We recently showed that at desensitized muscle nicotinic receptors, epibatidine selects by 300-fold between the two agonist binding sites. To determine whether receptors in the resting, activatible state show similar site selectivity, we studied epibatidine-induced activation of mouse fetal and adult receptors expressed in 293 HEK cells. Kinetic analysis of single- channel currents reveals that (-)-epibatidine binds with 15-fold selectivity to sites of adult receptors and 75-fold selectivity to sites of fetal receptors. For each receptor subtype, site selectivity arises solely from different rates of epibatidine dissociation from the two sites. To determine the structural basis for epibatidine selectivity, we introduced mutations into either the γ or the δ subunit and measured epibatidine binding and epibatidine-induced single-channel currents. Complexes formed by α and mutant γ(K34S + F172l) subunits bind epibatidine with increased affinity compared to αγ complexes, whereas the kinetics of α2βδγ(K34S+F172l) receptors reveal on change in affinity of the low-affinity site, but increased affinity of the high-affinity site. Conversely, complexes formed by α and mutant δ(S36K+I178F) subunits bind epibatidine with decreased affinity compared to αδ complexes, whereas the kinetics of α2βγδ(S36K+I178F) receptors show markedly reduced sensitivity to epibatidine. The overall data show that epibatidine activates muscle receptors by binding with high affinity to αγ and αε sites, but with low affinity to the αδ site.
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