[(3)H]Epibatidine photolabels non-equivalent amino acids in the agonist binding site of Torpedo and alpha4beta2 nicotinic acetylcholine receptors.
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.
Srivastava
Shouryadeep
S
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.
Hamouda
Ayman K
AK
Pandhare
Akash
A
Duddempudi
Phaneendra K
PK
Sanghvi
Mitesh
M
Cohen
Jonathan B
JB
Blanton
Michael P
MP
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
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