Abstract
The glycine receptor is a target for both alcohols and anesthetics, and certain amino acids in the alpha1 subunit transmembrane segments (TM) are critical for drug effects. Introducing larger amino acids at these positions increases the potency of glycine, suggesting that introducing larger residues, or drug molecules, into the drug-binding cavity facilitates channel opening. A possible mechanism for these actions is that the volume of the cavity expands and contracts during channel opening and closing. To investigate this hypothesis, mutations for amino acids in TM1 (I229C) and TM2 (G256C, T259C, V260C, M263C, T264C, S267C, S270C) and TM3 (A288C) were individually expressed in Xenopus laevis oocytes. The ability of sulfhydryl-specific alkyl methanethiosulfonate (MTS) compounds of different lengths to covalently react with introduced cysteines in both the closed and open states of the receptor was determined. S267C was accessible to short chain (C3-C8) MTS in both open and closed states, but was only accessible to longer chain (C10-C16) MTS compounds in the open state. Reaction with S267C was faster in the open state. I229C and A288C showed state-dependent reaction with MTS only in the presence of agonist. M263C and S270C were also accessible to MTS labeling. Mutated residues more intracellular than M263C did not react, indicating a floor of the cavity. These data demonstrate that the conformational changes accompanying channel gating increase accessibility to amino acids critical for drug action in TM1, TM2, and TM3, which may provide a mechanism by which alcohols and anesthetics can act on glycine (and likely other) receptors.
Highlights
The glycine receptor (GlyR)1 is a target for both alcohols and anesthetics
By use of this method, residues accessible in the presence and/or absence of neurotransmitter to sulfhydryl-specific reagents have been determined for TM2 in GABAA and acetylcholine receptors and for TM3 and the TM2–TM3 loop for GABAA receptors (10 –14)
We first tested the ability of MTS reagents of different lengths to covalently react with a cysteine introduced at amino acid residue 267 (S267C)
Summary
GlyR, glycine receptor; TM, transmembrane segment; MTS, methanethiosulfonate; pCMBSϪ, para-chloromercuribenzene sulfonate; GABA, ␥-aminobutyric acid; MBS, modified Barth’s solution; Me2SO, dimethyl sulfoxide; ANOVA, analysis of variance. MTS reagents rapidly react to form disulfide bonds with cysteines in the presence of water, and an irreversible change in receptor function is taken as evidence of disulfide bond formation. The glycine receptor is one of the most credible candidates for mediating immobility caused by volatile anesthetics [33] These studies raise the question of the mechanism by which occupation of this protein cavity by alcohols, anesthetics, or MTS reagents facilitates activation (or prevents inactivation) of the channel. We studied the ability of MTS reagents of different lengths to covalently react with these nine positions in both the open and closed conformations of the glycine receptor
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