Abstract

To define potential alcohol binding sites in the neuronal nicotinic acetylcholine receptor (nAChR) we used cysteine mutagenesis and sulfhydryl-specific labeling. The basis of this strategy is that covalent addition of an alkylthiol group to a cysteine in an alcohol binding site will mimic the action of an irreversibly bound alcohol. Each amino acid in the extracellular region of the second transmembrane segment of the nAChR subunit α2 was mutated to cysteine. The resulting α2 subunits were coexpressed with wild-type β4in Xenopus laevis oocytes, and the responses were studied using two-electrode voltage clamp. Of the 11 mutants tested, 2 fulfilled criteria for participation in an alcohol binding site: α2(L262C)β4 and α2(L263C)β4. Covalent binding of propanethiol to these cysteines did not change acetylcholine (ACh) affinity, but modified ACh maximal response in both cases: it increased for α2(L263C)β4 and decreased for α2(L262C)β4. The same modifications on ACh responses were obtained with ethanol on α2(L263C)β4 and octanol on α2(L262C)β4. This suggested that alcohol binding at L263 enhances receptor function, whereas binding at L262 inhibits function. We studied different n-alcohols (ethanol, butanol, pentanol, and octanol), as well as isoflurane and urethane, on these two mutants. Covalent binding of propanethiol to the cysteines revealed changes in the alcohol modulation consistent with an excitatory site (L263) or an inhibitory site (L262) being no longer accessible to alcohol. Thus, n-alcohols appear to act on both sites and their ability to enhance (short-chain), inhibit (long-chain), or produce no effect (intermediate-chain) depends upon their relative action at these two sites.