EPIBATIDINE BINDS WITH UNIQUE SITE AND STATE SELECTIVITY TO MUSCLE NICOTINIC ACETYLCHOLINE-RECEPTORS

Ligand binding sites in fetal (alpha(2) beta gamma delta) and adult (a lpha(2) beta delta epsilon) muscle acetylcholine receptors are formed by alpha delta, alpha gamma, or alpha epsilon subunit pairs. Each type of binding site shows unique ligand selectivity due to different cont ributions by the delta, gamma, or epsilon subunits. The present study compares epibatidine and carbamylcholine binding in terms of their sit e and state selectivities for muscle receptors expressed in human embr yonic kidney 293 cells. Measurements of binding to alpha gamma, alpha delta, and alpha epsilon intracellular complexes reveal opposite site selectivities between epibatidine and carbamylcholine; for epibatidine the rank order of affinities is alpha epsilon > alpha gamma > alpha d elta, whereas for carbamylcholine the rank order is alpha delta congru ent to alpha epsilon > alpha gamma. Because the relative affinities of intracellular complexes resemble those of receptors in the closed act ivable state, the results suggest that epibatidine binds with unique s ite selectivity in activating the muscle receptor. Measurements of bin ding at equilibrium show that both enantiomers of epibatidine bind to adult and fetal receptors with shallow but monophasic binding curves. However, when receptors are fully desensitized, epibatidine binds in a biphasic manner, with dissociation constants of the two components di ffering by more than 170-fold. Studies of subunit-omitted receptors (a lpha(2) beta delta(2), alpha(2) beta gamma(2) and alpha(2) beta epsilo n(2)) reveal that in the desensitized state, the alpha delta interface forms the low affinity epibatidine site, whereas the alpha gamma and alpha epsilon interfaces form high affinity sites. In contrast to epib atidine, carbamylcholine shows little site selectivity for desensitize d fetal or adult receptors. Thus epibatidine is a potentially valuable probe of acetylcholine receptor binding site structure and of element s that confer state-dependent selectivities of the binding sites.