No abstract

Neurotransmitters such as acetylcholine and GABA (gamma-aminobutyric acid) mediate rapid synaptic transmission by activating receptors belonging to the gene superfamily of ligand-gated ion channels (LGICs). These channels are pentameric proteins that function as signal transducers, converting chemical messages into electrical signals. Neurotransmitters activate LGICs by interacting with a ligand-binding site, triggering a conformational change in the protein that results in the opening of an ion channel. This process, which is known as 'gating', occurs rapidly and reversibly, but the molecular rearrangements involved are not well understood. Here we show that optimal gating in the GABA(A) receptor, a member of the LGIC superfamily, is dependent on electrostatic interactions between the negatively charged Asp 57 and Asp 149 residues in extracellular loops 2 and 7, and the positively charged Lys 279 residue in the transmembrane 2-3 linker region of the alpha1-subunit. During gating, Asp 149 and Lys 279 seem to move closer to one another, providing a potential mechanism for the coupling of ligand binding to opening of the ion channel.

Ethanol produces a wide variety of behavioral and physiological effects in the body, but exactly how it acts to produce these effects is still poorly understood. Although ethanol was long believed to act nonspecifically through the disordering of lipids in cell membranes, proteins are at the core of most current theories of its mechanisms of action. Although ethanol affects various biochemical processes such as neurotransmitter release, enzyme function, and ion channel kinetics, we are only beginning to understand the specific molecular sites to which ethanol molecules bind to produce these myriad effects. For most effects of ethanol characterized thus far, it is unknown whether the protein whose function is being studied actually binds ethanol, or if alcohol is instead binding to another protein that then indirectly affects the functioning of the protein being studied. In this Review, we describe criteria that should be considered when identifying alcohol binding sites and highlight a number of proteins for which there exists considerable molecular-level evidence for distinct ethanol binding sites.

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...

The mechanisms of general anesthesia in the central nervous system are finally yielding to molecular examination. As a result of research during the past several decades, a group of ligand-gated ion channels have emerged as plausible targets for general anesthetics. Molecular biology techniques have greatly accelerated attempts to classify ligand-gated ion channel sensitivity to general anesthetics, and have identified the sites of receptor subunits critical for anesthetic modulation using chimeric and mutated receptors. The experimental data have facilitated the construction of tenable molecular models for anesthetic binding sites, which in turn allows structural predictions to be tested. In vivo significance of a putative anesthetic target can now be examined by targeted gene manipulations in mice. In this review, we summarize from a molecular perspective recent advances in our understanding of mechanisms of action of general anesthetics on ligand-gated ion channels.