Academy of Sciences , 998 :138 , 2003
Abstract : Congenital myasthenic syndromes (CMS) stem from defects in presynaptic, synaptic, and postsynaptic proteins. The presynaptic CMS are associated with defects that curtail the evoked release of acetylcholine (ACh) quanta or ACh resynthesis. Defects in ACh resynthesis have now been traced to mutations in choline acetyltransferase. A synaptic CMS is caused by mutations in the collagenic tail subunit (ColQ) of the endplate species of acetylcholinesterase that prevent the tail subunit from associating with catalytic subunits or from becoming inserted into the synaptic basal lamina. Most postsynaptic CMS are caused by mutations in subunits of the acetylcholine receptor (AChR) that alter the kinetic properties or decrease the expression of AChR. The kinetic mutations increase or decrease the synaptic response to ACh and result in slow- and fast-channel syndromes, respectively. Most low-expressor mutations reside in the AChR epsilon subunit and are partially compensated by residual expression of the fetal-type gamma subunit. In a subset of CMS patients, endplate AChR deficiency is caused by mutations in rapsyn, a molecule that plays a critical role in concentrating AChR in the postsynaptic membrane.
ESTHER : Engel_2003_Ann.N.Y.Acad.Sci_998_138
PubMedSearch : Engel_2003_Ann.N.Y.Acad.Sci_998_138
PubMedID: 14592871

Title : Congenital myasthenic syndromes: A diverse array of molecular targets - Engel_2003_J.Neurocytol_32_1017
Author(s) : Engel AG , Ohno K , Sine SM
Ref : Journal of Neurocytology , 32 :1017 , 2003
Abstract : The neuromuscular junction (NMJ) has served as a prototype for understanding mechanisms underlying synaptic transmission over the past 50 years. More recently, analysis of congenital myasthenic syndromes (CMS) revealed a diverse array of molecular targets and delineated their contributions to synaptic function. Clinical, electrophysiologic and morphologic studies have paved the way for detecting CMS-related mutations in proteins such as choline acetyltransferase acetylcholinesterase, the acetylcholine receptor, rapsyn, and the voltage-gated sodium channel of the Na(v)1.4 type. Further studies of the mutant proteins have allowed us to correlate the effects of the mutations with predicted alterations in protein structure. In this review, we focus on the symptomatology of the CMS, consider the factors that impair neuromuscular transmission, survey the mutations that have been uncovered in the different synaptic proteins, and consider the functional implications of the identified mutations.
ESTHER : Engel_2003_J.Neurocytol_32_1017
PubMedSearch : Engel_2003_J.Neurocytol_32_1017
PubMedID: 15034283

Title : Congenital myasthenic syndromes: progress over the past decade - Engel_2003_Muscle.Nerve_27_4
Author(s) : Engel AG , Ohno K , Sine SM
Ref : Muscle & Nerve , 27 :4 , 2003
Abstract : Congenital myasthenic syndromes (CMS) stem from defects in presynaptic, synaptic basal lamina, and postsynaptic proteins. The presynaptic CMS are associated with defects that curtail the evoked release of acetylcholine (ACh) quanta or ACh resynthesis. Defects in ACh resynthesis have now been traced to mutations in choline acetyltransferase. A basal lamina CMS is caused by mutations in the collagenic tail subunit (ColQ) of the endplate species of acetylcholinesterase that prevent the tail subunit from associating with catalytic subunits or from becoming inserted into the synaptic basal lamina. Most postsynaptic CMS are caused by mutations in subunits of the acetylcholine receptor (AChR) that alter the kinetic properties or decrease the expression of AChR. The kinetic mutations increase or decrease the synaptic response to ACh and result in slow- and fast-channel syndromes, respectively. Most low-expressor mutations reside in the AChR epsilon subunit and are partially compensated by residual expression of the fetal type gamma subunit. In a subset of CMS patients, endplate AChR deficiency is caused by mutations in rapsyn, a molecule that plays a critical role in concentrating AChR in the postsynaptic membrane.
ESTHER : Engel_2003_Muscle.Nerve_27_4
PubMedSearch : Engel_2003_Muscle.Nerve_27_4
PubMedID: 12508290

Title : The nicotinic receptor ligand binding domain - Sine_2002_J.Neurobiol_53_431
Author(s) : Sine SM
Ref : Journal of Neurobiology , 53 :431 , 2002
Abstract : The ligand binding domain (LBD) of the nicotinic acetylcholine receptor has served as a prototype for understanding molecular recognition in the family of neurotransmitter-gated ion channels. During the past fifty years, studies progressed from fundamental electrophysiological analyses of ACh-evoked ion flow, to biochemical purification of the receptor protein, pharmacological measurements of ligand binding, molecular cloning of receptor subunits, site-directed mutagenesis combined with functional analysis and recently, atomic structural determination. The emerging picture of the nicotinic receptor LBD is a specialized pocket of aromatic and hydrophobic residues formed at interfaces between protein subunits that changes conformation to convert agonist binding into gating of an intrinsic ion channel.
ESTHER : Sine_2002_J.Neurobiol_53_431
PubMedSearch : Sine_2002_J.Neurobiol_53_431
PubMedID: 12436411

Title : Naturally occurring mutations at the acetylcholine receptor binding site independently alter ACh binding and channel gating - Sine_2002_J.Gen.Physiol_120_483
Author(s) : Sine SM , Shen XM , Wang HL , Ohno K , Lee WY , Tsujino A , Brengmann J , Bren N , Vajsar J , Engel AG
Ref : Journal of General Physiology , 120 :483 , 2002
Abstract : By defining functional defects in a congenital myasthenic syndrome (CMS), we show that two mutant residues, located in a binding site region of the acetylcholine receptor (AChR) epsilon subunit, exert opposite effects on ACh binding and suppress channel gating. Single channel kinetic analysis reveals that the first mutation, epsilon N182Y, increases ACh affinity for receptors in the resting closed state, which promotes sequential occupancy of the binding sites and discloses rate constants for ACh occupancy of the nonmutant alphadelta site. Studies of the analogous mutation in the delta subunit, deltaN187Y, disclose rate constants for ACh occupancy of the nonmutant alpha epsilon site. The second CMS mutation, epsilon D175N, reduces ACh affinity for receptors in the resting closed state; occupancy of the mutant site still promotes gating because a large difference in affinity is maintained between closed and open states. epsilon D175N impairs overall gating, however, through an effect independent of ACh occupancy. When mapped on a structural model of the AChR binding site, epsilon N182Y localizes to the interface with the alpha subunit, and epsilon D175 to the entrance of the ACh binding cavity. Both epsilon N182Y and epsilon D175 show state specificity in affecting closed relative to desensitized state affinities, suggesting that the protein chain harboring epsilon N182 and epsilon D175 rearranges in the course of receptor desensitization. The overall results show that key residues at the ACh binding site differentially stabilize the agonist bound to closed, open and desensitized states, and provide a set point for gating of the channel.
ESTHER : Sine_2002_J.Gen.Physiol_120_483
PubMedSearch : Sine_2002_J.Gen.Physiol_120_483
PubMedID: 12356851

Title : Mechanism of tacrine block at adult human muscle nicotinic acetylcholine receptors - Prince_2002_J.Gen.Physiol_120_369
Author(s) : Prince RJ , Pennington RA , Sine SM
Ref : Journal of General Physiology , 120 :369 , 2002
Abstract : We used single-channel kinetic analysis to study the inhibitory effects of tacrine on human adult nicotinic receptors (nAChRs) transiently expressed in HEK 293 cells. Single channel recording from cell-attached patches revealed concentration- and voltage-dependent decreases in mean channel open probability produced by tacrine (IC(50) 4.6 microM at -70 mV, 1.6 microM at -150 mV). Two main effects of tacrine were apparent in the open- and closed-time distributions. First, the mean channel open time decreased with increasing tacrine concentration in a voltage-dependent manner, strongly suggesting that tacrine acts as an open-channel blocker. Second, tacrine produced a new class of closings whose duration increased with increasing tacrine concentration. Concentration dependence of closed-times is not predicted by sequential models of channel block, suggesting that tacrine blocks the nAChR by an unusual mechanism. To probe tacrine's mechanism of action we fitted a series of kinetic models to our data using maximum likelihood techniques. Models incorporating two tacrine binding sites in the open receptor channel gave dramatically improved fits to our data compared with the classic sequential model, which contains one site. Improved fits relative to the sequential model were also obtained with schemes incorporating a binding site in the closed channel, but only if it is assumed that the channel cannot gate with tacrine bound. Overall, the best description of our data was obtained with a model that combined two binding sites in the open channel with a single site in the closed state of the receptor.
ESTHER : Prince_2002_J.Gen.Physiol_120_369
PubMedSearch : Prince_2002_J.Gen.Physiol_120_369
PubMedID: 12198092

Title : Lysine scanning mutagenesis delineates structural model of the nicotinic receptor ligand binding domain - Sine_2002_J.Biol.Chem_277_29210
Author(s) : Sine SM , Wang HL , Bren N
Ref : Journal of Biological Chemistry , 277 :29210 , 2002
Abstract : Nicotinic acetylcholine receptors (AChR) and their relatives mediate rapid chemical transmission throughout the nervous system, yet their atomic structures remain elusive. Here we use lysine scanning mutagenesis to determine the orientation of residue side chains toward core hydrophobic or surface hydrophilic environments and use this information to build a structural model of the ligand binding region of the AChR from adult human muscle. The resulting side-chain orientations allow assignment of residue equivalence between AChR subunits and an acetylcholine binding protein solved by x-ray crystallography, providing the foundation for homology modeling. The resulting structural model of the AChR provides a picture of the ACh binding site and predicts novel pairs of residues that stabilize subunit interfaces. The overall results suggest that lysine scanning can provide the basis for structural modeling of other members of the AChR superfamily as well as of other proteins with repeating structures delimiting a hydrophobic core.
ESTHER : Sine_2002_J.Biol.Chem_277_29210
PubMedSearch : Sine_2002_J.Biol.Chem_277_29210
PubMedID: 12011092

Title : Novel modulation of neuronal nicotinic acetylcholine receptors by association with the endogenous prototoxin lynx1 - Ibanez-Tallon_2002_Neuron_33_893
Author(s) : Ibanez-Tallon I , Miwa JM , Wang HL , Adams NC , Crabtree GW , Sine SM , Heintz N
Ref : Neuron , 33 :893 , 2002
Abstract : We previously identified lynx1 as a neuronal membrane molecule related to snake alpha-neurotoxins able to modulate nAChRs. Here, we show that lynx1 colocalizes with nAChRs on CNS neurons and physically associates with nAChRs. Single-channel recordings show that lynx1 promotes the largest of three current amplitudes elicited by ACh through alpha(4)beta(2) nAChRs and that lynx1 enhances desensitization. Macroscopic recordings quantify the enhancement of desensitization onset by lynx1 and further show that it slows recovery from desensitization and increases the EC(50). These experiments establish that direct interaction of lynx1 with nAChRs can result in a novel type of functional modulation and suggest that prototoxins may play important roles in vivo by modulating functional properties of their cognate CNS receptors.
ESTHER : Ibanez-Tallon_2002_Neuron_33_893
PubMedSearch : Ibanez-Tallon_2002_Neuron_33_893
PubMedID: 11906696

Title : The spectrum of congenital myasthenic syndromes - Engel_2002_Mol.Neurobiol_26_347
Author(s) : Engel AG , Ohno K , Sine SM
Ref : Molecular Neurobiology , 26 :347 , 2002
Abstract : The past decade saw remarkable advances in defining the molecular and genetic basis of the congenital myasthenic syndromes. These advances would not have been possible without antecedent clinical observations, electrophysiologic analysis, and careful morphologic studies that pointed to candidate genes or proteins. For example, a kinetic abnormality of the acetylcholine receptor (AChR) detected at the single channel level pointed to a kinetic mutation in an AChR subunit; endplate AChR deficiency suggested mutations residing in an AChR subunit or in rapsyn; absence of acetylcholinesterase (AChE) from the endplate predicted mutations in the catalytic or collagen-tailed subunit of this enzyme; and a history of abrupt episodes of apnea associated with a stimulation dependent decrease of endplate potentials and currents implicated proteins concerned with ACh resynthesis or vesicular filling. Discovery of mutations in endplate-specific proteins also prompted expression studies that afforded proof of pathogenicity, provided clues for rational therapy, lead to precise structure function correlations, and highlighted functionally significant residues or molecular domains that previous systematic mutagenesis studies had failed to detect. An overview of the spectrum of the congenital myasthenic syndromes suggests that most are caused by mutations in AChR subunits, and particularly in the epsilon subunit. Future studies will likely uncover new types of CMS that reside in molecules governing quantal release, organization of the synaptic basal lamina, and expression and aggregation of AChR on the postsynaptic junctional folds.
ESTHER : Engel_2002_Mol.Neurobiol_26_347
PubMedSearch : Engel_2002_Mol.Neurobiol_26_347
PubMedID: 12428764

Title : Subunit-selective contribution to channel gating of the M4 domain of the nicotinic receptor - Bouzat_2002_Biophys.J_82_1920
Author(s) : Bouzat C , Gumilar F , del Carmen Esandi M , Sine SM
Ref : Biophysical Journal , 82 :1920 , 2002
Abstract : The muscle nicotinic receptor (AChR) is a pentamer of four different subunits, each of which contains four transmembrane domains (M1-M4). We recently showed that channel opening and closing rates of the AChR depend on a hydrogen bond involving a threonine at position 14' of the M4 domain in the alpha-subunit. To determine whether residues in equivalent positions in non-alpha-subunits contribute to channel gating, we mutated deltaT14', betaT14', and epsilonS14' and evaluated changes in the kinetics of acetylcholine-activated currents. The mutation epsilonS14'A profoundly slows the rate of channel closing, an effect opposite to that produced by mutation of alphaT14'. Unlike mutations of alphaT14', epsilonS14'A does not affect the rate of channel opening. Mutations in deltaT14' and betaT14' do not affect channel opening or closing kinetics, showing that conserved residues are not functionally equivalent in all subunits. Whereas alphaT14'A and epsilonS14'A subunits contribute additively to the closing rate, they contribute nonadditively to the opening rate. Substitution of residues preserving the hydrogen bonding ability at position 14' produce nearly normal gating kinetics. Thus, we identify subunit-specific contributions to channel gating of equivalent residues in M4 and elucidate the underlying mechanistic and structural bases.
ESTHER : Bouzat_2002_Biophys.J_82_1920
PubMedSearch : Bouzat_2002_Biophys.J_82_1920
PubMedID: 11916850

