The Basic Property of Lys385 Is Important for Potentiation of the Human α1 Glycine Receptor by Ethanol

Ethanol alters the function of several members of the Cys-loop ligand-gated ion channel superfamily. Recent studies have shown that the sensitivity of the alpha 1 glycine receptor (GlyR) to ethanol can be affected by the state of G protein activation mediated by the interaction of G beta gamma with intracellular amino acids in the GlyR. Here, we evaluated the physicochemical property of Lys385 that contributes to ethanol modulation by using mutagenesis, patchclamp, and biochemical techniques. A conserved substitution (K385R) did not affect either the apparent glycine EC50 (40 +/- 1 versus 41 +/- 0.5 mu M) or the ethanol-induced potentiation (53 +/- 5 versus 46 +/- 5%) of the human alpha 1 GlyR. On the other hand, replacement of this residue with glutamic acid (K385E), an acidic amino acid, reduced the potentiation of the GlyR to 10 +/- 1%. Furthermore, mutations with a hydrophobic leucine (K385L), a hydrogen bond donor glutamine (K385Q), or a neutral residue (K385A) also reduced ethanol modulation. Finally, substitution by a large and hydrophobic residue (K385F) and deletion of 385 (Lys385_) reduced ethanol modulation to 10 +/- 4 and 17 +/- 0.4%, respectively. Experiments using dynamic cysteine substitution with a methanethiosulfonate reagent and homology modeling indicate that the basic property and the position of Lys385, probably because of its interaction with G beta gamma, is critical for ethanol potentiation of the receptor.