Author Report for: Stokes C

Homomeric alpha7 nicotinic acetylcholine receptors (nAChR) have an intrinsically low probability of opening that can be overcome by alpha7-selective positive allosteric modulators (PAMs), which bind at a site involving the second transmembrane domain (TM2). Mutation of a methionine that is unique to alpha7 at the 15' position of TM2 to leucine, the residue in most other nAChR subunits, largely eliminates the activity of such PAMs. We tested the effect of the reverse mutation (L15'M) in heteromeric nAChR receptors containing alpha4 and beta2, which are the nAChR subunits that are most abundant in the brain. Receptors containing these mutations were found to be strongly potentiated by the alpha7 PAM 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS) but insensitive to the alternative PAM 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)-urea. The presence of the mutation in the beta2 subunit was necessary and sufficient for TQS sensitivity. The primary effect of the mutation in the alpha4 subunit was to reduce responses to acetylcholine applied alone. Sensitivity to TQS required only a single mutant beta subunit, regardless of the position of the mutant beta subunit within the pentameric complex. Similar results were obtained when beta2L15'M was coexpressed with alpha2 or alpha3 and when the L15'M mutation was placed in beta4 and coexpressed with alpha2, alpha3, or alpha4. Functional receptors were not observed when beta1L15'M subunits were coexpressed with other muscle nAChR subunits. The unique structure-activity relationship of PAMs and the alpha4beta2L15'M receptor compared with alpha7 and the availability of high-resolution alpha4beta2 structures may provide new insights into the fundamental mechanisms of nAChR allosteric potentiation. SIGNIFICANCE STATEMENT: Heteromeric neuronal nAChRs have a relatively high initial probability of channel activation compared to receptors that are homomers of alpha7 subunits but are insensitive to PAMs, which greatly increase the open probability of alpha7 receptors. These features of heteromeric nAChR can be reversed by mutation of a single residue present in all neuronal heteromeric nAChR subunits to the sequence found in alpha7. Specifically, the mutation of the TM2 15' leucine to methionine in alpha subunits reduces heteromeric receptor channel activation, while the same mutation in neuronal beta subunits allows heteromeric receptors to respond to select alpha7 PAMs. The results indicate a key role for this residue in the functional differences in the two main classes of neuronal nAChRs.

Nicotine, the primary psychoactive component in tobacco smoke, produces its behavioral effects through interactions with neuronal nicotinic acetylcholine receptors (nAChRs). alpha4beta2 nAChRs are the most abundant in mammalian brain, and converging evidence shows that this subtype mediates the rewarding and reinforcing effects of nicotine. A number of rare variants in the CHRNA4 gene that encode the alpha4 nAChR subunit have been identified in human subjects and appear to be underrepresented in a cohort of smokers. We compared three of these variants (alpha4R336C, alpha4P451L, and alpha4R487Q) to the common variant to determine their effects on alpha4beta2 nAChR pharmacology. We examined [(3)H]epibatidine binding, interacting proteins, and phosphorylation of the alpha4 nAChR subunit with liquid chromatography and tandem mass spectrometry (LC-MS/MS) in HEK 293 cells and voltage-clamp electrophysiology in Xenopus laevis oocytes. We observed significant effects of the alpha4 variants on nAChR expression, subcellular distribution, and sensitivity to nicotine-induced receptor upregulation. Proteomic analysis of immunopurified alpha4beta2 nAChRs incorporating the rare variants identified considerable differences in the intracellular interactomes due to these single amino acid substitutions. Electrophysiological characterization in X. laevis oocytes revealed alterations in the functional parameters of activation by nAChR agonists conferred by these alpha4 rare variants, as well as shifts in receptor function after incubation with nicotine. Taken together, these experiments suggest that genetic variation at CHRNA4 alters the assembly and expression of human alpha4beta2 nAChRs, resulting in receptors that are more sensitive to nicotine exposure than those assembled with the common alpha4 variant. The changes in nAChR pharmacology could contribute to differences in responses to smoked nicotine in individuals harboring these rare variants.

