Volatile anaesthetics have historically been considered to act in a nonspecific manner on the central nervous system. More recent studies, however, have revealed that the receptors for inhibitory neurotransmitters such as gamma-aminobutyric acid (GABA) and glycine are sensitive to clinically relevant concentrations of inhaled anaesthetics. The function of GABA(A) and glycine receptors is enhanced by a number of anaesthetics and alcohols, whereas activity of the related GABA rho1 receptor is reduced. We have used this difference in pharmacology to investigate the molecular basis for modulation of these receptors by anaesthetics and alcohols. By using chimaeric receptor constructs, we have identified a region of 45 amino-acid residues that is both necessary and sufficient for the enhancement of receptor function. Within this region, two specific amino-acid residues in transmembrane domains 2 and 3 are critical for allosteric modulation of both GABA(A) and glycine receptors by alcohols and two volatile anaesthetics. These observations support the idea that anaesthetics exert a specific effect on these ion-channel proteins, and allow for the future testing of specific hypotheses of the action of anaesthetics.

Ligand-gated ion channels are a target for inhaled anesthetics and alcohols in the central nervous system. The inhibitory strychninesensitive glycine and ␥-aminobutyric acid type A receptors are positively modulated by anesthetics and alcohols, and site-directed mutagenesis techniques have identified amino acid residues important for the action of volatile anesthetics and alcohols in these receptors. A key question is whether these amino acids are part of an alcohol͞anesthetic-binding site. In the present study, we used an alkanethiol anesthetic to covalently label its binding site by mutating selected amino acids to cysteine. We demonstrated that the anesthetic propanethiol, or alternatively, propyl methanethiosulfonate, covalently binds to cysteine residues introduced into a specific second transmembrane site in glycine receptor and ␥-aminobutyric acid type A receptor subunits and irreversibly enhances receptor function. Moreover, upon permanent occupation of the site by propyl disulfide, the usual ability of octanol, enflurane, and isoflurane to potentiate the function of the ion channels was lost. This approach provides strong evidence that the actions of anesthetics in these receptors are due to binding at a single site. Despite their wide use, the mechanism of action of alcohols and general anesthetics remains controversial. In contrast to most other classes of drugs, which are either assumed or known to act on specific protein receptors, anesthetic action is often attributed to multiple nonspecific sites (1). Based on the relationship between the potencies of anesthetics and their lipid solubilities described by Meyer (2) and Overton (3), the lipid bilayer of neuronal membrane was long considered the primary target for general anesthesia. Even recent studies invoke this hypothesis (4), although other studies suggest proteins as the site of action of inhaled anesthetics and n-alcohols (5-7).A traditional approach to distinguish between these two alternatives would apply radioligand-binding assays with alcohols and inhalational anesthetics. However, the low affinities and rapid kinetics of these compounds makes such studies unfeasible (8). In the present study, we used a new approach, using anesthetic alcohol analogs that form covalent bonds at their site of action, to show that binding at a single site produced irreversible anesthetic-like effects. This goal was accomplished with propanethiol and with propyl methanethiosulfonate (PMTS); both compounds can form propyl disulfide bonds with cysteine residues introduced at specific sites in brain proteins (Fig. 1).Our candidate targets for the actions of the general anesthetics and n-alcohols were the main inhibitory receptors in spinal cord and brain: the strychnine-sensitive glycine receptor (GlyR) and the ␥-aminobutyric acid type A (GABA A ) receptor. Clinically relevant concentrations of volatile anesthetics and n-alcohols potentiate the action of glycine and GABA on these receptors (7,9,10,(11)(12)(13)(14). Site-directed mutagenesis techniques defined reg...

Alcohols in the homologous series of nalcohols increase in central nervous system depressant potency with increasing chain length until a ''cutoff'' is reached, after which further increases in molecular size no longer increase alcohol potency. A similar phenomenon has been observed in the regulation of ligand-gated ion channels by alcohols. Different ligand-gated ion channels exhibit radically different cutoff points, suggesting the existence of discrete alcohol binding pockets of variable size on these membrane proteins. The identification of amino acid residues that determine the alcohol cutoff may, therefore, provide information about the location of alcohol binding sites. Alcohol regulation of the glycine receptor is critically dependent on specific amino acid residues in transmembrane domains 2 and 3 of the ␣ subunit. We now demonstrate that these residues in the glycine ␣1 and the ␥-aminobutyric acid 1 receptors also control alcohol cutoff. By mutation of Ser-267 to Gln, it was possible to decrease the cutoff in the glycine ␣1 receptor, whereas mutation of Ile-307 and͞or Trp-328 in the ␥-aminobutyric acid 1 receptor to smaller residues increased the cutoff. These results support the existence of alcohol binding pockets in these membrane proteins and suggest that the amino acid residues present at these positions can control the size of the alcohol binding cavity.