Epibatidine Binds with Unique Site and State Selectivity to Muscle Nicotinic Acetylcholine Receptors

Abstract

Ligand binding sites in fetal (␣ 2 ␤␥␦) and adult (␣ 2 ␤␦⑀) muscle acetylcholine receptors are formed by ␣␦, ␣␥, or ␣⑀ subunit pairs. Each type of binding site shows unique ligand selectivity due to different contributions by the ␦, ␥, or ⑀ subunits. The present study compares epibatidine and carbamylcholine binding in terms of their site and state selectivities for muscle receptors expressed in human embryonic kidney 293 cells. Measurements of binding to ␣␥, ␣␦, and ␣⑀ intracellular complexes reveal opposite site selectivities between epibatidine and carbamylcholine; for epibatidine the rank order of affinities is ␣⑀ > ␣␥ > ␣␦, whereas for carbamylcholine the rank order is ␣␦ Х ␣⑀ > ␣␥. Because the relative affinities of intracellular complexes resemble those of receptors in the closed activable state, the results suggest that epibatidine binds with unique site selectivity in activating the muscle receptor. Measurements of binding 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 differing by more than 170-fold. Studies of subunitomitted receptors (␣ 2 ␤␦ 2 , ␣ 2 ␤␥ 2 , and ␣ 2 ␤⑀ 2 ) reveal that in the desensitized state, the ␣␦ interface forms the low affinity epibatidine site, whereas the ␣␥ and ␣⑀ interfaces form high affinity sites. In contrast to epibatidine, carbamylcholine shows little site selectivity for desensitized fetal or adult receptors. Thus epibatidine is a potentially valuable probe of acetylcholine receptor binding site structure and of elements that confer state-dependent selectivities of the binding sites.Epibatidine recently emerged as one of the most potent nicotinic receptor ligands thus far characterized. In addition to its femtomolar to picomolar affinity for certain neuronal acetylcholine receptor (AChR) 1 subtypes, epibatidine also induces analgesia and is approximately 200 times more potent than morphine as an analgesic (1-3). Despite the intense interest in the neuronal actions of epibatidine, its interactions with muscle-type AChR are not well characterized.Muscle-type AChRs are heteropentamers of homologous but functionally distinct subunits with compositions ␣ 2 ␤␥␦ in fetal and ␣ 2 ␤⑀␦ in adult muscle (4). Within the pentamer are two binding sites for acetylcholine (ACh); one is formed at the ␣␦ subunit interface, whereas the other is formed at the ␣␥ interface in the fetal receptor and at the ␣⑀ interface in the adult. Each type of binding site displays distinct selectivities for agonists and competitive antagonists. For example, carbamylcholine binds 30-fold less tightly to the ␣␥ binding site than to the ␣␦ and ␣⑀ binding sites owing to different contributions of the ␥, ␦, and ⑀ subunits (5, 6). Previous studies used ␥-␦ and ⑀-␦ subunit chimeras to identify residues in these subunits which confer site selectivity for curare, conotoxin M1, and carbamylcholi...

Epibatidine binds to four sites on the Torpedo nicotinic acetylcholine receptor

“…The novelty of this report is that the agonist Sine and colleagues [29,30] previously reported heterogeneity of epibatidine binding to the homologous fetal and adult muscle receptors with apparent affinities that are generally consistent with those reported here. In these earlier studies, affinities for epibatidine isomers were measured indirectly by their ability to inhibit the initial rate of [ 125 I]-α-BTx binding and thus no direct determination of the number of epibatidine sites could be made.…”

“…However, in the case of epibatidine binding to the intracellular dimers or subunit-depleted receptors, the Hill coefficients were less than one, suggesting that there may be heterogeneity of binding sites within each interface. These earlier results may be explained, as suggested [29], by the formation of a mixed population of receptor complexes or by the presence of different receptor conformations. However, based on the above evidence for four epibatidine sites on each native Torpedo receptor, we suggest that there are two epibatidine binding sites associated with each of the α−γ and α−δ interfaces.…”

“…In these earlier studies, affinities for epibatidine isomers were measured indirectly by their ability to inhibit the initial rate of [ 125 I]-α-BTx binding and thus no direct determination of the number of epibatidine sites could be made. By studying epibatidine binding to intracellular complexes of subunit pairs (αγ, αδ, αε) and subunit-deficient receptors (α 2 βγ 2 , α 2 βε 2 ,α 2 βδ 2 ) expressed on the cell surface, convincing evidence was provided to suggest that the higher affinity epibatidine binding was conferred by a site lying at the α−γ (or α−ε) interface and that lower affinity binding could be attributed to a site at the α−δ interface [29]. However, in the case of epibatidine binding to the intracellular dimers or subunit-depleted receptors, the Hill coefficients were less than one, suggesting that there may be heterogeneity of binding sites within each interface.…”

Models of the extracellular domain of the nicotinic receptors and of agonist- and Ca 2+ -binding sites

“…4 shows that the tyrosine aryl faces the binding pocket and could reinforce the interaction with the ligand and therefore decrease the free energy of the complex. The same difference has been shown for the affinity towards epibatine of the mouse ␣␥-and ␣␦-binding sites (50).…”

[3H]Epibatidine Photolabels Non-equivalent Amino Acids in the Agonist Binding Site of Torpedo and α4β2 Nicotinic Acetylcholine Receptors

“…Although epibatidine may bind with 3-4-fold higher affinity to one of the two sites in the Torpedo nAChR, as does ACh (42), it did not have the high selectivity between the sites seen in the mouse muscle nAChR (Ͼ170-fold higher affinity for the ␣-␥ than the ␣-␦ site in the desensitized state (43) 195 ) ABS by what must be the same epibatidine photoreactive intermediate, we used computational methods to predict favored epibatidine docking orientations in the ABS in homology models of the Torpedo and ␣4␤2 nAChRs based on the structure of the epibatidine-bound form of Aplysia AChBP (12). When epibatidine was docked in the Torpedo ␣-␥ ABS, a single binding orientation was highly favored (Fig.…”