Title : Identification of residues at the alpha and epsilon subunit interfaces mediating species selectivity of Waglerin-1 for nicotinic acetylcholine receptors - Molles_2002_J.Biol.Chem_277_5433
Author(s) : Molles BE , Rezai P , Kline EF , McArdle JJ , Sine SM , Taylor P
Ref : Journal of Biological Chemistry , 277 :5433 , 2002
Abstract : Waglerin-1 (Wtx-1) is a 22-amino acid peptide that is a competitive antagonist of the muscle nicotinic receptor (nAChR). We find that Wtx-1 binds 2100-fold more tightly to the alpha-epsilon than to the alpha-delta binding site interface of the mouse nAChR. Moreover, Wtx-1 binds 100-fold more tightly to the alpha-epsilon interface from mouse nAChR than that from rat or human sources. Site-directed mutagenesis of residues differing in the extracellular domains of rat and mouse epsilon subunits indicates that residues 59 and 115 mediate the species difference in Wtx-1 affinity. Mutation of residues 59 (Asp in mouse, Glu in rat epsilon) and 115 (Tyr in mouse, Ser in rat epsilon) converts Wtx-1 affinity for the alpha-epsilon interface of one species to that of the other species. Studies of different mutations at position 59 indicate both steric and electrostatic contributions to Wtx-1 affinity, whereas at position 115, both aromatic and polar groups contribute to affinity. The human nAChR also has lower affinity for Wtx-1 than mouse nAChR, but unlike rat nAChR, residues in both alpha and epsilon subunits mediate the affinity difference. In human nAChR, polar residues (Ser-187 and Thr-189) confer low affinity, whereas in mouse nAChR aromatic residues (Trp-187 and Phe-189) confer high affinity. The overall results show that non-conserved residues at the nAChR binding site, although not crucial for activation by ACh, govern the potency of neuromuscular toxins.
ESTHER : Molles_2002_J.Biol.Chem_277_5433
PubMedSearch : Molles_2002_J.Biol.Chem_277_5433
PubMedID: 11724791

Title : Residues in the epsilon subunit of the nicotinic acetylcholine receptor interact to confer selectivity of waglerin-1 for the alpha-epsilon subunit interface site - Molles_2002_Biochemistry_41_7895
Author(s) : Molles BE , Tsigelny I , Nguyen PD , Gao SX , Sine SM , Taylor P
Ref : Biochemistry , 41 :7895 , 2002
Abstract : Waglerin-1 (Wtx-1) is a 22-amino acid peptide that competitively antagonizes muscle nicotinic acetylcholine receptors (nAChRs). Previous work demonstrated that Wtx-1 binds to mouse nAChRs with higher affinity than receptors from rats or humans, and distinguished residues in alpha and epsilon subunits that govern the species selectivity. These studies also showed that Wtx-1 binds selectively to the alpha-epsilon binding site with significantly higher affinity than to the alpha-delta binding site. Here we identify residues at equivalent positions in the epsilon, gamma, and delta subunits that govern Wtx-1 selectivity for one of the two binding sites on the nAChR pentamer. Using a series of chimeric and point mutant subunits, we show that residues Gly-57, Asp-59, Tyr-111, Tyr-115, and Asp-173 of the epsilon subunit account predominantly for the 3700-fold higher affinity of the alpha-epsilon site relative to that of the alpha-gamma site. Similarly, we find that residues Lys-34, Gly-57, Asp-59, and Asp-173 account predominantly for the high affinity of the alpha-epsilon site relative to that of the alpha-delta site. Analysis of combinations of point mutations reveals that Asp-173 in the epsilon subunit is required together with the remaining determinants in the epsilon subunit to achieve Wtx-1 selectivity. In particular, Lys-34 interacts with Asp-173 to confer high affinity, resulting in a DeltaDeltaG(INT) of -2.3 kcal/mol in the epsilon subunit and a DeltaDeltaG(INT) of -1.3 kcal/mol in the delta subunit. Asp-173 is part of a nonhomologous insertion not found in the acetylcholine binding protein structure. The key role of this insertion in Wtx-1 selectivity indicates that it is proximal to the ligand binding site. We use the binding and interaction energies for Wtx-1 to generate structural models of the alpha-epsilon, alpha-gamma, and alpha-delta binding sites containing the nonhomologous insertion.
ESTHER : Molles_2002_Biochemistry_41_7895
PubMedSearch : Molles_2002_Biochemistry_41_7895
PubMedID: 12069578

Title : Pairwise electrostatic interactions between alpha-neurotoxins and gamma, delta, and epsilon subunits of the nicotinic acetylcholine receptor - Osaka_2000_J.Biol.Chem_275_5478
Author(s) : Osaka H , Malany S , Molles BE , Sine SM , Taylor P
Ref : Journal of Biological Chemistry , 275 :5478 , 2000
Abstract : alpha-Neurotoxins bind with high affinity to alpha-gamma and alpha-delta subunit interfaces of the nicotinic acetylcholine receptor. Since this high affinity complex likely involves a van der Waals surface area of approximately 1200 A(2) and 25-35 residues on the receptor surface, analysis of side chains should delineate major interactions and the orientation of bound alpha-neurotoxin. Three distinct regions on the gamma subunit, defined by Trp(55), Leu(119), Asp(174), and Glu(176), contribute to alpha-toxin affinity. Of six charge reversal mutations on the three loops of Naja mossambica mossambica alpha-toxin, Lys(27) --> Glu, Arg(33) --> Glu, and Arg(36) --> Glu in loop II reduce binding energy substantially, while mutations in loops I and III have little effect. Paired residues were analyzed by thermodynamic mutant cycles to delineate electrostatic linkages between the six alpha-toxin charge reversal mutations and three key residues on the gamma subunit. Large coupling energies were found between Arg(33) at the tip of loop II and gammaLeu(119) (-5.7 kcal/mol) and between Lys(27) and gammaGlu(176) (-5.9 kcal/mol). gammaTrp(55) couples strongly to both Arg(33) and Lys(27), whereas gammaAsp(174) couples minimally to charged alpha-toxin residues. Arg(36), despite strong energetic contributions, does not partner with any gamma subunit residues, perhaps indicating its proximity to the alpha subunit. By analyzing cationic, neutral and anionic residues in the mutant cycles, interactions at gamma176 and gamma119 can be distinguished from those at gamma55.
ESTHER : Osaka_2000_J.Biol.Chem_275_5478
PubMedSearch : Osaka_2000_J.Biol.Chem_275_5478
PubMedID: 10681526

Title : Orientation of alpha-neurotoxin at the subunit interfaces of the nicotinic acetylcholine receptor - Malany_2000_Biochemistry_39_15388
Author(s) : Malany S , Osaka H , Sine SM , Taylor P
Ref : Biochemistry , 39 :15388 , 2000
Abstract : The alpha-neurotoxins are three-fingered peptide toxins that bind selectively at interfaces formed by the alpha subunit and its associating subunit partner, gamma, delta, or epsilon of the nicotinic acetylcholine receptor. Because the alpha-neurotoxin from Naja mossambica mossambica I shows an unusual selectivity for the alpha gamma and alpha delta over the alpha epsilon subunit interface, residue replacement and mutant cycle analysis of paired residues enabled us to identify the determinants in the gamma and delta sequences governing alpha-toxin recognition. To complement this approach, we have similarly analyzed residues on the alpha subunit face of the binding site dictating specificity for alpha-toxin. Analysis of the alpha gamma interface shows unique pairwise interactions between the charged residues on the alpha-toxin and three regions on the alpha subunit located around residue Asp(99), between residues Trp(149) and Val(153), and between residues Trp(187) and Asp(200). Substitutions of cationic residues at positions between Trp(149) and Val(153) markedly reduce the rate of alpha-toxin binding, and these cationic residues appear to be determinants in preventing alpha-toxin binding to alpha 2, alpha 3, and alpha 4 subunit containing receptors. Replacement of selected residues in the alpha-toxin shows that Ser(8) on loop I and Arg(33) and Arg(36) on the face of loop II, in apposition to loop I, are critical to the alpha-toxin for association with the alpha subunit. Pairwise mutant cycle analysis has enabled us to position residues on the concave face of the three alpha-toxin loops with respect to alpha and gamma subunit residues in the alpha-toxin binding site. Binding of NmmI alpha-toxin to the alpha gamma interface appears to have dominant electrostatic interactions not seen at the alpha delta interface.
ESTHER : Malany_2000_Biochemistry_39_15388
PubMedSearch : Malany_2000_Biochemistry_39_15388
PubMedID: 11112524

Title : Fundamental gating mechanism of nicotinic receptor channel revealed by mutation causing a congenital myasthenic syndrome - Wang_2000_J.Gen.Physiol_116_449
Author(s) : Wang HL , Ohno K , Milone M , Brengman JM , Evoli A , Batocchi AP , Middleton LT , Christodoulou K , Engel AG , Sine SM
Ref : Journal of General Physiology , 116 :449 , 2000
Abstract : We describe the genetic and kinetic defects in a congenital myasthenic syndrome due to the mutation epsilonA411P in the amphipathic helix of the acetylcholine receptor (AChR) epsilon subunit. Myasthenic patients from three unrelated families are either homozygous for epsilonA411P or are heterozygous and harbor a null mutation in the second epsilon allele, indicating that epsilonA411P is recessive. We expressed human AChRs containing wild-type or A411P epsilon subunits in 293HEK cells, recorded single channel currents at high bandwidth, and determined microscopic rate constants for individual channels using hidden Markov modeling. For individual wild-type and mutant channels, each rate constant distributes as a Gaussian function, but the spread in the distributions for channel opening and closing rate constants is greatly expanded by epsilonA411P. Prolines engineered into positions flanking residue 411 of the epsilon subunit greatly increase the range of activation kinetics similar to epsilonA411P, whereas prolines engineered into positions equivalent to epsilonA411 in beta and delta subunits are without effect. Thus, the amphipathic helix of the epsilon subunit stabilizes the channel, minimizing the number and range of kinetic modes accessible to individual AChRs. The findings suggest that analogous stabilizing structures are present in other ion channels, and possibly allosteric proteins in general, and that they evolved to maintain uniformity of activation episodes. The findings further suggest that the fundamental gating mechanism of the AChR channel can be explained by a corrugated energy landscape superimposed on a steeply sloped energy well.
ESTHER : Wang_2000_J.Gen.Physiol_116_449
PubMedSearch : Wang_2000_J.Gen.Physiol_116_449
PubMedID: 10962020

Title : Pairwise interactions between neuronal alpha(7) acetylcholine receptors and alpha-conotoxin PnIB - Quiram_2000_J.Biol.Chem_275_4889
Author(s) : Quiram PA , McIntosh JM , Sine SM
Ref : Journal of Biological Chemistry , 275 :4889 , 2000
Abstract : This work uses alpha-conotoxin PnIB to probe the agonist binding site of neuronal alpha(7) acetylcholine receptors. We mutated the 13 non-cysteine residues in CTx PnIB, expressed alpha(7)/5-hydroxytryptamine-3 homomeric receptors in 293 HEK cells, and measured binding of each mutant toxin to the expressed receptors by competition against the initial rate of (125)I-alpha-bungarotoxin binding. The results reveal that residues Ser-4, Leu-5, Pro-6, Pro-7, Ala-9, and Leu-10 endow CTx PnIB with affinity for alpha(7)/5-hydroxytryptamine-3 receptors; side chains of these residues cluster in a localized region within the three-dimensional structure of CTx PnIB. We next mutated key residues in the seven loops of alpha(7) that converge at subunit interfaces to form the agonist binding site. The results reveal predominant contributions by residues Trp-149 and Tyr-93 in alpha(7) and smaller contributions by Ser-34, Arg-186, Tyr-188, and Tyr-195. To identify pairwise interactions that stabilize the receptor-conotoxin complex, we measured binding of receptor and toxin mutations and analyzed the results by double mutant cycles. The results reveal a single dominant interaction between Leu-10 of CTx PnIB and Trp-149 of alpha(7) that anchors the toxin to the binding site. We also find weaker interactions between Pro-6 of CTx PnIB and Trp-149 and between both Pro-6 and Pro-7 and Tyr-93 of alpha(7). The overall results demonstrate that a localized hydrophobic region in CTx PnIB interacts with conserved aromatic residues on one of the two faces of the alpha(7) binding site.
ESTHER : Quiram_2000_J.Biol.Chem_275_4889
PubMedSearch : Quiram_2000_J.Biol.Chem_275_4889
PubMedID: 10671525

Title : Hydrophobic pairwise interactions stabilize alpha-conotoxin MI in the muscle acetylcholine receptor binding site - Bren_2000_J.Biol.Chem_275_12692
Author(s) : Bren N , Sine SM
Ref : Journal of Biological Chemistry , 275 :12692 , 2000
Abstract : The present work delineates pairwise interactions underlying the nanomolar affinity of alpha-conotoxin MI (CTx MI) for the alpha-delta site of the muscle acetylcholine receptor (AChR). We mutated all non-cysteine residues in CTx MI, expressed the alpha(2)betadelta(2) pentameric form of the AChR in 293 human embryonic kidney cells, and measured binding of the mutant toxins by competition against the initial rate of (125)I-alpha-bungarotoxin binding. The CTx MI mutations P6G, A7V, G9S, and Y12T all decrease affinity for alpha(2)betadelta(2) pentamers by 10,000-fold. Side chains at these four positions localize to a restricted region of the known three-dimensional structure of CTx MI. Mutations of the AChR reveal major contributions to CTx MI affinity by Tyr-198 in the alpha subunit and by the selectivity determinants Ser-36, Tyr-113, and Ile-178 in the delta subunit. By using double mutant cycles analysis, we find that Tyr-12 of CTx MI interacts strongly with all three selectivity determinants in the delta subunit and that deltaSer-36 and deltaIle-178 are interdependent in stabilizing Tyr-12. We find additional strong interactions between Gly-9 and Pro-6 in CTx MI and selectivity determinants in the delta subunit, and between Ala-7 and Pro-6 and Tyr-198 in the alpha subunit. The overall results reveal the orientation of CTx MI when bound to the alpha-delta interface and show that primarily hydrophobic interactions stabilize the complex.
ESTHER : Bren_2000_J.Biol.Chem_275_12692
PubMedSearch : Bren_2000_J.Biol.Chem_275_12692
PubMedID: 10777563