GAT107, the (+)-enantiomer of racemic 4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide, is a strong positive allosteric modulator (PAM) of alpha7 nicotinic acetylcholine receptor (nAChR) activation by orthosteric agonists with intrinsic allosteric agonist activities. The direct activation produced by GAT107 in electrophysiological studies is observed only as long as GAT107 is freely diffusible in solution, although the potentiating activity primed by GAT107 can persist for over 30 min after drug washout. Direct activation is sensitive to alpha7 nAChR antagonist methyllycaconitine, although the primed potentiation is not. The data are consistent with GAT107 activity arising from two different sites. We show that the coupling between PAMs and the binding of orthosteric ligands requires tryptophan 55 (Trp-55), which is located at the subunit interface on the complementary surface of the orthosteric binding site. Mutations of Trp-55 increase the direct activation produced by GAT107 and reduce or prevent the synergy between allosteric and orthosteric binding sites, so that these mutants can also be directly activated by other PAMs such as PNU-120596 and TQS, which do not activate wild-type alpha7 in the absence of orthosteric agonists. We identify Tyr-93 as an essential element for orthosteric activation, because Y93C mutants are insensitive to orthosteric agonists but respond to GAT107. Our data show that both orthosteric and allosteric activation of alpha7 nAChR require cooperative activity at the interface between the subunits in the extracellular domain. These cooperative effects rely on key aromatic residues, and although mutations of Trp-55 reduce the restraints placed on the requirement for orthosteric agonists, Tyr-93 can conduct both orthosteric activation and desensitization among the subunits.

RATIONALE: Cigarette smoking is one of the most serious health problems worldwide and people trying to stop smoking have high rates of relapse. Zebrafish (Danio rerio), by combining pharmacological and behavioral assays, is a promising animal model for rapidly screening new compounds to induce smoking cessation. OBJECTIVES: This study aims to identify possible acetylcholine nicotinic receptors (nAChRs) involved in mediating nicotine (NIC)-induced conditioned place preference (CPP) in zebrafish and investigate the effect of the CC4 and CC26 cytisine derivatives in reducing NIC-induced CPP.
METHODS: CPP was evaluated using a two-compartment chamber, and the zebrafish were given CC4 (0.001-5 mg/kg), CC26 (0.001-1 mg/kg), cytisine (0.1-2.5 mg/kg), and varenicline (1-10 mg/kg) alone or with NIC (0.001 mg/kg). Swimming activity was evaluated using a square observational chamber. The affinity of the nicotinic ligands for native zebrafish brain nAChRs was evaluated by binding studies using [(3)H]-Epibatidine (Epi) and [(125)I]-alphaBungarotoxin (alphaBgtx) radioligands, and their subtype specificity was determined by means of electrophysiological assay of oocyte-expressed alpha4beta2 and alpha7 subtypes.
RESULTS: CC4 and CC26 induced CPP with an inverted U-shaped dose-response curve similar to that of NIC. However, when co-administered with NIC, they blocked its reinforcing or slightly aversive effect. Binding and electrophysiological studies showed that this effect was due to binding to high-affinity heteromeric but not alpha7-containing receptors.
CONCLUSIONS: We have further characterized CC4 and identified a new compound (CC26) that may be active in inducing smoking cessation. Zebrafish is a very useful model for screening new compounds that can affect the rewarding properties of NIC.

The homomeric alpha7 nicotinic acetylcholine receptor is a well-studied therapeutic target, though its characteristically rapid desensitization complicates the development of drugs with specific agonist effects. Moreover, some experimental compounds such as GTS-21 (2,4diMeOBA), a derivative of the alpha7-selective partial agonist benzylidene anabaseine (BA), produce a prolonged residual desensitization (RD) in which the receptor remains non-activatable long after the drug has been removed from extracellular solution. In contrast, the desensitization caused by GTS-21's dihydroxy metabolite (2,4diOHBA) is relatively short-lived. RD is hypothetically due to stable binding of the ligand to the receptor in its desensitized state. We can attribute the reduction in RD to a single BA hydroxyl group on the 4' benzylidene position. Computational prediction derived from homology modeling showed the serine36 (S36) residue of alpha7 as a reasonable candidate for point-to-point interaction between BA compounds and the receptor. Through evaluating the activity of BA and simple derivatives on wild-type and mutant alpha7 receptors, it was observed that the drug-receptor pairs which were capable of hydrogen bonding at residue 36 exhibited significantly less stable desensitization. Further experiments involving the type II positive allosteric modulator (PAM) PNU-120596 showed that the various BA compounds' preference to induce either a PAM-sensitive (D(s)) or PAM-insensitive (D(i)) desensitized state is concentration dependent and suggested that both states are destabilized by S36 H-bonding. These results indicate that the fine-tuning of agonists for specific interaction with S36 can facilitate the development of therapeutics with targeted effects on ion channel desensitization properties and conformational state stability.

