[3H]Epibatidine Photolabels Non-equivalent Amino Acids in the Agonist Binding Site of Torpedo and α4β2 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)beta gamma delta and expressed human alpha 4 beta 2 nAChRs with [H-3]epibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of alpha Tyr(198) in agonist binding site Segment C of the principal (+) face in both alpha subunits and of gamma Leu(109) and gamma Tyr(117) in Segment E of the complementary (-) face, with no labeling detected in the delta subunit. For affinity-purified alpha 4 beta 2 nAChRs, [H-3]epibatidine photolabeled alpha 4Tyr(195) (equivalent to Torpedo alpha Tyr(190)) in Segment C as well as beta 2Val(111) and beta 2Ser(113) in Segment E (equivalent to Torpedo gamma Leu(109) and gamma Tyr(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 alpha 4 beta 2 nAChR.