Title : The extracellular linker of muscle acetylcholine receptor channels is a gating control element - Grosman_2000_J.Gen.Physiol_116_327
Author(s) : Grosman C , Salamone FN , Sine SM , Auerbach A
Ref : Journal of General Physiology , 116 :327 , 2000
Abstract : We describe the functional consequences of mutations in the linker between the second and third transmembrane segments (M2-M3L) of muscle acetylcholine receptors at the single-channel level. Hydrophobic mutations (Ile, Cys, and Phe) placed near the middle of the linker of the alpha subunit (alphaS269) prolong apparent openings elicited by low concentrations of acetylcholine (ACh), whereas hydrophilic mutations (Asp, Lys, and Gln) are without effect. Because the gating kinetics of the alphaS269I receptor (a congenital myasthenic syndrome mutant) in the presence of ACh are too fast, choline was used as the agonist. This revealed an approximately 92-fold increased gating equilibrium constant, which is consistent with an approximately 10-fold decreased EC(50) in the presence of ACh. With choline, this mutation accelerates channel opening approximately 28-fold, slows channel closing approximately 3-fold, but does not affect agonist binding to the closed state. These ratios suggest that, with ACh, alphaS269I acetylcholine receptors open at a rate of approximately 1.4 x 10(6) s(-1) and close at a rate of approximately 760 s(-1). These gating rate constants, together with the measured duration of apparent openings at low ACh concentrations, further suggest that ACh dissociates from the diliganded open receptor at a rate of approximately 140 s(-1). Ile mutations at positions flanking alphaS269 impair, rather than enhance, channel gating. Inserting or deleting one residue from this linker in the alpha subunit increased and decreased, respectively, the apparent open time approximately twofold. Contrary to the alphaS269I mutation, Ile mutations at equivalent positions of the beta, straightepsilon, and delta subunits do not affect apparent open-channel lifetimes. However, in beta and straightepsilon, shifting the mutation one residue to the NH(2)-terminal end enhances channel gating. The overall results indicate that this linker is a control element whose hydrophobicity determines channel gating in a position- and subunit-dependent manner. Characterization of the transition state of the gating reaction suggests that during channel opening the M2-M3L of the alpha subunit moves before the corresponding linkers of the beta and straightepsilon subunits.
ESTHER : Grosman_2000_J.Gen.Physiol_116_327
PubMedSearch : Grosman_2000_J.Gen.Physiol_116_327
PubMedID: 10962011

Title : Subunit interface selectivity of the alpha-neurotoxins for the nicotinic acetylcholine receptor - Osaka_1999_J.Biol.Chem_274_9581
Author(s) : Osaka H , Malany S , Kanter J , Sine SM , Taylor P
Ref : Journal of Biological Chemistry , 274 :9581 , 1999
Abstract : Peptide toxins selective for particular subunit interfaces of the nicotinic acetylcholine receptor have proven invaluable in assigning candidate residues located in the two binding sites and for determining probable orientations of the bound peptide. We report here on a short alpha-neurotoxin from Naja mossambica mossambica (NmmI) that, similar to other alpha-neurotoxins, binds with high affinity to alphagamma and alphadelta subunit interfaces (KD approximately 100 pM) but binds with markedly reduced affinity to the alphaepsilon interface (KD approximately 100 nM). By constructing chimeras composed of portions of the gamma and epsilon subunits and coexpressing them with wild type alpha, beta, and delta subunits in HEK 293 cells, we identify a region of the subunit sequence responsible for the difference in affinity. Within this region, gammaPro-175 and gammaGlu-176 confer high affinity, whereas Thr and Ala, found at homologous positions in epsilon, confer low affinity. To identify an interaction between gammaGlu-176 and residues in NmmI, we have examined cationic residues in the central loop of the toxin and measured binding of mutant toxin-receptor combinations. The data show strong pairwise interactions or coupling between gammaGlu-176 and Lys-27 of NmmI and progressively weaker interactions with Arg-33 and Arg-36 in loop II of this three-loop toxin. Thus, loop II of NmmI, and in particular the face of this loop closest to loop III, appears to come into close apposition with Glu-176 of the gamma subunit surface of the binding site interface.
ESTHER : Osaka_1999_J.Biol.Chem_274_9581
PubMedSearch : Osaka_1999_J.Biol.Chem_274_9581
PubMedID: 10092644

Title : Mutation causing congenital myasthenia reveals acetylcholine receptor beta\/delta subunit interaction essential for assembly - Quiram_1999_J.Clin.Invest_104_1403
Author(s) : Quiram PA , Ohno K , Milone M , Patterson MC , Pruitt NJ , Brengman JM , Sine SM , Engel AG
Ref : J Clinical Investigation , 104 :1403 , 1999
Abstract : We describe a severe postsynaptic congenital myasthenic syndrome with marked endplate acetylcholine receptor (AChR) deficiency caused by 2 heteroallelic mutations in the beta subunit gene. One mutation causes skipping of exon 8, truncating the beta subunit before its M1 transmembrane domain, and abolishing surface expression of pentameric AChR. The other mutation, a 3-codon deletion (beta426delEQE) in the long cytoplasmic loop between the M3 and M4 domains, curtails but does not abolish expression. By coexpressing beta426delEQE with combinations of wild-type subunits in 293 HEK cells, we demonstrate that beta426delEQE impairs AChR assembly by disrupting a specific interaction between beta and delta subunits. Studies with related deletion and missense mutants indicate that secondary structure in this region of the beta subunit is crucial for interaction with the delta subunit. The findings imply that the mutated residues are positioned at the interface between beta and delta subunits and demonstrate contribution of this local region of the long cytoplasmic loop to AChR assembly.
ESTHER : Quiram_1999_J.Clin.Invest_104_1403
PubMedSearch : Quiram_1999_J.Clin.Invest_104_1403
PubMedID: 10562302

Title : Congenital myasthenic syndromes: recent advances - Engel_1999_Arch.Neurol_56_163
Author(s) : Engel AG , Ohno K , Sine SM
Ref : Archives of Neurology , 56 :163 , 1999
Abstract : Congenital myasthenic syndromes (CMS) can arise from presynaptic, synaptic, or postsynaptic defects. Mutations of the acetylcholine receptor (AChR) that increase or decrease the synaptic response to acetylcholine (ACh) are a common cause of the postsynaptic CMS. An increased response occurs in the slow-channel syndromes. Here, dominant mutations in different AChR subunits and in different domains of the subunits prolong the activation episodes of AChR by either delaying channel closure or increasing the affinity of AChR for ACh. A decreased synaptic response to ACh occurs with recessive, loss-of-function mutations. Missense mutations in the low-affinity, fast-channel syndrome and in a disorder associated with mode-switching kinetics of AChR result in brief activation episodes and reduce the probability of channel opening. Mutations causing premature termination of the translational chain or missense mutations preventing the assembly or glycosylation of AChR curtail the expression of AChR. These mutations are concentrated in the epsilon subunit, probably because substitution of the fetal gamma for the adult epsilon subunit can rescue humans from fatal null mutations in epsilon. Recent molecular genetic studies have also elucidated the pathogenesis of the CMS caused by absence of the asymmetric form of acetylcholinesterase from the synaptic basal lamina. Endplate acetylcholinesterase deficiency is now known to be caused by mutations in the collagenic tail subunit of the asymmetric enzyme that prevents the association of the collagenic tail subunit with the catalytic subunit or its insertion into the basal lamina.
ESTHER : Engel_1999_Arch.Neurol_56_163
PubMedSearch : Engel_1999_Arch.Neurol_56_163
PubMedID: 10025421

Title : Acetylcholine receptor M3 domain: stereochemical and volume contributions to channel gating - Wang_1999_Nat.Neurosci_2_226
Author(s) : Wang HL , Milone M , Ohno K , Shen XM , Tsujino A , Batocchi AP , Tonali P , Brengman J , Engel AG , Sine SM
Ref : Nat Neurosci , 2 :226 , 1999
Abstract : By defining the functional defect in a congenital myasthenic syndrome (CMS), we show that the third transmembrane domain (M3) of the muscle acetylcholine receptor governs the speed and efficiency of gating of its channel. The clinical phenotype of this CMS results from the mutation V285I in M3 of the alpha subunit, which attenuates endplate currents, accelerates their decay and causes abnormally brief acetylcholine-induced single-channel currents. Kinetic analysis of engineered alpha V285I receptors demonstrated a predominant effect on channel gating, with abnormally slow opening and rapid closing rates. Analysis of site-directed mutations revealed stereochemical and volume-dependent contributions of alpha V285 to channel gating. Thus, we demonstrate a functional role for the M3 domain as a key component of the nicotinic acetylcholine receptor channel-gating mechanism.
ESTHER : Wang_1999_Nat.Neurosci_2_226
PubMedSearch : Wang_1999_Nat.Neurosci_2_226
PubMedID: 10195214

Title : Acetylcholine and epibatidine binding to muscle acetylcholine receptors distinguish between concerted and uncoupled models - Prince_1999_J.Biol.Chem_274_19623
Author(s) : Prince RJ , Sine SM
Ref : Journal of Biological Chemistry , 274 :19623 , 1999
Abstract : The muscle acetylcholine receptor (AChR) has served as a prototype for understanding allosteric mechanisms of neurotransmitter-gated ion channels. The phenomenon of cooperative agonist binding is described by the model of Monod et al. (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118; MWC model), which requires concerted switching of the two binding sites between low and high affinity states. The present study examines binding of acetylcholine (ACh) and epibatidine, agonists with opposite selectivity for the two binding sites of mouse muscle AChRs. We expressed either fetal or adult AChRs in 293 HEK cells and measured agonist binding by competition against the initial rate of 125I-alpha-bungarotoxin binding. We fit predictions of the MWC model to epibatidine and ACh binding data simultaneously, taking as constants previously determined parameters for agonist binding and channel gating steps, and varying the agonist-independent parameters. We find that the MWC model describes the apparent dissociation constants for both agonists but predicts Hill coefficients that are far too steep. An Uncoupled model, which relaxes the requirement of concerted state transitions, accurately describes binding of both ACh and epibatidine and provides parameters for agonist-independent steps consistent with known aspects of AChR function.
ESTHER : Prince_1999_J.Biol.Chem_274_19623
PubMedSearch : Prince_1999_J.Biol.Chem_274_19623
PubMedID: 10391899

Title : Pairwise interactions between neuronal alpha7 acetylcholine receptors and alpha-conotoxin ImI - Quiram_1999_J.Biol.Chem_274_19517
Author(s) : Quiram PA , Jones JJ , Sine SM
Ref : Journal of Biological Chemistry , 274 :19517 , 1999
Abstract : The present work uses alpha-conotoxin ImI (CTx ImI) to probe the neurotransmitter binding site of neuronal alpha7 acetylcholine receptors. We identify key residues in alpha7 that contribute to CTx ImI affinity, and use mutant cycles analysis to identify pairs of residues that stabilize the receptor-conotoxin complex. We first mutated key residues in the seven known loops of alpha7 that converge at the subunit interface to form the ligand binding site. The mutant subunits were expressed in 293 HEK cells, and CTx ImI binding was measured by competition against the initial rate of 125I-alpha-bungarotoxin binding. The results reveal a predominant contribution by Tyr-195 in alpha7, accompanied by smaller contributions by Thr-77, Tyr-93, Asn-111, Gln-117, and Trp-149. Based upon our previous identification of bioactive residues in CTx ImI, we measured binding of receptor and toxin mutations and analyzed the results using thermodynamic mutant cycles. The results reveal a single dominant interaction between Arg-7 of CTx ImI and Tyr-195 of alpha7 that anchors the toxin to the binding site. We also find multiple weak interactions between Asp-5 of CTx ImI and Trp-149, Tyr-151, and Gly-153 of alpha7, and between Trp-10 of CTx ImI and Thr-77 and Asn-111 of alpha7. The overall results establish the orientation of CTx ImI as it bridges the subunit interface and demonstrate close approach of residues on opposing faces of the alpha7 binding site.
ESTHER : Quiram_1999_J.Biol.Chem_274_19517
PubMedSearch : Quiram_1999_J.Biol.Chem_274_19517
PubMedID: 10391883

Title : Probing the structure of the ligand binding site on the muscle nicotinic receptor with Waglerin peptides -
Author(s) : Molles BE , Kline EF , Sine SM , McArdle JJ , Taylor P
Ref : Journal de Physiologie (Paris) , 92 :470 , 1998

Title : Binding orientation of thealpha-neurotoxins with the nicotinic acetylcholine receptor -
Author(s) : Osaka H , Ackermann EJ , Kanter J , Tsigelny I , Sine SM , Taylor P
Ref : Journal de Physiologie (Paris) , 92 :476 , 1998

Title : Molecular dissection of subunit interfaces in the nicotinic acetylcholine receptor - Sine_1998_J.Physiol.Paris_92_101
Author(s) : Sine SM , Bren N , Quiram PA
Ref : Journal de Physiologie (Paris) , 92 :101 , 1998
Abstract : Ligand binding sites in the muscle nicotinic acetylcholine receptor are generated by pairs of alpha and non-alpha subunits. The non-alpha subunits, gamma, delta and epsilon, contribute significantly to overall affinity of agonists and antagonists, and confer selectivity of these ligands for the two binding sites. By constructing chimeras composed of segments of the various non-alpha subunits and determining ligand selectivity, we have identified four loops, well separated in the linear sequence, that contribute to the non-alpha portion of the binding site. Studies of point mutations in these loops and labeling of engineered cysteines show that the peptide backbones of each non-alpha subunit fold into similar basic scaffolds. Studies of mutations of the peptide antagonists alpha-conotoxin M1 and ImI reveal pairs of residues in the binding site and the toxin that stabilize the complex.
ESTHER : Sine_1998_J.Physiol.Paris_92_101
PubMedSearch : Sine_1998_J.Physiol.Paris_92_101
PubMedID: 9782451