Partial agonist therapies for the treatment of nicotine addiction and dependence depend on both agonistic and antagonistic effects of the ligands, and side effects associated with other nAChRs greatly limit the efficacy of nicotinic partial agonists. We evaluated the in vitro pharmacological properties of four partial agonists, two current smoking cessation drugs, varenicline and cytisine, and two novel bispidine compounds, BPC and BMSP, by using defined nAChR subtypes expressed in Xenopus laevis oocytes and human embryonic kidney 293 cells. Similar to varenicline and cytisine, BPC and BMSP are partial agonists of alpha4beta2 nAChRs, although BMSP produced very little activation of these receptors. Unlike varenicline and cytisine, BPC and BMSP showed desired low activity. BPC produced mecamylamine-sensitive steady-state activation of alpha4* receptors that was not evident with BMSP. We evaluated the modulation of alpha4*- and alpha7-mediated responses in rat lateral geniculate nucleus (LGN) neurons and hippocampal stratum radiatum (SR) interneurons, respectively. The LGN neurons were sensitive to a very low concentration of varenicline, and the SR interneuron responses were also sensitive to varenicline at a submicromolar concentration. Although 300 nM BPC strongly inhibited the ACh-evoked responses of LGN neurons, it did not inhibit the alpha7 currents of SR interneurons. Similar results were observed with 300 nM BMSP. Additionally, the bispidine compounds were efficacious in the mouse tail suspension test, demonstrating that they affect receptors in the brain when delivered systemically. Our data indicate that BPC and BMSP are promising alpha4beta2* partial agonists for pharmacotherapeutics.

Zebrafish (Danio rerio) have been used to study multiple effects of nicotine, for example on cognition, locomotion, and stress responses, relying on the assumption that pharmacological tools will operate similarly upon molecular substrates in the fish and mammalian systems. We have cloned the zebrafish nicotinic acetylcholine receptor (nAChR) subunits and expressed key nAChR subtypes in Xenopus oocytes including neuronal (alpha4beta2, alpha2beta2, alpha3beta4, and alpha7) and muscle (alpha1beta1(b)varepsilondelta) nAChR. Consistent with studies of mammalian nAChR, nicotine was relatively inactive on muscle-type receptors, having both low potency and efficacy. It had high efficacy but low potency for alpha7 receptors, and the best potency and good efficacy for alpha4beta2 receptors. Cytisine, a key lead compound for the development of smoking cessation agents, is a full agonist for both mammalian alpha7 and alpha3beta4 receptors, but a full agonist only for the fish alpha7, with surprisingly low efficacy for alpha3beta4. The efficacy of cytisine for alpha4beta2 was somewhat greater than typically reported for mammalian alpha4beta2. The ganglionic blocker mecamylamine was most potent for blocking alpha3beta4 receptors, least potent for alpha7, and roughly equipotent for the muscle receptors and the beta2-containing nAChR. However, the block of beta2-containing receptors was slowly reversible, consistent with effective targeting of these CNS-type receptors in vivo. Three prototypical alpha7-selective agonists, choline, tropane, and 4OH-GTS-21, were tested, and these agents were observed to activate both fish alpha7 and alpha4beta2 nAChR. Our data therefore indicate that while some pharmacological tools used in zebrafish may function as expected, others will not.

Drug development for nicotinic acetylcholine receptors (nAChR) is challenged by subtype diversity arising from variations in subunit composition. On-target activity for neuronal heteromeric receptors is typically associated with CNS receptors that contain alpha4 and other subunits, while off-target activity could be associated with ganglionic-type receptors containing alpha3beta4 binding sites and other subunits, including beta4, beta2, alpha5, or alpha3 as a structural subunit in the pentamer. Additional interest in alpha3 beta4 alpha5-containing receptors arises from genome-wide association studies linking these genes, and a single nucleotide polymorphism (SNP) in alpha5 in particular, to lung cancer and heavy smoking. While alpha3 and beta4 readily form receptors in expression system such as the Xenopus oocyte, since alpha5 is not required for function, simple co-expression approaches may under-represent alpha5-containing receptors. We used a concatamer of human alpha3 and beta4 subunits to form ligand-binding domains, and show that we can force the insertions of alternative structural subunits into the functional pentamers. These alpha3beta4 variants differ in sensitivity to ACh, nicotine, varenicline, and cytisine. Our data indicated lower efficacy for varenicline and cytisine than expected for beta4-containing receptors, based on previous studies of rodent receptors. We confirm that these therapeutically important alpha4 receptor partial agonists may present different autonomic-based side-effect profiles in humans than will be seen in rodent models, with varenicline being more potent for human than rat receptors and cytisine less potent. Our initial characterizations failed to find functional effects of the alpha5 SNP. However, our data validate this approach for further investigations.

