title: Physostigmine and galanthamine bind in the presence of agonist at the canonical and noncanonical subunit interfaces of a nicotinic acetylcholine receptor.
abstract: 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-α) surface of the transmitter binding sites (γTyr-111/γTyr-117/δTyr172). They also photolabeled δTyr-212 at the δ-β subunit interface and γTyr-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 γTyr-111, γTyr-117, δTyr-212, and γTyr-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 α-γ 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 δ-β interface (3) in a location equivalent to the benzodiazepine binding site in GABA(A) receptors.
authors.affiliation: Department of Neurobiology and PhD Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Ayman K
authors.initials: AK
authors.lastname: Hamouda
authors.forename: Tilia
authors.initials: T
authors.lastname: Kimm
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Cohen
mesh_terms: D000465:Algorithms; D000818:Animals; D001665:Binding Sites; D018679:Cholinergic Agonists / Q000494:pharmacology*; D002800:Cholinesterase Inhibitors / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005702:Galantamine / Q000378:metabolism*; D006868:Hydrolysis; D066298:In Vitro Techniques; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000737:chemistry; D010777:Photochemistry; D010830:Physostigmine / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011869:Radioligand Assay; D011963:Receptors, GABA-A / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014101:Torpedo
chemical_list: D018679:Cholinergic Agonists; D002800:Cholinesterase Inhibitors; D007473:Ion Channels; D011963:Receptors, GABA-A; D011978:Receptors, Nicotinic; D005702:Galantamine; D010830:Physostigmine; D010447:Peptide Hydrolases