Title : Epibatidine activates muscle acetylcholine receptors with unique site selectivity - Prince_1998_Biophys.J_75_1817
Author(s) : Prince RJ , Sine SM
Ref : Biophysical Journal , 75 :1817 , 1998
Abstract : We recently showed that at desensitized muscle nicotinic receptors, epibatidine selects by 300-fold between the two agonist binding sites. To determine whether receptors in the resting, activatible state show similar site selectivity, we studied epibatidine-induced activation of mouse fetal and adult receptors expressed in 293 HEK cells. Kinetic analysis of single-channel currents reveals that (-)-epibatidine binds with 15-fold selectivity to sites of adult receptors and 75-fold selectivity to sites of fetal receptors. For each receptor subtype, site selectivity arises solely from different rates of epibatidine dissociation from the two sites. To determine the structural basis for epibatidine selectivity, we introduced mutations into either the gamma or the delta subunit and measured epibatidine binding and epibatidine-induced single-channel currents. Complexes formed by alpha and mutant gamma(K34S+F172I) subunits bind epibatidine with increased affinity compared to alphagamma complexes, whereas the kinetics of alpha2betadeltagamma(K34S+F172I) receptors reveal no change in affinity of the low-affinity site, but increased affinity of the high-affinity site. Conversely, complexes formed by alpha and mutant delta(S36K+I178F) subunits bind epibatidine with decreased affinity compared to alphadelta complexes, whereas the kinetics of alpha2betagammadelta(S36K+I178F) and alpha2betaepsilondelta(S36K+I178F) receptors show markedly reduced sensitivity to epibatidine. The overall data show that epibatidine activates muscle receptors by binding with high affinity to alphagamma and alphaepsilon sites, but with low affinity to the alphadelta site.
ESTHER : Prince_1998_Biophys.J_75_1817
PubMedSearch : Prince_1998_Biophys.J_75_1817
PubMedID: 9746523

Title : Epibatidine binds with unique site and state selectivity to muscle nicotinic acetylcholine receptors - Prince_1998_J.Biol.Chem_273_7843
Author(s) : Prince RJ , Sine SM
Ref : Journal of Biological Chemistry , 273 :7843 , 1998
Abstract : Ligand binding sites in fetal (alpha2betagammadelta) and adult (alpha2betadeltaepsilon) muscle acetylcholine receptors are formed by alphadelta, alphagamma, or alphaepsilon subunit pairs. Each type of binding site shows unique ligand selectivity due to different contributions by the delta, gamma, or epsilon subunits. The present study compares epibatidine and carbamylcholine binding in terms of their site and state selectivities for muscle receptors expressed in human embryonic kidney 293 cells. Measurements of binding to alphagamma, alphadelta, and alphaepsilon intracellular complexes reveal opposite site selectivities between epibatidine and carbamylcholine; for epibatidine the rank order of affinities is alphaepsilon > alphagamma > alphadelta, whereas for carbamylcholine the rank order is alphadelta congruent with alphaepsilon > alphagamma. Because the relative affinities of intracellular complexes resemble those of receptors in the closed activable state, the results suggest that epibatidine binds with unique site selectivity in activating the muscle receptor. Measurements of binding at equilibrium show that both enantiomers of epibatidine bind to adult and fetal receptors with shallow but monophasic binding curves. However, when receptors are fully desensitized, epibatidine binds in a biphasic manner, with dissociation constants of the two components differing by more than 170-fold. Studies of subunit-omitted receptors (alpha2betadelta2, alpha2betagamma2, and alpha2betaepsilon2) reveal that in the desensitized state, the alphadelta interface forms the low affinity epibatidine site, whereas the alphagamma and alphaepsilon interfaces form high affinity sites. In contrast to epibatidine, carbamylcholine shows little site selectivity for desensitized fetal or adult receptors. Thus epibatidine is a potentially valuable probe of acetylcholine receptor binding site structure and of elements that confer state-dependent selectivities of the binding sites.
ESTHER : Prince_1998_J.Biol.Chem_273_7843
PubMedSearch : Prince_1998_J.Biol.Chem_273_7843
PubMedID: 9525877

Title : Identification of residues in the neuronal alpha7 acetylcholine receptor that confer selectivity for conotoxin ImI - Quiram_1998_J.Biol.Chem_273_11001
Author(s) : Quiram PA , Sine SM
Ref : Journal of Biological Chemistry , 273 :11001 , 1998
Abstract : To identify residues in the neuronal alpha7 acetylcholine subunit that confer high affinity for the neuronal-specific toxin conotoxin ImI (CTx ImI), we constructed alpha7-alpha1 chimeras containing segments of the muscle alpha1 subunit inserted into equivalent positions of the neuronal alpha7 subunit. To achieve high expression in 293 human embryonic kidney cells and formation of homo-oligomers, we joined the extracellular domains of each chimera to the M1 junction of the 5-hydroxytryptamine-3 (5HT-3) subunit. Measurements of CTx ImI binding to the chimeric receptors reveal three pairs of residues in equivalent positions of the primary sequence that confer high affinity of CTx ImI for alpha7/5HT-3 over alpha1/5HT-3 homo-oligomers. Two of these pairs, alpha7Trp55/alpha1Arg55 and alpha7Ser59/alpha1Gln59, are within one of the four loops that contribute to the traditional non-alpha subunit face of the muscle receptor binding site. The third pair, alpha7Thr77/alpha1Lys77, is not within previously described loops of either the alpha or non-alpha faces and may represent a new loop or an allosterically coupled loop. Exchanging these residues between alpha1 and alpha7 subunits exchanges the affinities of the binding sites for CTx ImI, suggesting that the alpha7 and alpha1 subunits, despite sequence identity of only 38%, share similar protein scaffolds.
ESTHER : Quiram_1998_J.Biol.Chem_273_11001
PubMedSearch : Quiram_1998_J.Biol.Chem_273_11001
PubMedID: 9556580

Title : Structural elements in alpha-conotoxin ImI essential for binding to neuronal alpha7 receptors - Quiram_1998_J.Biol.Chem_273_11007
Author(s) : Quiram PA , Sine SM
Ref : Journal of Biological Chemistry , 273 :11007 , 1998
Abstract : The neuronal-specific toxin alpha-conotoxin ImI (CTx ImI) has the sequence Gly-Cys-Cys-Ser-Asp-Pro-Arg-Cys-Ala-Trp-Arg-Cys-NH2, in which each cysteine forms a disulfide bridge to produce a constrained two-loop structure. To investigate the structural basis for bioactivity we mutated individual residues in CTx ImI and determined bioactivity. Bioactivity of the toxins was determined by their competition against 125I-labeled alpha-bungarotoxin binding to homomeric receptors containing alpha7 sequence in the major extracellular domain and 5HT-3 sequence elsewhere. The results reveal two regions in CTx ImI essential for binding to the alpha7/5HT-3 receptor. The first is the triad Asp-Pro-Arg in the first loop, where conservative mutations of each residue diminish affinity by 2-3 orders of magnitude. The second region is the lone Trp in the second loop, where an aromatic side chain is required. The overall results suggest that within the triad of the first loop, Pro positions the flanking Asp and Arg for optimal interaction with one portion of the binding site, while within the second loop, Trp stabilizes the complex through its aromatic ring.
ESTHER : Quiram_1998_J.Biol.Chem_273_11007
PubMedSearch : Quiram_1998_J.Biol.Chem_273_11007
PubMedID: 9556581

Title : Mode switching kinetics produced by a naturally occurring mutation in the cytoplasmic loop of the human acetylcholine receptor epsilon subunit - Milone_1998_Neuron_20_575
Author(s) : Milone M , Wang HL , Ohno K , Prince R , Fukudome T , Shen XM , Brengman JM , Griggs RC , Sine SM , Engel AG
Ref : Neuron , 20 :575 , 1998
Abstract : We describe the genetic and kinetic defects in a congenital myasthenic syndrome caused by heteroallelic mutations of the acetylcholine receptor (AChR) epsilon subunit gene. The mutations are an in-frame duplication of six residues in the long cytoplasmic loop (epsilon1254ins18) and a cysteine-loop null mutation (epsilonC128S). The epsilon1254 ins18 mutation causes mode switching in the kinetics of receptor activation in which three modes activate slowly and inactivate rapidly. The epsilon1245ins18-AChR at the endplate shows abnormally brief activation episodes during steady state agonist application and appears electrically silent during the synaptic response to acetylcholine. The phenotypic consequences are endplate AChR deficiency, simplification of the postsynaptic region, and compensatory expression of fetal AChR that restores electrical activity at the endplate and rescues the phenotype.
ESTHER : Milone_1998_Neuron_20_575
PubMedSearch : Milone_1998_Neuron_20_575
PubMedID: 9539130

Title : Congenital myasthenic syndromes. New insights from molecular genetic and patch-clamp studies -
Author(s) : Engel AG , Ohno K , Milone M , Sine SM
Ref : Annals of the New York Academy of Sciences , 841 :140 , 1998
PubMedID: 9668233

Title : Residues at the subunit interfaces of the nicotinic acetylcholine receptor that contribute to alpha-conotoxin M1 binding - Sugiyama_1998_Mol.Pharmacol_53_787
Author(s) : Sugiyama N , Marchot P , Kawanishi C , Osaka H , Molles BE , Sine SM , Taylor P
Ref : Molecular Pharmacology , 53 :787 , 1998
Abstract : The two binding sites in the pentameric nicotinic acetylcholine receptor of subunit composition alpha2 beta gamma delta are formed by nonequivalent alpha-gamma and alpha-delta subunit interfaces, which produce site selectivity in the binding of agonists and antagonists. We show by sedimentation analysis that 125I-alpha-conotoxin M1 binds with high affinity to the alpha-delta subunit dimers, but not to alpha-gamma dimers, nor to alpha, gamma, and delta monomers, a finding consistent with alpha-conotoxin M1 selectivity for the alpha delta interface in the intact receptor measured by competition against alpha-bungarotoxin binding. We also extend previous identification of alpha-conotoxin M1 determinants in the gamma and delta subunits to the alpha subunit interface by mutagenesis of conserved residues in the alpha subunit. Most mutations of the alpha subunit affect affinity similarly at the two sites, but Tyr93Phe, Val188Lys, Tyr190Thr, Tyr198Thr, and Asp152Asn affect affinity in a site-selective manner. Mutant cycle analysis reveals only weak or no interactions between mutant alpha and non-alpha subunits, indicating that side chains of the alpha subunit do not interact with those of the gamma or delta subunits in stabilizing alpha-conotoxin M1. The overall findings suggest different binding configurations of alpha-conotoxin M1 at the alpha-delta and alpha-gamma binding interfaces.
ESTHER : Sugiyama_1998_Mol.Pharmacol_53_787
PubMedSearch : Sugiyama_1998_Mol.Pharmacol_53_787
PubMedID: 9547372

Title : Congenital myasthenic syndromes: experiments of nature - Engel_1998_J.Physiol.Paris_92_113
Author(s) : Engel AG , Ohno K , Sine SM
Ref : Journal de Physiologie (Paris) , 92 :113 , 1998
Abstract : Congenital myasthenic syndromes (CMS) can arise from presynaptic, synaptic, or postsynaptic defects. Recent studies indicate that mutations in the acetylcholine receptor (AChR) subunit genes are a common cause of the postsynaptic CMS. The mutations, which increase or decrease the response to acetylcholine, are experiments of nature that highlight functionally significant domains of the AChR.
ESTHER : Engel_1998_J.Physiol.Paris_92_113
PubMedSearch : Engel_1998_J.Physiol.Paris_92_113
PubMedID: 9782453

Title : Toxins selective for subunit interfaces as probes of nicotinic acetylcholine receptor structure - Taylor_1998_J.Physiol.Paris_92_79
Author(s) : Taylor P , Osaka H , Molles BE , Sugiyama N , Marchot P , Ackermann EJ , Malany S , McArdle JJ , Sine SM , Tsigelny I
Ref : Journal de Physiologie (Paris) , 92 :79 , 1998
Abstract : The pentameric structure of the nicotinic acetylcholine receptor with two of the five subunit interfaces serving as ligand binding sites offers an opportunity to distinguish features on the surfaces of the subunits and their ligand specificity characteristics. This problem has been approached through the study of assembly of subunits and binding characteristics of selective peptide toxins. The receptor, with its circular order of homologous subunits (alpha gamma alpha delta beta), assembles in only one arrangement, and through mutagenesis, the residues governing assembly can be ascertained. Selectivity of certain toxins is sufficient to readily distinguish between sites at the alpha gamma and alpha delta interfaces. By interchanging residues on the gamma and delta subunits, and ascertaining how they interact with the alpha-subunit, determinants forming the binding sites can be delineated. The alpha-conotoxins, which contain two disulfide loops and 12-14 amino acids, show a 10,000-fold preference for the alpha delta over the alpha gamma subunit interface with alpha epsilon falling between the two. The waglerins, as 22-24 amino acid peptides with a single core disulfide loop, show a 2000-fold preference for alpha epsilon over the alpha gamma and alpha delta interfaces. Finally, the 6700 Da short alpha-neurotoxin from N. mossambica mossambica shows a 10,000-fold preference for the alpha gamma and alpha delta interfaces over alpha epsilon. Selective mutagenesis enables one to also distinguish alpha-neurotoxin binding at the alpha gamma and alpha delta subunits. This information, when coupled with homology modeling of domains and site-directed residue modification, reveals important elements of receptor structure and conformation.
ESTHER : Taylor_1998_J.Physiol.Paris_92_79
PubMedSearch : Taylor_1998_J.Physiol.Paris_92_79
PubMedID: 9782448

Title : A model of the nicotinic receptor extracellular domain based on sequence identity and residue location - Tsigelny_1997_Biophys.J_73_52
Author(s) : Tsigelny I , Sugiyama N , Sine SM , Taylor P
Ref : Biophysical Journal , 73 :52 , 1997
Abstract : We have modeled the extracellular domains of individual subunits (amino acids 31-200) in the nicotinic acetylcholine receptor using sequence homology with copper binding proteins of known crystal structure, plastocyanin and pseudoazurin, and data from recent site-specific mutagenesis, antibody mapping, and site-directed labelling studies. These data formed an initial model that was refined using molecular dynamics and mechanics as well as electrostatic and solvation energy calculations. The sequences between residues 31 and 164 in the alpha 1-subunit and corresponding residues in homologous receptor subunits show similarity with the core sequence of the cation binding site in plastocyanin and pseudoazurin, a region in the template proteins characterized by multiple hairpin loops. In addition to defining the subunit interfaces that comprise the site for agonist and competitive antagonist binding in more detail, the findings show that negatively charged residues cluster in domains arranged to diminish electrostatic free energy of the complex. Electrostatic factors also appear to distinguish the ligand binding interfaces, alpha gamma and alpha delta, from the other three interfaces on the pentameric receptor.
ESTHER : Tsigelny_1997_Biophys.J_73_52
PubMedSearch : Tsigelny_1997_Biophys.J_73_52
PubMedID: 9199771

