Molecular mechanisms of alcohol and volatile anesthetic modulation of glycine receptor function
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Alcohol and volatile anesthetics enhance the function of the glycine receptor (GlyR), an inhibitory neurotransmitter-gated ion channel. Increasing evidence suggests that this enhancement results from direct interactions of these drugs with a binding pocket located between the transmembrane domains of each of the five subunits that compose a GlyR. We hypothesized that only one of the five binding pockets per receptor pentamer need be occupied by drug in order to achieve enhancement of receptor function. To test this hypothesis, a serine to cysteine point mutation at binding pocket residue 267 (S267C) was utilized for its ability to be covalently labeled by the thiol reagent propyl methanethiosulfonate (PMTS). By coinjecting Xenopus oocytes with increasing ratios of wt:S267C cDNAs, heteromeric GlyRs containing a single S267C subunit were expressed. Using two-electrode voltage clamp electrophysiology, we observed that PMTS labeling viii of these receptors resulted in irreversible enhancement of receptor currents, indicating that drug occupancy of one binding pocket per receptor pentamer is sufficient to enhance GlyR function. We next investigated the possibility that binding pocket residues might directly influence the channel gate by examining the effects of point mutations at two such residues on channel kinetics. Single-channel recordings of an alanine 288 to tryptophan (A288W) mutation in HEK 293 cells revealed a large increase in the stability of the channel open state, while single-channel recordings of an S267 to glutamine (S267Q) mutation demonstrated a large destabilization of the channel open state. Despite significant evidence that volatile anesthetics enhance GlyR function, the single-channel kinetics underlying this modulation remain unknown. We examined halothane and isoflurane modulation of wild type GlyR single-channel activity in outside-out patches pulled from transiently transfected HEK 293 cells. Analysis revealed that these volatile anesthetics enhance receptor function by stabilizing the channel open state, increasing the mean duration of bursts of openings, and increasing the mean number of openings per burst. These data indicate that volatile anesthetics enhance GlyR function through kinetic mechanisms similar to those by which allosteric modulators enhance GABAA receptor function.