Mapping the structural requirements for nicotinic acetylcholine receptor activation by using tethered alkyltrimethylammonium agonists and antagonists

A molecule as simple in structure as tetramethylammonium gates the nicotinic acetylcholine receptor ( nAChR) with high efficacy. To compare the structure of the nAChR transmitter binding site in the open channel state with that of the ACh binding protein, we determined the efficacy of nAChR gating by -S(CH2)(n)N(CH3)(3)(+) (n = 1-4) tethered to substituted cysteines at positions in the alpha subunits or gamma and delta subunits predicted to contribute to the ACh binding sites in mutant Torpedo nAChRs expressed in Xenopus oocytes. For tethered thiocholine [-S(CH2)(2)N(CH3)(3)(+)], we previously reported that within alpha 195-201 gating was observed only at alpha Y198C while at alpha Y93C it acted as an antagonist. We now show that within alpha 191-194, thiocholine activates when tethered at alpha Cys192 or alpha Cys193. Thiocholine also activates when tethered at alpha Y190C or alpha W149C in nAChRs containing beta subunit mutation (beta L257S) that destabilizes the closed channel, but not from gamma W55C/delta W57C, where longer adducts can activate. When tethered at positions in binding site segment E, thiocholine activates only from gamma L119C/delta L121C, where the shorter -S(CH2)(1)N(CH3)(3)(+) acts as an antagonist. Longer adducts tethered at gamma L109C/delta L111C or gamma L119C/delta L121C also activate, but less efficiently. The length requirements for efficient gating by tethered agonists agree closely with predictions based upon the structure of the agonist site in a nAChR homology model derived from the ACh binding protein structure, which suggests that this structure is an excellent model of the nAChR agonist binding site in the open channel conformation. The inability of thiocholine to activate from alpha Y93C, which is not predicted by the model, is discussed in terms of the structure of the nAChR in the closed state.