Title : Identification of equivalent residues in the gamma, delta, and epsilon subunits of the nicotinic receptor that contribute to alpha-bungarotoxin binding - Sine_1997_J.Biol.Chem_272_23521
Author(s) : Sine SM
Ref : Journal of Biological Chemistry , 272 :23521 , 1997
Abstract : Cysteine was introduced from residues 116 to 121 of the gamma subunit of the fetal mouse acetylcholine receptor, and the mutant receptors were treated with methanethiosulfonate reagents and examined for changes in ligand binding properties. Of the 18 combinations of mutant and reagent, only receptors harboring gammaL119C treated with the quaternary ammonium reagent MTSET (trimethylammonium-ethyl methanethiosulfonate) show a decreased number of alpha-bungarotoxin (alpha-btx) sites. The decrease of 50% suggests that alpha-btx binding to the site harboring gammaL119C is blocked. Analysis of binding of the site-selective ligands dimethyl-d-tubocurarine (DMT) and alpha-conotoxin M1 (CTX) confirm specificity of modification for the site harboring gammaL119C. Cysteines placed at equivalent positions of the delta and epsilon subunits also lead to selective loss of alpha-btx binding following MTSET treatment. gammaL119C receptors treated with the primary amine reagent MTSEA (aminoethyl methanethiosulfonate) retain alpha-btx binding to both sites but show reduced affinity for DMT and CTX at the modified site. Lysine mutagenesis of Leugamma119, Leudelta121, and Leuepsilon119 mimics MTSEA treatment, whereas mutagenesis of Thralpha119 and Glnbeta119 is without effect, demonstrating subunit and residue specificity of MTSEA modification. MTSET modification of nearby gammaY117C does not block alpha-btx binding but markedly diminishes affinity for DMT and CTX. The overall findings indicate a localized point of interaction between alpha-btx and the modified gammaL119C, deltaL121C, and epsilonL119C.
ESTHER : Sine_1997_J.Biol.Chem_272_23521
PubMedSearch : Sine_1997_J.Biol.Chem_272_23521
PubMedID: 9295287

Title : Mutation in the M1 domain of the acetylcholine receptor alpha subunit decreases the rate of agonist dissociation - Wang_1997_J.Gen.Physiol_109_757
Author(s) : Wang HL , Auerbach A , Bren N , Ohno K , Engel AG , Sine SM
Ref : Journal of General Physiology , 109 :757 , 1997
Abstract : We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) alpha subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing alpha N217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC50 to lower agonist concentrations for alpha N217K. The apparent affinity of ACh binding, measured by competition against the rate of 125I-alpha-bungarotoxin binding, is also enhanced 20-fold by alpha N217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the beta, epsilon, or delta subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.
ESTHER : Wang_1997_J.Gen.Physiol_109_757
PubMedSearch : Wang_1997_J.Gen.Physiol_109_757
PubMedID: 9222901

Title : Slow-channel myasthenic syndrome caused by enhanced activation, desensitization, and agonist binding affinity attributable to mutation in the M2 domain of the acetylcholine receptor alpha subunit - Milone_1997_J.Neurosci_17_5651
Author(s) : Milone M , Wang HL , Ohno K , Fukudome T , Pruitt JN , Bren N , Sine SM , Engel AG
Ref : Journal of Neuroscience , 17 :5651 , 1997
Abstract : We describe a novel genetic and kinetic defect in a slow-channel congenital myasthenic syndrome. The severely disabled propositus has advanced endplate myopathy, prolonged and biexponentially decaying endplate currents, and prolonged acetylcholine receptor (AChR) channel openings. Genetic analysis reveals the heterozygous mutation alphaV249F in the propositus and mosaicism for alphaV249F in the asymptomatic father. Unlike mutations described previously in the M2 transmembrane domain, alphaV249F is located N-terminal to the conserved leucines and is not predicted to face the channel lumen. Expression of the alphaV249F AChR in HEK fibroblasts demonstrates increased channel openings in the absence of ACh, prolonged openings in its presence, enhanced steady-state desensitization, and nanomolar rather than micromolar affinity of one of the two binding sites in the resting activatable state. Thus, neuromuscular transmission is compromised because cationic overloading leads to degenerating junctional folds and loss of AChR, because an increased fraction of AChR is desensitized in the resting state, and because physiological rates of stimulation elicit additional desensitization and depolarization block of transmission.
ESTHER : Milone_1997_J.Neurosci_17_5651
PubMedSearch : Milone_1997_J.Neurosci_17_5651
PubMedID: 9221765

Title : Identification of residues in the adult nicotinic acetylcholine receptor that confer selectivity for curariform antagonists - Bren_1997_J.Biol.Chem_272_30793
Author(s) : Bren N , Sine SM
Ref : Journal of Biological Chemistry , 272 :30793 , 1997
Abstract : We identify residues in the epsilon and delta subunits of the adult nicotinic acetylcholine receptor that give the alphaepsilon and alphadelta binding sites different affinities for the curariform antagonist dimethyl d-tubocurarine (DMT). By constructing epsilon-delta subunit chimeras, coexpressing them with complementary subunits, and measuring DMT binding, we identify two pairs of residues, Ileepsilon58/Hisdelta60 and Aspepsilon59/Aladelta61, responsible for DMT site selectivity in the adult receptor. The two determinants contribute approximately equally to the binding site and interact in contributing to the site. Exchange of these residues from one subunit to the other exchanges the affinities of the resulting binding sites. These determinants in the adult receptor are far from those that confer site selectivity in the fetal receptor; determinants in the fetal receptor are Ilegamma116/Valdelta118, Tyrgamma117/Thrdelta119, and Sergamma161/Lysdelta163. Thus, alternative residues confer DMT selectivity in fetal and adult acetylcholine receptors.
ESTHER : Bren_1997_J.Biol.Chem_272_30793
PubMedSearch : Bren_1997_J.Biol.Chem_272_30793
PubMedID: 9388220

Title : Congenital myasthenic syndromes due to heteroallelic nonsense\/missense mutations in the acetylcholine receptor epsilon subunit gene: identification and functional characterization of six new mutations - Ohno_1997_Hum.Mol.Genet_6_753
Author(s) : Ohno K , Quiram PA , Milone M , Wang HL , Harper MC , Pruitt JN, 2nd , Brengman JM , Pao L , Fischbeck KH , Crawford TO , Sine SM , Engel AG
Ref : Hum Mol Genet , 6 :753 , 1997
Abstract : We describe and functionally characterize six mutations of the acetylcholine receptor (AChR) epsilon subunit gene in three congenital myasthenic syndrome patients. Endplate studies demonstrated severe endplate AChR deficiency, dispersed endplate regions and well preserved junctional folds in all three patients. Electrophysiologic studies were consistent with expression of the fetal gamma-AChR at the endplates in one patient, prolongation of some channel events in another and gamma-AChR expression as well as some shorter than normal channel events in still another. Genetic analysis revealed two recessive and heteroallelic epsilon subunit gene mutations in each patient. One mutation in each (epsilonC190T [epsilon R64X], epsilon 127ins5 and epsilon 553del 7) generates a nonsense codon that predicts truncation of the epsilon subunit in its N-terminal, extracellular domain; and one mutation in each generates a missense codon (epsilon R147L, epsilon P245L and epsilon R311W). None of the mutations was detected in 100 controls. Expression studies in HEK cells indicate that the three nonsense mutations are null mutations and that surface expression of AChRs harboring the missense mutations is significantly reduced. Kinetic analysis of AChRs harboring the missense mutations show that epsilon R147L is kinetically benign, epsilon P245L prolongs burst open duration 2-fold by slowing the rate of channel closing and epsilon R311W shortens burst duration 2-fold by slowing the rate of channel opening and speeding the rate of ACh dissociation. The modest changes in activation kinetics are probably overshadowed by reduced expression of the missense mutations. The consequences of the endplate AChR deficiency are mitigated by persistent expression of gamma-AChR, changes in the release of transmitter quanta and appearance of multiple endplate regions on the muscle fiber.
ESTHER : Ohno_1997_Hum.Mol.Genet_6_753
PubMedSearch : Ohno_1997_Hum.Mol.Genet_6_753
PubMedID: 9158150

Title : Molecular dissection of subunit interfaces in the acetylcholine receptor. Identification of residues that determine agonist selectivity - Prince_1996_J.Biol.Chem_271_25770
Author(s) : Prince RJ , Sine SM
Ref : Journal of Biological Chemistry , 271 :25770 , 1996
Abstract : Agonists and antagonists select between the alphagamma and ns31744adelta binding sites of the fetal muscle acetylcholine receptor owing to different contributions by the gamma and delta subunits. To identify determinants of selectivity for agonists, we constructed a panel of gamma-delta subunit chimeras, co-expressed them with the alpha subunit in 293 HEK cells, and measured carbamylcholine binding affinity of intracellular complexes. Wild-type alphadelta complexes bind carbamylcholine about 30-fold more tightly than alphagamma complexes. This degree of selectivity is similar to that of the resting state of the receptor determined by kinetic analysis of single-channel events. We identify a primary set of determinants of selectivity, Lysgamma34/Serdelta36 and Phegamma172/Iledelta178, and a secondary set, Glugamma57/Aspdelta59 and Cysgamma115/Tyrdelta117. The contributions of all four determinants are subunit-dependent and are modified by interaction with one another. Coexpression of point mutant subunits with complementary wild-type subunits to form cell surface pentamers shows that Lysgamma34/Serdelta36 and Phegamma172/Iledelta178 contribute in a manner consistent with affecting selectivity of the resting state of the receptor, while Glugamma57 appears to contribute to the affinity of the desensitized state. The four determinants either coincide with or are close to residues known to contribute to the acetylcholine binding site. These results suggest that a minimum of four loops in the gamma and delta subunits contribute to the agonist binding site.
ESTHER : Prince_1996_J.Biol.Chem_271_25770
PubMedSearch : Prince_1996_J.Biol.Chem_271_25770
PubMedID: 8824205

Title : Asymmetric contribution of the conserved disulfide loop to subunit oligomerization and assembly of the nicotinic acetylcholine receptor - Fu_1996_J.Biol.Chem_271_31479
Author(s) : Fu DX , Sine SM
Ref : Journal of Biological Chemistry , 271 :31479 , 1996
Abstract : The acetylcholine receptor (AChR) at the motor synapse is a pentamer of homologous subunits with the composition alpha2betagammadelta. Owing to the circular arrangement of subunits that forms a central ion channel, each subunit interface contains contributions from opposite faces of each subunit, designated + and -. Common to all subunits of the AChR and members of its superfamily is a disulfide loop formed between cysteines 128 and 142 of the major extracellular domain. To gain insight into the structural contribution of the disulfide loop and its possible location, we mutated the invariant proline at position 136 to glycine (P136G) and examined subunit assembly. When introduced into any AChR subunit, P136G disrupted assembly by affecting the - face of the subunit, suggesting equivalent positioning of the loop in each subunit and localization to the - face. Also, the contribution of the loop in the overall assembly process differed for each subunit. In the beta and gamma subunits, P136G prevented assembly of higher order heteroligomers, whereas in the alpha and delta subunits, P136G prevented transport of assembled pentamers to the cell surface. The results demonstrate asymmetry in the contribution of the disulfide loop to formation of subunit interfaces, and that the loop in each subunit contributes at different stages of assembly.
ESTHER : Fu_1996_J.Biol.Chem_271_31479
PubMedSearch : Fu_1996_J.Biol.Chem_271_31479
PubMedID: 8940161

Title : End-plate acetylcholine receptor deficiency due to nonsense mutations in the epsilon subunit - Engel_1996_Ann.Neurol_40_810
Author(s) : Engel AG , Ohno K , Bouzat C , Sine SM , Griggs RC
Ref : Annals of Neurology , 40 :810 , 1996
Abstract : We describe a congenital myasthenic syndrome associated with severe end-plate (EP) acetylcholine receptor (AChR) deficiency not associated with an EP myopathy, and with evidence of immature AChR, containing the gamma instead of the epsilon subunit (gamma-AChR) at the EPs. Molecular genetic analysis of AChR-subunit genes revealed two mutations in the epsilon-subunit gene: insertion of a thymine after epsilon nucleotide 1101 (epsilon 11O1insT) that generates a nonsense codon directly, and insertion of a guanine after epsilon nucleotide 1293 (epsilon 1293insG) that generates three missense codons followed by a nonsense codon. Each mutation predicts truncation of the epsilon subunit at the level of the long cytoplasmic loop, between the third (M3) and fourth (M4) membrane spanning domains. The propositus' asymptomatic son carries epsilon 1293G, indicating that the two mutations are heteroallelic. Expression of AChR harboring either mutation in human embryonic kidney (HEK) fibroblasts was markedly reduced. Single-channel activity recorded from HEK cells expressing epsilon 11O1insT-AChR was infrequent but resembled activity of wild-type AChR channels in amplitude and open duration. No channel activity could be recorded from HEK cells expressing epsilon 1293insG-AChR. Expression of gamma-AChR at the EPs may serve as the means of phenotypic rescue from potentially fatal nonsense mutations in the epsilon-subunit gene.
ESTHER : Engel_1996_Ann.Neurol_40_810
PubMedSearch : Engel_1996_Ann.Neurol_40_810
PubMedID: 8957026

