Forman S A, Zhou Q
Department of Anesthesia and Critical Care, Massachusetts General Hospital, Boston 02114, USA.
Alcohol Clin Exp Res. 2000 Sep;24(9):1363-8.
Ethanol (EtOH) inhibition and enhancement of ligand-gated ion channel functions may be due to direct interactions with sites on these membrane proteins. Peripheral nicotinic receptors have pore sites that long-chain alcohols inhibit but EtOH does not, either because of weak binding (low affinity) or inability to impair ion translocation (low efficacy). We tested whether nicotinic pore mutations that increase hydrophobicity and/or size can create a site where EtOH inhibits at physiological concentrations.
We studied recombinant expressed mouse muscle receptors with patch-clamp electrophysiology to measure EtOH effects both on single-channel conductance and on multichannel currents elicited with rapid agonist superfusion. We studied pairs of mutants with similar residue sizes but different hydrophobicitics at position alpha252 to determine if size or hydrophobicity determines EtOH sensitivity.
Inhibition of wild-type currents is seen at EtOH concentrations >300 mM. Receptors that contain the alphaS252I (serine to isoleucine) mutation are significantly inhibited by 100 mM EtOH. Adding a second homologous mutation on the beta subunit (betaT263I) further enhances sensitivity and creates receptors that are inhibited significantly by 50 mM EtOH. The open-state conductance of single nicotinic channels is reduced in the presence of EtOH, which closely parallels EtOH inhibition of multichannel currents. In two isosteric mutant pairs at position alpha252, only hydrophobic side-chains significantly increase receptor sensitivity to EtOH.
Wild-type nicotinic receptors have a very low affinity for EtOH, but only one or two mutations in the pore site create receptors inhibited by physiological EtOH concentrations. Ethanol inhibition is due primarily to channel interactions in the open state. At amino acid alpha252, side-chain hydrophobicity, not size, determines receptor sensitivity to EtOH inhibition. We propose that similar sites may exist within the pores of other EtOH-sensitive ion channels, such as NMDA receptors and neuronal nicotinic receptors.
