The structure of the alcohol and anesthetic binding site in the strychnine-sensitive glycine receptor



Journal Title

Journal ISSN

Volume Title



The inhibitory glycine receptor is a target for both alcohols and volatile anesthetics. The function of strychnine-sensitive glycine receptors is enhanced in the presence of clinically relevant concentrations of ethanol and longer chain alcohols as well as volatile anesthetics. Site-directed mutagenesis techniques have identified residues in transmembrane segment (TM) one (I229), two (S267) and three (A288) that mediate the effects of alcohols and anesthetics, and drug binding is hypothesized to involve all amino acids from all four transmembrane segments. Here, by the use of crosslinking studies in receptors expressed in Xenopus laevis oocytes, we determined that S267 and A288 are near-neighbors that face one another in three-dimensional space. This provided an improved model of orientation for these two transmembrane segments and provides insight towards the location and role of the TM2-TM3 interface. Second, changes in the accessibility to the binding cavity and changes in the volume of this binding cavity were examined during channel gating for amino acids in all four TMs using mutagenesis and sulfhydryl-specific reagents of different lengths. S267C was accessible to short chain (C3-C8) methanethiosulfonate (MTS) compounds 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. Mutated residues more intracellular than M263C in TM2 did not react, indicating a floor of the cavity. I229C and A288C showed state-dependent reaction with MTS only in the presence of agonist. Reaction of propyl MTS with A288C receptors abolished the effect of the anesthetic isoflurane, providing strong support that A288C is contributing to a binding site for alcohols and anesthetics. Four of twelve mutants tested in TM4 (W407C, I409C, Y410C and K411C) reacted with propyl MTS, providing information on which amino acids were in water-accessible positions and thus possible candidates for involvement in alcohol and anesthetic binding. These data demonstrate that the conformational changes accompanying channel gating increase accessibility to amino acids critical for drug action, which may provide a mechanism by which alcohols and anesthetics can act on glycine (and likely other) receptors.