Title : New mutations in acetylcholine receptor subunit genes reveal heterogeneity in the slow-channel congenital myasthenic syndrome - Engel_1996_Hum.Mol.Genet_5_1217
Author(s) : Engel AG , Ohno K , Milone M , Wang HL , Nakano S , Bouzat C , Pruitt JN, 2nd , Hutchinson DO , Brengman JM , Bren N , Sieb JP , Sine SM
Ref : Hum Mol Genet , 5 :1217 , 1996
Abstract : Mutations in genes encoding the epsilon, delta, beta and alpha subunits of the end plate acetylcholine (ACh) receptor (AChR) are described and functionally characterized in three slow-channel congenital myasthenic syndrome patients. All three had prolonged end plate currents and AChR channel opening episodes and an end plate myopathy with loss of AChR from degenerating junctional folds. Genetic analysis revealed heterozygous mutations: epsilon L269F and delta Q267E in Patient 1, beta V266M in Patient 2, and alpha N217K in Patient 3 that were not detected in 100 normal controls. Patients 1 and 2 have no similarly affected relatives; in Patient 3, the mutation cosegregates with the disease in three generations. epsilon L269F, delta Q267E and beta V266M occur in the second and alpha N217K in the first transmembrane domain of AChR subunits; all have been postulated to contribute to the lining of the upper half of the channel lumen and all but delta Q267E are positioned toward the channel lumen, and introduce an enlarged side chain. Expression studies in HEK cells indicate that all of the mutations express normal amounts of AChR. epsilon L269F, beta V266M, and alpha N217K slow the rate of channel closure in the presence of ACh and increase apparent affinity for ACh; epsilon L269F and alpha N217K enhance desensitization, and epsilon L269F and beta V266M cause pathologic channel openings in the absence of ACh, rendering the channel leaky, delta Q267E has none of these effects and is therefore a rare polymorphism or a benign mutation. The end plate myopathy stems from cationic overloading of the postsynaptic region. The safety margin of neuromuscular transmission is compromised by AChR loss from the junctional folds and by a depolarization block owing to temporal summation of prolonged end plate potentials at physiologic rates of stimulation.
ESTHER : Engel_1996_Hum.Mol.Genet_5_1217
PubMedSearch : Engel_1996_Hum.Mol.Genet_5_1217
PubMedID: 8872460

Title : Congenital myasthenic syndrome caused by decreased agonist binding affinity due to a mutation in the acetylcholine receptor epsilon subunit - Ohno_1996_Neuron_17_157
Author(s) : Ohno K , Wang HL , Milone M , Bren N , Brengman JM , Nakano S , Quiram P , Pruitt JN , Sine SM , Engel AG
Ref : Neuron , 17 :157 , 1996
Abstract : We describe the genetic and kinetic defects for a low-affinity fast channel disease of the acetylcholine receptor (AChR) that causes a myasthenic syndrome. In two unrelated patients with very small miniature end plate (EP) potentials, but with normal EP AChR density and normal EP ultrastructure, patch-clamp studies demonstrated infrequent AChR channel events, diminished channel reopenings during ACh occupancy, and resistance to desensitization by ACh. Each patient had two heteroallelic AChR epsilon subunit gene mutations: a common epsilon P121L mutation, a signal peptide mutation (epsilon G-8R) (patient 1), and a glycosylation consensus site mutation (epsilon S143L) (patient 2). AChR expression in HEK fibroblasts was normal with epsilon P121L but was markedly reduced with the other mutations. Therefore, epsilon P121L defines the clinical phenotype. Studies of the engineered epsilon P121L AChR revealed a markedly decreased rate of channel opening, little change in affinity of the resting state for ACh, but reduced affinity of the open channel and desensitized states.
ESTHER : Ohno_1996_Neuron_17_157
PubMedSearch : Ohno_1996_Neuron_17_157
PubMedID: 8755487

Title : Molecular dissection of subunit interfaces in the acetylcholine receptor: identification of determinants of alpha-conotoxin M1 selectivity - Sine_1995_Neuron_15_205
Author(s) : Sine SM , Kreienkamp HJ , Bren N , Maeda RK , Taylor P
Ref : Neuron , 15 :205 , 1995
Abstract : The acetylcholine receptor from vertebrate skeletal muscle is a pentamer of homologous subunits with composition alpha 2 beta gamma delta. Its two ligand binding sites, formed at alpha-gamma and alpha-delta interfaces, differ in their affinities for agonists and competitive antagonists, owing to different contributions of the gamma and delta subunits. To identify portions of the gamma and delta subunits that contribute to the binding sites, the experiments described here use gamma-delta subunit chimeras and site-specific mutants to determine the basis of the 10,000-fold selectivity of conotoxin M1 for the sites. Three distinct regions of the extracellular domain were found to contribute to conotoxin M1 selectivity, each containing a single residue responsible for the contribution of that region. Residues K34, S111, and F172 of the gamma subunit confer low affinity to the alpha-gamma binding site, whereas the corresponding residues of the delta subunit, S36, Y113, and I178, confer high affinity to the alpha-delta site. Identification of three separate determinants of ligand selectivity suggests a limited model of the folding pattern of the extracellular domain of the subunits.
ESTHER : Sine_1995_Neuron_15_205
PubMedSearch : Sine_1995_Neuron_15_205
PubMedID: 7619523

Title : Mutation of the acetylcholine receptor alpha subunit causes a slow-channel myasthenic syndrome by enhancing agonist binding affinity - Sine_1995_Neuron_15_229
Author(s) : Sine SM , Ohno K , Bouzat C , Auerbach A , Milone M , Pruitt JN , Engel AG
Ref : Neuron , 15 :229 , 1995
Abstract : In five members of a family and another unrelated person affected by a slow-channel congenital myasthenic syndrome (SCCMS), molecular genetic analysis of acetylcholine receptor (AChR) subunit genes revealed a heterozygous G to A mutation at nucleotide 457 of the alpha subunit, converting codon 153 from glycine to serine (alpha G153S). Electrophysiologic analysis of SCCMS end plates revealed prolonged decay of miniature end plate currents and prolonged activation episodes of single AChR channels. Engineered mutant AChR expressed in HEK fibroblasts exhibited prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Single-channel kinetic analysis of engineered alpha G153S AChR revealed a markedly decreased rate of ACh dissociation, which causes the mutant AChR to open repeatedly during ACh occupancy. In addition, ACh binding measurements combined with the kinetic analysis indicated increased desensitization of the mutant AChR. Thus, ACh binding affinity can dictate the time course of the synaptic response, and alpha G153 contributes to the low binding affinity for ACh needed to speed the decay of the synaptic response.
ESTHER : Sine_1995_Neuron_15_229
PubMedSearch : Sine_1995_Neuron_15_229
PubMedID: 7619526

Title : Intersubunit contacts governing assembly of the mammalian nicotinic acetylcholine receptor - Kreienkamp_1995_Neuron_14_635
Author(s) : Kreienkamp HJ , Maeda RK , Sine SM , Taylor P
Ref : Neuron , 14 :635 , 1995
Abstract : Through specific intersubunit contacts, the four subunits of the nicotinic acetylcholine receptor assemble into an alpha 2 beta gamma delta pentamer. The specificity of subunit association leads to formation of proper ligand binding sites and to transport of assembled pentamers to the cell surface. To identify determinants of subunit association, we constructed chimeric subunits, transfected them into HEK 293 cells, and studied their association with wild-type subunits. We used beta gamma chimeras to determine sequences that associate with the alpha subunit to form a ligand binding site and found residues 21-131 of the gamma subunit sufficient to form the site. Residues 51-131 of the beta subunit do not form a binding site, but do promote surface expression of pentamers; of these residues, R117 is key for surface expression. We studied formation of tetramers by alpha and gamma subunits and dimers by alpha and delta subunits, and used gamma delta chimeras to identify sequences that result in either dimers or tetramers. The conserved residues I145 and T150 of the gamma subunit promote alpha gamma alpha gamma tetramer formation, whereas the corresponding residues in the delta subunit, K145 and K150, allow only alpha delta dimer formation.
ESTHER : Kreienkamp_1995_Neuron_14_635
PubMedSearch : Kreienkamp_1995_Neuron_14_635
PubMedID: 7695910

Title : Congenital myasthenic syndrome caused by prolonged acetylcholine receptor channel openings due to a mutation in the M2 domain of the epsilon subunit - Ohno_1995_Proc.Natl.Acad.Sci.U.S.A_92_758
Author(s) : Ohno K , Hutchinson DO , Milone M , Brengman JM , Bouzat C , Sine SM , Engel AG
Ref : Proc Natl Acad Sci U S A , 92 :758 , 1995
Abstract : In a congenital myasthenic syndrome with a severe endplate myopathy, patch-clamp studies revealed markedly prolonged acetylcholine receptor (AChR) channel openings. Molecular genetic analysis of AChR subunit genes demonstrated a heterozygous adenosine-to-cytosine transversion at nucleotide 790 in exon 8 of the epsilon-subunit gene, predicting substitution of proline for threonine at codon 264 and no other mutations in the entire coding sequences of genes encoding the alpha, beta, delta, and epsilon subunits. Genetically engineered mutant AChR expressed in a human embryonic kidney fibroblast cell line also exhibited markedly prolonged openings in the presence of agonist and even opened in its absence. The Thr-264-->Pro mutation in the epsilon subunit involves a highly conserved residue in the M2 domain lining the channel pore and is likely to disrupt the putative M2 alpha-helix. Our findings indicate that a single mutation at a critical site can greatly alter AChR channel kinetics, leading to a congenital myasthenic syndrome. This observation raises the possibility that mutations involving subunits of other ligand-gated channels may also exist and be the basis of various other neurologic or psychiatric disorders.
ESTHER : Ohno_1995_Proc.Natl.Acad.Sci.U.S.A_92_758
PubMedSearch : Ohno_1995_Proc.Natl.Acad.Sci.U.S.A_92_758
PubMedID: 7531341

Title : Conserved tyrosines in the alpha subunit of the nicotinic acetylcholine receptor stabilize quaternary ammonium groups of agonists and curariform antagonists - Sine_1994_J.Biol.Chem_269_8808
Author(s) : Sine SM , Quiram P , Papanikolaou F , Kreienkamp HJ , Taylor P
Ref : Journal of Biological Chemistry , 269 :8808 , 1994
Abstract : Studies with site-directed labeling reagents have identified residues near the ligand binding pocket of the nicotinic acetylcholine receptor. Among these residues are three conserved tyrosines, Tyr-93, Tyr-190, and Tyr-198 of the alpha subunit. Previous studies combined mutagenesis, expression in Xenopus oocytes, and dose-response analysis to examine contributions of these tyrosines to agonist affinity. In this study, we prepared a series of mutants at each position, expressed them in 293 HEK cells, and studied binding of agonists and antagonists to mutant receptors on intact cells. We show that all three tyrosines contribute to binding of agonists, and that each tyrosine contributes roughly equally to the binding energy. Although the contributions are roughly equivalent, the nature of the contribution is not equivalent at each position. For Tyr-93 and Tyr-190 the aromatic hydroxyl is essential, whereas for Tyr-198 aromaticity of the side chain is essential. Nearly identical results were obtained for the elementary quaternary ligand tetramethylammonium, indicating that these tyrosines contribute to stabilization of the quaternary ammonium portion of agonist. Tyr-190 and Tyr-198 also contribute to binding of the competitive antagonist dimethyl-d-tubocurarine; the side chain specificity for binding supports tyrosine interactions with one of two quaternary ammonium groups in dimethyl-d-tubocurarine. Y190F, in addition to altering binding affinity, also affects the equilibrium between activatable and desensitized receptor states.
ESTHER : Sine_1994_J.Biol.Chem_269_8808
PubMedSearch : Sine_1994_J.Biol.Chem_269_8808
PubMedID: 8132615

Title : Competitive antagonists bridge the alpha-gamma subunit interface of the acetylcholine receptor through quaternary ammonium-aromatic interactions - Fu_1994_J.Biol.Chem_269_26152
Author(s) : Fu DX , Sine SM
Ref : Journal of Biological Chemistry , 269 :26152 , 1994
Abstract : We recently demonstrated that conserved tyrosines Tyr198 of the alpha subunit and Tyr117 of the gamma subunit of the acetylcholine receptor stabilize binding of the curariform antagonist dimethyl-d-tubocurarine (DMT). To test the hypothesis that DMT interacts directly with these tyrosines, and therefore bridges the alpha-gamma subunit interface, we introduced point mutations into these key positions and expressed one or both mutant subunits in alpha 2 beta gamma 2 acetylcholine receptors in 293 HEK cells. Binding of DMT, measured by competition against the initial rate of 125I-alpha-bungarotoxin binding, shows high affinity for aromatic mutations, reduced affinity for polar mutations, and lowest affinity for arginine mutations. Similar side chain dependences were observed for both Tyr alpha 198 and Tyr gamma 117, indicating interaction of these residues with two symmetrical chemical groups in DMT. Two more bisquaternary antagonists, pancuronium and gallamine, show side chain dependences similar to that of DMT, indicating that the primary stabilizing interactions are aromatic-quaternary in both subunits. For the rigid ligands DMT and pancuronium, co-expressing mutant alpha and gamma subunits revealed independent contributions by each determinant, but strict independence was not observed for the flexible ligand gallamine. The free energy contributed by each aromatic-quaternary interaction was estimated to be 2-4 kcal/mol, as determined from the free energy difference between aromatic and alkyl hydroxyl mutations. Our results suggest that bis-quaternary competitive antagonists bridge the alpha-gamma subunit interface by fitting into a pocket bounded by tyrosines at positions 198 of the alpha subunit and 117 of the gamma subunit.
ESTHER : Fu_1994_J.Biol.Chem_269_26152
PubMedSearch : Fu_1994_J.Biol.Chem_269_26152
PubMedID: 7929328

Title : Structural basis of the different gating kinetics of fetal and adult acetylcholine receptors - Bouzat_1994_Neuron_13_1395
Author(s) : Bouzat C , Bren N , Sine SM
Ref : Neuron , 13 :1395 , 1994
Abstract : Structure-function studies have identified key functional motifs in the acetylcholine receptor, including residues that contribute to the ion channel and to the ligand-binding sites. Little is known, however, about determinants of channel gating kinetics. To identify structural correlates of gating, we examined the structural basis of the fetal-to-adult decrease in channel open time conferred by the presence of the epsilon subunit in place of the gamma subunit. By constructing chimeras composed of segments of the epsilon and gamma subunits, we show that the main determinant of this kinetic change is a 30 residue segment of a predicted amphipathic helix located between transmembrane domains M3 and M4. Further subdividing the amphipathic helix revealed that either multiple residues or its overall conformation confers this regulation of channel kinetics. We also show that L440 and M442, conserved residues within M4 of the gamma subunit, contribute to long duration openings characteristic of the fetal receptor.
ESTHER : Bouzat_1994_Neuron_13_1395
PubMedSearch : Bouzat_1994_Neuron_13_1395
PubMedID: 7993630

