Hamouda_2013_J.Neurosci_33_485

Galanthamine and physostigmine are clinically used cholinomimetics that both inhibit acetylcholinesterase and also interact directly with and potentiate nAChRs. As with most nAChR-positive allosteric modulators, the location and number of their binding site(s) within nAChRs are unknown. In this study, we use the intrinsic photoreactivities of [(3)H]physostigmine and [(3)H]galanthamine upon irradiation at 312 nm to directly identify amino acids contributing to their binding sites in the Torpedo californica nAChR. Protein sequencing of fragments isolated from proteolytic digests of [(3)H]physostigmine- or [(3)H]galanthamine-photolabeled nAChR establish that, in the presence of agonist (carbamylcholine), both drugs photolabeled amino acids on the complementary (non-alpha) surface of the transmitter binding sites (gammaTyr-111/gammaTyr-117/deltaTyr172). They also photolabeled deltaTyr-212 at the delta-beta subunit interface and gammaTyr-105 in the vestibule of the ion channel, with photolabeling of both residues enhanced in the presence of agonist. Furthermore, [(3)H]physostigmine photolabeling of gammaTyr-111, gammaTyr-117, deltaTyr-212, and gammaTyr-105 was inhibited in the presence of nonradioactive galanthamine. The locations of the photolabeled amino acids in the nAChR structure and the results of computational docking studies provide evidence that, in the presence of agonist, physostigmine and galanthamine bind to at least three distinct sites in the nAChR extracellular domain: at the alpha-gamma interface (1) in the entry to the transmitter binding site and (2) in the vestibule of the ion channel near the level of the transmitter binding site, and at the delta-beta interface (3) in a location equivalent to the benzodiazepine binding site in GABA(A) receptors.