Mutation in neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes blocks ethanol action.

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Amino acids (AAs) in the extracellular portion of the transmembrane domain of several inhibitory ligand-gated ion channels participate in an alcohol binding site. To extend these studies to neuronal nicotinic acetylcholine receptors (nAChRs), we focused on an AA (L262) located in the same region of the second transmembrane domain of the alpha2 subunit of neuronal nAChRs. Single-point mutation of alpha2L262 was carried out, the resulting alpha2 subunits co-expressed with wild-type beta4 subunits in Xenopus laevis oocytes, and studied using two-electrode voltage clamp. Ethanol enhancement of ACh responses was diminished [alpha2(L262F)beta4] or abolished [alpha2(L262G)beta4, alpha2(L2625)beta4 and alpha2(L262A)beta4]. Mutation of the homologous AA in beta4 [beta4(L258A)] did not modify the ethanol modulation and the mutation in alpha2 was dominant, because ethanol did not enhance ACh responses in alpha2(L262A)beta4(L258A) nAChRs. n-Alcohols (ethanol through octanol) were applied to alpha2(L262A)beta4 nAChRs. As described previously for other nAChRs, short-chain alcohols enhanced, intermediate-chain alcohols had no effect and long-chain alcohols inhibited ACh responses in the wild-type receptor. For alpha2(L262A)beta4 nAChRs the alcohol enhancing effect was absent, and the alcohol inhibitory action was increased. Although this suggests removal of an alcohol enhancing site through mutagenesis, we cannot rule out the enhancement of action at an alcohol inhibitory site.