Title : Glycosylation sites selectively interfere with alpha-toxin binding to the nicotinic acetylcholine receptor - Kreienkamp_1994_J.Biol.Chem_269_8108
Author(s) : Kreienkamp HJ , Sine SM , Maeda RK , Taylor P
Ref : Journal of Biological Chemistry , 269 :8108 , 1994
Abstract : Sequence analysis reveals unique features in the alpha-subunit of nicotinic acetylcholine receptors from the alpha-toxin-resistant cobra and mongoose. Included are N-linked glycosylation signals just amino-terminal to the Tyr190, Cys192-Cys193 region of the ligand binding domain, substitution of Trp187 and Phe189 by non-aromatic residues and alteration of the proline sequence Pro194-X-X-Pro197. Glycosylation signals were inserted into the toxin-sensitive mouse alpha-subunit by the mutations F189N and W187N/F189T. The F189N alpha-subunit, when transfected with beta, gamma and delta, showed a 140-fold loss of alpha-bungarotoxin affinity, whereas the W187N/F189T double mutation exhibited a divergence in alpha-toxin affinities at the two sites, one class showing a 600-fold and the other showing an 11-fold reduction. The W187N mutant and the double mutant F189N/S191A lacking the requisite glycosylation signals exhibited little alteration in affinity, as did the P194L and P197H mutations. The glycosylation sites had little or no influence on binding of toxins of intermediate (alpha-conotoxin, 1500 Da) or small mass (lophotoxin, 500 Da) and of the agonist, carbamylcholine. The two sites for the binding of alpha-conotoxin M1 have widely divergent dissociation constants of 2.1 and 14,800 nM. Expression of alpha/gamma- and alpha/delta-subunit pairs indicated that the high and low affinity sites are formed by the alpha/delta and alpha/gamma contacts, respectively.
ESTHER : Kreienkamp_1994_J.Biol.Chem_269_8108
PubMedSearch : Kreienkamp_1994_J.Biol.Chem_269_8108
PubMedID: 7907588

Title : Molecular dissection of subunit interfaces in the acetylcholine receptor: identification of residues that determine curare selectivity - Sine_1993_Proc.Natl.Acad.Sci.U.S.A_90_9436
Author(s) : Sine SM
Ref : Proc Natl Acad Sci U S A , 90 :9436 , 1993
Abstract : The acetylcholine receptor from vertebrate skeletal muscle is a transmembrane channel that binds nerve-released acetylcholine to elicit rapid transport of small cations. Composed of two alpha subunits and one beta, one gamma, and one delta subunit, the receptor is a cooperative protein containing two sites that bind agonists, curariform antagonists, and snake alpha-toxins. Until recently the two binding sites were thought to reside entirely within each of the two alpha subunits, but affinity labeling and expression studies have demonstrated contributions by the gamma and delta subunits. Affinity labeling and mutagenesis studies have identified residues of the alpha subunit that contribute to the binding site, but the corresponding gamma- and delta-subunit residues remain unknown. By making gamma-delta chimeras and following the nearly 100-fold difference in curare affinity for the two binding sites, the present work identified residues of the gamma and delta subunits likely to be near the binding site. Two sets of binding determinants were identified in homologous positions of the gamma and delta subunits. The determinants lie on either side of a disulfide loop found within the major extracellular domain of the subunits. This loop is common to all acetylcholine, gamma-aminobutyrate, and glycine receptor subunits.
ESTHER : Sine_1993_Proc.Natl.Acad.Sci.U.S.A_90_9436
PubMedSearch : Sine_1993_Proc.Natl.Acad.Sci.U.S.A_90_9436
PubMedID: 8415719

Title : Gamma- and delta-subunits regulate the affinity and the cooperativity of ligand binding to the acetylcholine receptor - Sine_1991_J.Biol.Chem_266_19369
Author(s) : Sine SM , Claudio T
Ref : Journal of Biological Chemistry , 266 :19369 , 1991
Abstract : The acetylcholine receptor (AChR) from vertebrate skeletal muscle is a pentamer composed of two ligand-binding alpha-subunits and one beta-, gamma-, and delta-subunit. To examine the functional roles of the non-alpha-subunits, we have expressed, in stable cell lines, AChRs lacking either a gamma- or a delta-subunit. Most previous work has examined how these changes in subunit composition affect single channel properties. Here, we take advantage of the stable expression system to produce large amounts of AChR and, for the first time, examine ligand binding to altered AChRs on intact cells. The changes in subunit composition affect both ligand affinity and cooperativity of the receptor, suggesting important roles for the gamma- and delta-subunits, both in shaping the ligand binding site and maintaining cooperative interactions between alpha-subunits.
ESTHER : Sine_1991_J.Biol.Chem_266_19369
PubMedSearch : Sine_1991_J.Biol.Chem_266_19369
PubMedID: 1680865

Title : Stable expression of the mouse nicotinic acetylcholine receptor in mouse fibroblasts. Comparison of receptors in native and transfected cells - Sine_1991_J.Biol.Chem_266_13679
Author(s) : Sine SM , Claudio T
Ref : Journal of Biological Chemistry , 266 :13679 , 1991
Abstract : A stable cell line expressing mouse acetylcholine receptors (AChRs), named AM4, was established by cotransfecting into NIH 3T3 fibroblasts, alpha-, beta-, gamma-, and delta-subunit cDNAs plus the neor gene by calcium phosphate precipitation. Surface AChRs on AM4 cells contain all four subunits, sediment as a single approximately 9 S peak on sucrose gradients, and have the same ratio of alpha- to beta-subunits as surface AChRs from mouse BC3H-1 cells. The surface AChRs exhibit pharmacological properties identical to those obtained for BC3H-1 cells, including the association and dissociation rates of alpha-bungarotoxin, a low affinity and cooperative instantaneous dose-response curve, cooperative steady state agonist binding and desensitization, cooperative enhancement of agonist binding affinity by local anesthetics, and distinct affinities for curariform antagonists. Patch clamp measurements on AM4 cells reveal AChR single channel properties identical to those obtained from BC3H-1 cells, including a single class of channels with a conductance of 56 pS, short and long duration openings at low and high agonist concentrations, brief and intermediate closed duration components at low agonist concentrations, and six distinct closed duration components at high agonist concentrations. The biochemical, pharmacological, and single channel measurements indicate at least 95% of the surface AChRs on AM4 cells are alpha 2 beta gamma delta pentamers.
ESTHER : Sine_1991_J.Biol.Chem_266_13679
PubMedSearch : Sine_1991_J.Biol.Chem_266_13679
PubMedID: 1713208

Title : Activation of Torpedo acetylcholine receptors expressed in mouse fibroblasts. Single channel current kinetics reveal distinct agonist binding affinities - Sine_1990_J.Gen.Physiol_96_395
Author(s) : Sine SM , Claudio T , Sigworth FJ
Ref : Journal of General Physiology , 96 :395 , 1990
Abstract : The experiments described examine single channel currents recorded through Torpedo acetylcholine receptor channels stably expressed by a mouse fibroblast cell line. Closed-duration histograms were constructed from currents elicited by 0.5-300 microM acetylcholine (ACh). The concentration dependence of closed durations is well described by a four-state linear scheme with the addition of open-channel block by ACh. Analysis of closed durations measured at low concentrations gives estimates of the rate of opening of doubly liganded receptors, beta, the rate of dissociation of ACh from doubly liganded receptors, k-2, and the rate of channel closing, alpha. The rate of ACh dissociation from singly liganded receptors, k-1, is then deduced from closed-duration histograms obtained at intermediate ACh concentrations. With k-1, k-2 and beta determined, the rates of ACh association, k+1 and k+2, are estimated from fitting closed-duration histograms obtained over a range of high ACh concentrations. A complete set of rate constants is presented for three experimental conditions: (a) Ca2(+)-free extracellular solution containing 1 mM free Mg2+ at 22 degrees C, (b) Ca2(+)-free solution at 12 degrees C, and (c) extracellular Ca2+ and Mg2+, both at 0.5 mM, at 22 degrees C. For all three conditions the dissociation constant for the first agonist binding site is approximately 100-fold lower than that for the second site. The different affinities are due primarily to different dissociation rates. Both the association and dissociation rates depend strongly on temperature. At 22 degrees C ACh associates at diffusion-limited rates, whereas at 12 degrees C association is 30- to 60-fold slower. Also slowed at 12 degrees C are beta (4-fold), k-2 (3-fold), k-1 (25-fold), and alpha (15-fold). In contrast to the activation rate constants, those for ACh-induced block decrease only twofold between 22 and 12 degrees C. Changing from a Ca2(+)-free to a Ca2(+)-containing extracellular solution does not affect k+1 and k+2, but increases beta (twofold) and decreases k-2, k-1, and alpha (all twofold). Spectral analysis of single channel currents supports the parameter estimates obtained from fitting the open- and closed-duration histograms, and improves resolution of brief channel blockages produced by ACh.
ESTHER : Sine_1990_J.Gen.Physiol_96_395
PubMedSearch : Sine_1990_J.Gen.Physiol_96_395
PubMedID: 1698917

Title : Functional properties of human skeletal muscle acetylcholine receptors expressed by the TE671 cell line - Sine_1988_J.Biol.Chem_263_18052
Author(s) : Sine SM
Ref : Journal of Biological Chemistry , 263 :18052 , 1988
Abstract : Functional properties of acetylcholine receptors from intact TE671 human medulloblastoma cells were examined using tracer ion flux, ligand competition against 125I-labeled alpha-bungarotoxin binding, and single channel recording measurements. 125I-Labeled alpha-bungarotoxin binds to surface receptors with the forward rate constant 1.8 X 10(5) M-1 s-1 and dissociates with the rate constant 4.6 X 10(-5) s-1, at 21 degrees C; the apparent dissociation constant is 2.6 X 10(-10) M. alpha-Bungarotoxin binds to at least two sites/receptor, but blocks agonist-induced 22Na+ uptake when bound to only one site. The reversible antagonists, dimethyl-d-tubocurarine and gallamine, occupy two sites which exhibit nearly equivalent affinities, but block agonist-induced uptake by occupying only one site. Strong agonists activate rapid sodium uptake with relatively low affinity, but desensitize with a much higher affinity; among agonists, the ratio of low to high affinity dissociation constants ranges from 1600 to 4000. By using the estimated dissociation constants, the allosteric model of Monod, Wyman, and Changeux (MWC) can be fitted to the concentration dependencies of both steady-state agonist occupancy and desensitization. The fitting analysis discloses an allosteric constant of 3 X 10(-5), which is the ratio of activatable to desensitized receptors in the absence of agonist. The rate of recovery from desensitization can exceed the rate of onset of desensitization elicited by low concentrations of agonist, further supporting the general MWC framework. Single channel recordings show that the channel opening probability is greater than 0.7 at high agonist concentrations. Favorable channel opening is shown to only slightly oppose strong desensitization.
ESTHER : Sine_1988_J.Biol.Chem_263_18052
PubMedSearch : Sine_1988_J.Biol.Chem_263_18052
PubMedID: 2461364

Title : Genetic reconstitution of functional acetylcholine receptor channels in mouse fibroblasts - Claudio_1987_Science_238_1688
Author(s) : Claudio T , Green WN , Hartman DS , Hayden D , Paulson HL , Sigworth FJ , Sine SM , Swedlund A
Ref : Science , 238 :1688 , 1987
Abstract : Foreign genes can be stably integrated into the genome of a cell by means of DNA-mediated gene transfer techniques, and large quantities of homogenous cells that continuously express these gene products can then be isolated. Such an expression system can be used to study the functional consequences of introducing specific mutations into genes and to study the expressed protein in the absence of cellular components with which it is normally in contact. All four Torpedo acetylcholine receptor (AChR) subunit complementary DNA's were introduced into the genome of a mouse fibroblast cell by DNA-mediated gene transfer. A clonal cell line that stably produced high concentrations of correctly assembled cell surface AChR's and formed proper ligand-gated ion channels was isolated. With this new expression system, recombinant DNA, biochemical, pharmacological, and electrophysiological techniques were combined to study Torpedo AChR's in a single intact system. The physiological and pharmacological profiles of Torpedo AChR's expressed in mouse fibroblast cells differ in some details from those described earlier, and may provide a more accurate reflection of the properties of this receptor in its natural environment.
ESTHER : Claudio_1987_Science_238_1688
PubMedSearch : Claudio_1987_Science_238_1688
PubMedID: 3686008

Title : Data transformations for improved display and fitting of single-channel dwell time histograms - Sigworth_1987_Biophys.J_52_1047
Author(s) : Sigworth FJ , Sine SM
Ref : Biophysical Journal , 52 :1047 , 1987
Abstract : A.L. Blatz and K.L. Magleby (1986a. J. Physiol. [Lond.]. 378:141-174) have demonstrated the usefulness of plotting histograms with a logarithmic time axis to display the distributions of dwell times from recordings of single ionic channels. We derive here the probability density function (pdf) corresponding to logarithmically binned histograms. Plotted on a logarithmic time scale the pdf is a peaked function with an invariant width; this and other properties of the pdf, coupled with a variance-stabilizing (square root) transformation for the ordinate, greatly simplify the interpretation and manual fitting of distributions containing multiple exponential components. We have also examined the statistical errors in estimation, by the maximum-likelihood method, of kinetic parameters from logarithmically binned data. Using binned data greatly accelerates the fitting procedure and introduces significant errors only for bins spaced more widely than 8-16 per decade.
ESTHER : Sigworth_1987_Biophys.J_52_1047
PubMedSearch : Sigworth_1987_Biophys.J_52_1047
PubMedID: 2447968