A series of arylidene anabaseines were synthesized to probe the functional impact of hydrogen bonding on human alpha7 nicotinic acetylcholine receptor (nAChR) activation and desensitization. The aryl groups were either hydrogen bond acceptors (furans), donors (pyrroles), or neither (thiophenes). These compounds were tested against a series of point mutants of the ligand-binding domain residue Gln-57, a residue hypothesized to be proximate to the aryl group of the bound agonist and a putative hydrogen bonding partner. Q57K, Q57D, Q57E, and Q57L were chosen to remove the dual hydrogen bonding donor/acceptor ability of Gln-57 and replace it with hydrogen bond donating, hydrogen bond accepting, or nonhydrogen bonding ability. Activation of the receptor was compromised with hydrogen bonding mismatches, for example, pairing a pyrrole with Q57K or Q57L, or a furan anabaseine with Q57D or Q57E. Ligand co-applications with the positive allosteric modulator PNU-120596 produced significantly enhanced currents whose degree of enhancement was greater for 2-furans or -pyrroles than for their 3-substituted isomers, whereas the nonhydrogen bonding thiophenes failed to show this correlation. Interestingly, the PNU-120596 agonist co-application data revealed that for wild-type alpha7 nAChR, the 3-furan desensitized state was relatively stabilized compared with that of 2-furan, a reversal of the relationship observed with respect to the barrier for entry into the desensitized state. These data highlight the importance of hydrogen bonding on the receptor-ligand state, and suggest that it may be possible to fine-tune features of agonists that mediate state selection in the nAChR.

A large number of structurally diverse ligands have been produced to selectively target alpha7 nicotinic acetylcholine receptors (nAChRs). We applied the method of scanning cysteine accessibility mutations (SCAM) to the ligand-binding domain of the alpha7 nAChR to identify subdomains of particular importance to the binding and subsequent activation by select agonists. We evaluated the activity of four structurally distinct alpha7 agonists on wild-type human alpha7 and 44 targeted mutants expressed in Xenopus oocytes. Responses were measured prior and subsequent to the application of the sulfhydryl reagent methanethiosulfonate ethylammonium (MTSEA). One mutant (C116S) served as a Cys-null control, and the additional mutants were made in the C116S background. In many cases, the insertion of free cysteines into the agonist-binding site had a negative effect on function, with 12 of 44 mutants showing no detectable responses to ACh, and with only 19 of the 44 mutants showing sufficiently large responses to permit further study. Several of the cysteine mutations, including W55C, showed selectively reduced responses to the largest agonist tested, 2-methoxy,4-hydroxy-benzylidene anabaseine. Interestingly, although homology models suggest that most of the introduced cysteine mutations should have had good solvent accessibility, application of MTSEA had no effect or produced only modest changes in the agonist response profile of most mutants. Consistent with previous studies implicating W55 to play important roles in agonist activation, MTSEA treatment further decreased the functional responses of W55C to all the test agonists. While the cysteine mutation at L119 itself had relatively little effect on receptor function, treatment of L119C receptors with MTSEA or alternative cationic sulfhydryl reagents profoundly decreased activation by all agonists tested, suggesting a general block of gating. The homologous mutation in heteromeric nAChRs produced similar results, provided that the mutation was placed in the beta subunit complementary surface of the ligand-binding domain. Structural models locate the L119 residue directly across the subunit interface from the C-loop of the primary face of the binding domain. Our data suggest that a covalent modification of L119C by MTSEA or other cationic reagents might block the binding of even small agonists such as TMA through electrostatic interactions. Reaction of L119C with small non-polar reagents increases activation by small agonists but can block the access of large ligands such as benzylidene anabaseines to the ligand-binding domain.

