title: [(3)H]Epibatidine photolabels non-equivalent amino acids in the agonist binding site of Torpedo and alpha4beta2 nicotinic acetylcholine receptors.
abstract: Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo alpha(2)betagammadelta and expressed human alpha4beta2 nAChRs with [(3)H]epibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of alphaTyr(198) in agonist binding site Segment C of the principal (+) face in both alpha subunits and of gammaLeu(109) and gammaTyr(117) in Segment E of the complementary (-) face, with no labeling detected in the delta subunit. For affinity-purified alpha4beta2 nAChRs, [(3)H]epibatidine photolabeled alpha4Tyr(195) (equivalent to Torpedo alphaTyr(190)) in Segment C as well as beta2Val(111) and beta2Ser(113) in Segment E (equivalent to Torpedo gammaLeu(109) and gammaTyr(111), respectively). Consideration of the location of the photolabeled amino acids in homology models of the nAChRs based upon the acetylcholine-binding protein structure and the results of ligand docking simulations suggests that epibatidine binds in a single preferred orientation within the alpha-gamma transmitter binding site, whereas it binds in two distinct orientations in the alpha4beta2 nAChR.
authors.affiliation: Department of Pharmacology and Neuroscience and the Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA.
authors.forename: Shouryadeep
authors.initials: S
authors.lastname: Srivastava
authors.forename: Ayman K
authors.initials: AK
authors.lastname: Hamouda
authors.forename: Akash
authors.initials: A
authors.lastname: Pandhare
authors.forename: Phaneendra K
authors.initials: PK
authors.lastname: Duddempudi
authors.forename: Mitesh
authors.initials: M
authors.lastname: Sanghvi
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Cohen
authors.forename: Michael P
authors.initials: MP
authors.lastname: Blanton
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D000596:Amino Acids / Q000737:chemistry*; D018712:Analgesics, Non-Narcotic / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray / Q000379:methods; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008954:Models, Biological; D011485:Protein Binding; D011725:Pyridines / Q000494:pharmacology*; D011978:Receptors, Nicotinic / Q000378:metabolism*; D014101:Torpedo; D014443:Tyrosine / Q000737:chemistry
chemical_list: D000596:Amino Acids; D018712:Analgesics, Non-Narcotic; D019086:Bridged Bicyclo Compounds, Heterocyclic; D011725:Pyridines; D011978:Receptors, Nicotinic; C403531:nicotinic receptor alpha4beta2; D014443:Tyrosine; C082748:epibatidine; D000109:Acetylcholine