Title : Activation of acetylcholine receptors on clonal mammalian BC3H-1 cells by high concentrations of agonist - Sine_1987_J.Physiol_385_325
Author(s) : Sine SM , Steinbach JH
Ref : The Journal of Physiology , 385 :325 , 1987
Abstract : 1. Currents were recorded through acetylcholine (ACh) receptor channels on clonal BC3H-1 cells in the presence of high concentrations of ACh (20-1000 microM) and carbamylcholine (180-1000 microM). 2. Channel openings at high agonist concentrations occur in clusters separated by long silent periods (seconds). Clusters, in turn, show groups of closely spaced openings separated by relatively long (hundreds of milliseconds) closed periods. The closed periods between clusters and between groups within clusters are thought to reflect two desensitized states (Sakmann, Patlak & Neher, 1980). 3. Openings within groups consist largely of long-duration openings. An excess of brief-duration openings is seen at all high agonist concentrations; most brief openings occur as isolated, solitary openings. 4. The distribution of closed periods within groups shows four exponential components with time constants separated by several fold over the range of 50 microseconds to 50 ms. 5. The distribution of closed periods within groups is analysed as a function of agonist concentration, to estimate rate constants for transitions in a hypothetical reaction scheme for receptor activation. One or two of these components (depending on agonist and agonist concentration) appear to reflect agonist binding and channel gating. It is hypothesized that the other closed-period components within groups at high agonist concentrations result from additional states of doubly liganded receptors which have closed ion channels. 6. With ACh as agonist the data indicate that binding and activation saturate at concentrations above 130 microM. The data are quantitatively consistent with measurements made at low concentrations of ACh (Sine & Steinbach, 1986b), and indicate that a four-state linear scheme is able to describe major features of ACh-receptor activation on BC3H-1 cells. The channel opening rate is estimated to be about 450 s-1 and the closing rate about 35 s-1 (-70 mV, 11 degrees C). The concentration dependence of closed durations suggests that some positive co-operativity exists in agonist binding. The dissociation constant with one ACh molecule bound is about 50 microM, and that with two bound is about 10 microM (for an ACh receptor with a closed channel). 7. Saturation is not observed with carbamylcholine, even at 1 mM. The data are consistent with data obtained at low concentrations of carbamylcholine, and are in over-all agreement with the interpretation of data obtained with ACh. The affinity for carbamylcholine is estimated to be about 20-fold lower than with ACh.
ESTHER : Sine_1987_J.Physiol_385_325
PubMedSearch : Sine_1987_J.Physiol_385_325
PubMedID: 2443668

Title : Function of nicotinic acetylcholine receptors - Steinbach_1987_Soc.Gen.Physiol.Ser_41_19
Author(s) : Steinbach JH , Sine SM
Ref : Soc Gen Physiol Ser , 41 :19 , 1987
Abstract : In summary, the AChRs on BC3H-1 cells behave in a fashion consistent with the major properties expected of muscle nicotinic AChRs. The results we have discussed here provide a quantitative description of some of the steps in receptor activation, and give a general picture of the functional states the AChR adopts. The results provide a framework for biochemical and ultrastructural studies of the AChR as well. One clear problem is that the receptor with an open channel is a very low probability state at equilibrium, at any concentration. A second problem is the postulated existence of closed channel states associated with brief-duration openings. It is not known what the probability is that a receptor is in such a state, so even "resting" receptors at low agonist concentrations may be in several functional states. At high agonist concentrations the receptor population goes through several transient states before reaching the final equilibrium state. At 11 degrees C, with a high concentration of ACh (greater than 0.3 mM) receptors with open channels appear rapidly (rate approximately 500 sec-1). Initially, there is a high probability that a receptor is both doubly liganded and has an open channel (0.9, Fig. 6). However, the predesensitized state develops rapidly (approximately 30 sec-1), and eventually the probability reaches about 0.4 that doubly liganded receptors are predesensitized. The short-lived desensitized state develops at a rate of about 3 sec-1. After a second or so, the distribution is about 0.3:0.2:0.5 for AChRs between receptors with open channels, predesensitized receptors, and short-lived desensitized receptors. Long-lived desensitization develops at a rate of about 0.5 sec-1, but at equilibrium the probability that a receptor is in this state is above 0.99. For AChRs on BC3H-1 cells, and most likely all AChRs, biochemical and structural studies need to be made in a narrow time window to catch an appreciable fraction of the receptors in any of the transient functional states described. Only time-resolved biochemical studies will provide the structural information necessary to work out the relationship between structure and function for the ACh receptor and give some substance to the ghostly kinetic states necessary to describe channel function.
ESTHER : Steinbach_1987_Soc.Gen.Physiol.Ser_41_19
PubMedSearch : Steinbach_1987_Soc.Gen.Physiol.Ser_41_19
PubMedID: 2436310

Title : Acetylcholine receptor activation by a site-selective ligand: nature of brief open and closed states in BC3H-1 cells - Sine_1986_J.Physiol_370_357
Author(s) : Sine SM , Steinbach JH
Ref : The Journal of Physiology , 370 :357 , 1986
Abstract : Single-channel currents were recorded through acetylcholine receptor channels of clonal BC3H-1 muscle cells activated by the curare-like compound, DMT binds selectively to the two alpha-neurotoxin-binding sites on these receptors, with apparent dissociation constants differing by about 1000-fold (Sine & Taylor, 1981). Receptor channels do not open with DMT bound only to the high-affinity site, but only at DMT concentrations at which both high- and low-affinity sites are occupied. Open-duration histograms are not single exponentials, but are described by the sums of two (or three) exponentials. Both brief- and long-duration openings are observed in the presence of 3 microM-DMT, and are seen at the same relative frequency up to 80 microM-DMT. Long-duration openings are interrupted by brief closures with a mean duration of 50 microseconds and which occur at a frequency of 50-60 per second of open time. These temporal characteristics closely parallel those of the brief closures observed with the full agonists, acetylcholine, carbamylcholine, and suberyldicholine. Raised concentrations of DMT apparently block open channels in a voltage-dependent fashion. It is concluded that both brief- and long-duration openings arise from receptors with two molecules of DMT bound. Furthermore, brief closures in general do not appear to reflect receptor activation processes. Instead, they seem to arise through entry to a closed state with properties independent of the agonist, but characteristic of open channels.
ESTHER : Sine_1986_J.Physiol_370_357
PubMedSearch : Sine_1986_J.Physiol_370_357
PubMedID: 2420977

Title : Activation of acetylcholine receptors on clonal mammalian BC3H-1 cells by low concentrations of agonist - Sine_1986_J.Physiol_373_129
Author(s) : Sine SM , Steinbach JH
Ref : The Journal of Physiology , 373 :129 , 1986
Abstract : The patch-clamp technique was used to examine the activation of single acetylcholine receptor channels of clonal BC3H-1 mouse muscle cells. Single-channel currents were activated by low concentrations of the strong agonists acetylcholine (ACh, 50-100 nM), carbamylcholine (1-2 microM), and suberyldicholine (30-50 nM). At low agonist concentrations channel openings occur as isolated short-duration openings and as bursts of longer duration openings separated by brief closed periods. Two distinct types of brief closed periods separate long duration openings: brief closures (mean duration, 50 microseconds) and intermediate closures (mean duration, 0.5-1.0 ms). The kinetic properties of intermediate closures depend on the agonist, suggesting that they reflect receptor reopening from the closed state leading to the open state. Properties of brief closures, in contrast, are independent of the agonist, indicating that they result from an additional closed state leading away from the pathway producing the open state. A receptor activation scheme is proposed which accounts for the observed closed states, and transition rate estimates are presented for steps within the proposed scheme. The channel opening rate, beta, differs several-fold for the agonists studied (200-1400 s-1) and is comparable to the dissociation rate, k-2 (900 s-1). The dissociation rate is similar for the three agonists studied. The channel closing rate, alpha, is much slower than the opening rate (20-60 s-1). The probability is high that a doubly liganded channel is in the open state and depends on the agonist (0.75-0.97). Beta increases and alpha decreases at more negative membrane potentials, whereas k-2 shows little potential dependence.
ESTHER : Sine_1986_J.Physiol_373_129
PubMedSearch : Sine_1986_J.Physiol_373_129
PubMedID: 2427693

Title : Activation of a nicotinic acetylcholine receptor - Sine_1984_Biophys.J_45_175
Author(s) : Sine SM , Steinbach JH
Ref : Biophysical Journal , 45 :175 , 1984
Abstract : We studied activation of the nicotinic acetylcholine (ACh) receptor on cells of a mouse clonal muscle cell line (BC3H1). We analyzed single-channel currents through outside-out patches elicited with various concentrations of acetylcholine (ACh), carbamylcholine (Carb) and suberyldicholine (Sub). Our goal is to determine a likely reaction scheme for receptor activation by agonist and to determine values of rate constants for transitions in that scheme. Over a wide range of agonist concentrations the open-time duration histograms are not described by single exponential functions, but are well-described by the sum of two exponentials, a brief-duration and a long-duration component. At high concentration, channel openings occur in groups and these groups contain an excess number of brief openings. We conclude that there are two open states of the ACh receptor with different mean open times and that a single receptor may open to either open state. The concentration dependence of the numbers of brief and long openings indicates that brief openings do not result from the opening of channels of receptors which have only one agonist molecule bound to them. Closed-time duration histograms exhibit a major brief component at low concentrations. We have used the method proposed by Colquhoun and Sakmann (1981) to analyze these brief closings and to extract estimates for the rates of channel opening (beta) and agonist dissociation (k-2). We find that this estimate of beta does not predict our closed-time histograms at high agonist concentration (ACh: 30-300 microM; Carb: 300-1,000 microM). We conclude that brief closings at low agonist concentrations do not result solely from transitions between the doubly-liganded open and the doubly-liganded closed states. Instead, we postulate the existence of a second closed-channel state coupled to the open state.
ESTHER : Sine_1984_Biophys.J_45_175
PubMedSearch : Sine_1984_Biophys.J_45_175
PubMedID: 6324901

Title : Agonists block currents through acetylcholine receptor channels - Sine_1984_Biophys.J_46_277
Author(s) : Sine SM , Steinbach JH
Ref : Biophysical Journal , 46 :277 , 1984
Abstract : We have examined the effects of high concentrations of cholinergic agonists on currents through single acetylcholine receptor (AChR) channels on clonal BC3H1 cells. We find that raised concentrations of acetylcholine (ACh; above 300 microM) or carbamylcholine (Carb; above 1,000 microM) produce a voltage- and concentration-dependent reduction in the mean single-channel current. Raised concentrations of suberyldicholine (Sub; above 3 microM) produce a voltage- and concentration-dependent increase in the number of brief duration low-conductance interruptions of open-channel currents. These observations can be quantitatively described by a model in which agonist molecules enter and transiently occlude the ion-channel of the AChR.
ESTHER : Sine_1984_Biophys.J_46_277
PubMedSearch : Sine_1984_Biophys.J_46_277
PubMedID: 6478036

Title : Local anesthetics and histrionicotoxin are allosteric inhibitors of the acetylcholine receptor. Studies of clonal muscle cells -
Author(s) : Sine SM , Taylor P
Ref : Journal of Biological Chemistry , 257 :8106 , 1982
PubMedID: 7085658

Title : Relationship between reversible antagonist occupancy and the functional capacity of the acetylcholine receptor -
Author(s) : Sine SM , Taylor P
Ref : Journal of Biological Chemistry , 256 :6692 , 1981
PubMedID: 7240238

Title : The relationship between agonist occupation and the permeability response of the cholinergic receptor revealed by bound cobra alpha-toxin - Sine_1980_J.Biol.Chem_255_10144
Author(s) : Sine SM , Taylor P
Ref : Journal of Biological Chemistry , 255 :10144 , 1980
Abstract : The decrement in functional capacity of the nicotinic receptor on intact BC3H-1 cells has been simultaneously compared with the fractional occupation of the receptor by cobra alpha-toxin. A parabolic, concave inward relationship between the fractional occupation of receptors by alpha-toxin and the decrement in permeability response is observed when the latter is tested over a range of agonist concentrations. Since alpha-toxin binding appears equivalent at each site on the receptor, the observed relationship is accommodated by a model where activation of a permeability response requires agonist occupation of two toxin-binding sites per functional receptor. Furthermore, the binding of alpha-toxin and agonist appears to be mutually exclusive, but occupation of either of the two sites by alpha-toxin is sufficient to block the functional capacity of the receptor. Consistent with this model, when a major fraction of sites is occupied by alpha-toxin, the concentration dependence for either carbamylcholine-mediated activation or desensitization of the remaining functional receptors is not detectably altered and retains positive cooperativity. In contrast, progressive occupation of the available sites by alpha-toxin leads to a decrease in apparent affinity and a corresponding loss of positive cooperation for agonist occupation functions generated upon instantaneous or following equilibrium exposure to the agonist. At high degrees of fractional occupancy to alpha-toxin, where the dominant species capable of binding agonist would contain a single bound toxin molecule, the Hill coefficient for the equilibrium occupation function for full agonists falls from a value of 1.4 to 0.7. By contrast, the binding isotherms for antagonists which typically exhibit values less than 1.0 are not altered following fractional irreversible occupation by alpha-toxin. Thus, the two binding sites on the receptor oligomer are not intrinsically equivalent for the binding of agonists and reversible antagonists. A scheme for desensitization of the receptor is presented which incorporates both nonequivalence in the two agonist binding-sites and the maintenance of symmetry in the receptor states undergoing transitions.
ESTHER : Sine_1980_J.Biol.Chem_255_10144
PubMedSearch : Sine_1980_J.Biol.Chem_255_10144
PubMedID: 7430118

Title : Studies on vitamin D and its analogs. VI. 3-deoxy-A-homovitamin D3, a model synthesis -
Author(s) : Sine SM , Conklin TE , Okamura WH
Ref : J Org Chem , 39 :3797 , 1974
PubMedID: 4377157

Title : Vitamin D in solution: conformations of vitamin D3, 1alpha,25-dihydroxyvitamin D3, and dihydrotachysterol3 - Wing_1974_Science_186_939
Author(s) : Wing RM , Okamura WH , Pirio MR , Sine SM , Norman AW
Ref : Science , 186 :939 , 1974
Abstract : Solution conformations of the A and seco B rings of vitamin D(3), 1(alpha), 25-dihydroxyvitamin D(3), 1(alpha)-hydroxyvitamin D(3), and dihydrotachysterol(3) have been established by high resolution, 300-megahertz proton magnetic resonance spectroscopy. The A ring of these steroids is dynamically equilibrated between two chair conformers. For vitamin D(3), 1(alpha)-hydroxyvitamin D(3), and 1(alpha),25-dihydroxyvitamin D(3) the relative proportions of the two conformers are 1 : 1, whereas dihydrotachysterol3 exists principally as only one conformer. Thus, the substituent groups on the A ring may be either equatorially or axially oriented, and suggests a refinement of the existing topological model for vitamin D hormonal activity.
ESTHER : Wing_1974_Science_186_939
PubMedSearch : Wing_1974_Science_186_939
PubMedID: 4377759