We have identified a means by which agonist-evoked responses of nicotinic receptors can be conditionally eliminated. Modification of alpha7L119C mutants by the sulfhydryl reagent 2-aminoethyl methanethiosulfonate (MTSEA) reduces responses to acetylcholine (ACh) by more than 97%, whereas corresponding mutations in muscle-type receptors produce effects that depend on the specific subunits mutated and ACh concentration. We coexpressed alpha7L119C subunits with pseudo wild-type alpha7C116S subunits, as well as ACh-insensitive alpha7Y188F subunits with wild-type alpha7 subunits in Xenopus laevis oocytes using varying ratios of cRNA. When mutant alpha7 cRNA was coinjected at a 5:1 ratio with wild-type cRNA, net charge responses to 300 microM ACh were retained by alpha7L119C-containing mutants after MTSEA modification and by the ACh-insensitive Y188F-containing mutants, even though the expected number of ACh-sensitive wild-type binding sites would on average be fewer than two per receptor. Responses of muscle-type receptors with one MTSEA-sensitive subunit were reduced at low ACh concentrations, but much less of an effect was observed when ACh concentrations were high (1 mM), indicating that saturation of a single binding site with agonist can evoke strong activation of nicotinic ACh receptors. Single-channel patch clamp analysis revealed that the burst durations of fetal wild-type and alpha1beta1gammadeltaL121C receptors were equivalent until the alpha1beta1gammadeltaL121C mutants were exposed to MTSEA, after which the majority (81%) of bursts were brief (<=2 ms). The longest duration events of the receptors modified at only one binding site were similar to the long bursts of native receptors traditionally associated with the activation of receptors with two sites containing bound agonists.

Homomeric alpha7 nicotinic acetylcholine receptors represent an important and complex pharmaceutical target. They can be activated by structurally diverse agonists and are highly likely to enter and remain in desensitized states at rates determined by the structures of the agonists. To identify structural elements regulating this function, we introduced reactive cysteines into the alpha7 ligand-binding domain allowing us to bind sulfhydryl-reactive (SH) agonist analogs or control reagents onto specific positions in the ligand binding domain. We identified four alpha7 mutants (S36C, L38C, W55C, and L119C) in which the tethering of the SH reagents blocked further acetylcholine-evoked activation of the receptor. However, after selective reaction with SH agonist analogs, the type II allosteric modulator N-(5-chloro-2,4-dimethoxyphenyl)-N'-(5-methyl-3-isoxazolyl-3-isoxazolyl)-urea (PNU-120596) could reactivate L119C and W55C mutants and receptors with a reduced or modified C-loop. Modified S36C and L38C mutants were insensitive to reactivation by PNU-120596, whether they were reacted with agonist analogs or alternative SH reagents. Molecular modeling showed that in the W55C and L119C mutants, the ammonium pharmacophore of the agonist analog methanethiosulfonate-ethyltrimethylammonium would be in a similar but nonidentical position underneath the C-loop. The orientation assumed by the ligand tethered to 119C was approximately 3-fold more sensitive to PNU-120596 than the alternative pose at 55C. Our results support the hypothesis that a single ligand can bind within the receptor in different ways and, depending on the specific binding pose, may variously promote activation or desensitization, or, alternatively, function as a competitive antagonist. This insight may provide a new approach for drug development.

Extensive molecular docking, molecular dynamics simulations, and binding free energy calculations have been performed to understand how alpha7-specific agonists of nicotinic acetylcholine receptor (nAChR), including AR-R17779 (1), GTS-21 (4), and 4-OH-GTS-21 (5), interact with the alpha7 receptor, leading to important new insights into the receptor-agonist binding. In particular, the cationic head of 4 and 5 has favorable hydrogen bonding and cation-pi interactions with residue Trp149. The computational results have also led us to better understand the roles of Gln117 and other residues in the receptor binding with agonists. The computational predictions are supported by data obtained from wet experimental tests. The new insights into the binding and structure-activity relationship obtained from this study should be valuable for future rational design of more potent and selective agonists of the alpha7 receptor.

