Specific binding sites for alcohols and anesthetics on ligand-gated ion channels

Abstract

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...

A gating mechanism proposed from a simulation of a human α7 nicotinic acetylcholine receptor

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