Heterologous expression systems have increased the feasibility of developing selective ligands to target nicotinic acetylcholine receptor (nAChR) subtypes. However, the alpha6 subunit, a component in nAChRs that mediates some of the reinforcing effects of nicotine, is not easily expressed in systems such as the Xenopus oocyte. Certain aspects of alpha6-containing receptor pharmacology have been studied by using chimeric subunits containing the alpha6 ligand-binding domain. However, these chimeras would not be sensitive to an alpha6-selective channel blocker; therefore we developed an alpha6 chimera (alpha4/6) that has the transmembrane and intracellular domains of alpha6 and the extracellular domain of alpha4. We examined the pharmacological properties of alpha4/6-containing receptors and other important nAChR subtypes, including alpha7, alpha4beta2, alpha4beta4, alpha3beta4, alpha3beta2, and alpha3beta2beta3, as well as receptors containing alpha6/3 and alpha6/4 chimeras. Our data show that the absence or presence of the beta4 subunit is an important factor for sensitivity to the ganglionic blocker mecamylamine, and that dihydro-beta-erythroidine is most effective on subtypes containing the alpha4 subunit extracellular domain. Receptors containing the alpha6/4 subunit are sensitive to alpha-conotoxin PIA, while receptors containing the reciprocal alpha4/6 chimera are insensitive. In experiments with novel antagonists of nicotine-evoked dopamine release, the alpha4/6 chimera indicated that structural rigidity was a key element of compounds that could result in selectivity for noncompetitive inhibition of alpha6-containing receptors. Our data extend the information available on prototypical nAChR antagonists, and establish the alpha4/6 chimera as a useful new tool for screening drugs as selective nAChR antagonists.

An alpha7 nicotinic acetylcholine receptor sequence was cloned from Rhesus monkey (Macaca mulatta). This clone differs from the mature human alpha7 nicotinic acetylcholine receptor in only four amino acids, two of which are in the extracellular domain. The monkey alpha7 nicotinic receptor was characterized in regard to its functional responses to acetylcholine, choline, cytisine, and the experimental alpha7-selective agonists 4OH-GTS-21, TC-1698, and AR-R17779. For all of these agonists, the EC(50) for activation of monkey receptors was uniformly higher than for human receptors. In contrast, the potencies of mecamylamine and MLA for inhibiting monkey and human alpha7 were comparable. Acetylcholine and 4OH-GTS-21 were used to probe the significance of the single point differences in the extracellular domain. Mutants with the two different amino acids in the extracellular domain of the monkey receptor changed to the corresponding sequence of the human receptor had responses to these agonists that were not significantly different in EC(50) from wild-type human alpha7 nicotinic receptors. Monkey alpha7 nicotinic receptors have a serine at residue 171, while the human receptors have an asparagine at this site. Monkey S171N mutants were more like human alpha7 nicotinic receptors, while mutations at the other site (K186R) had relatively little effect. These experiments point toward the basic utility of the monkey receptor as a model for the human alpha7 nicotinic receptor, albeit with the caveat that these receptors will vary in their agonist concentration dependency. They also point to the potential importance of a newly identified sequence element for modeling the specific amino acids involved with receptor activation.

The alpha7 nAChR-selective partial agonist 3-(2,4-dimethoxybenzylidene)anabaseine (GTS-21) is more efficacious and potent for rat receptors than for human alpha7 receptors. Four single amino acid differences exist between human and rat alpha7 in the agonist binding site, two in the C loop, and one each in the E and F loops. Reciprocal mutations were made in these three domains and evaluated in Xenopus laevis oocytes. Mutations in the C and F loops significantly increased the efficacy of GTS-21 for the human receptor mutants but not to the level of the wild-type, and reciprocal mutations in rat alpha7 did not decrease responses to GTS-21. Whereas mutations in the E loop alone were without effect, the E- and F-loop mutations together increased GTS-21 efficacy and potency for human receptors, but the EF mutations in the rat receptors decreased the GTS-21 potency without changing the efficacy. The only mutants that showed a full reversal of the efficacy differences between human and rat alpha7 contained complete exchange of all four sites in the C, E, and F loops or just the sites in the C and F loops. However, the reversal of the potency ratio seen with the EF mutants was not evident in the CEF mutants. Our data therefore indicate that the pharmacological differences between rat and human alpha7 receptors are caused by reciprocal differences in sites within and around the binding site. Specific features in the agonist molecule itself are also identified that interact with these structural features of the receptor agonist binding site.