The TM2 6′ Position of GABA_A Receptors Mediates Alcohol Inhibition

Abstract

Ionotropic GABA A receptors (GABA A Rs), which mediate inhibitory neurotransmission in the central nervous system, are implicated in the behavioral effects of alcohol and alcoholism. Site-directed mutagenesis studies support the presence of discrete molecular sites involved in alcohol enhancement and, more recently, inhibition of GABA A Rs. We used Xenopus laevis oocytes to investigate the 6Ј position in the second transmembrane region of GABA A Rs as a site influencing alcohol inhibition. We asked whether modification of the 6Ј position by substitution with larger residues or methanethiol labeling [using methyl methanethiosulfonate (MMTS)] of a substituted cysteine, reduced GABA action and/or blocked further inhibition by alcohols. Labeling of the 6Ј position in either ␣2 or ␤2 subunits reduced responses to GABA. In addition, methanol and ethanol potentiation increased after MMTS labeling or substitution with tryptophan or methionine, consistent with elimination of an inhibitory site for these alcohols. Specific alcohols, but not the anesthetic etomidate, competed with MMTS labeling at the 6Ј position. We verified a role for the 6Ј position in previously tested ␣2␤2 as well as more physiologically relevant ␣2␤2␥2s GABA A Rs. Finally, we built a novel molecular model based on the invertebrate glutamate-gated chloride channel receptor, a GABA A R homolog, revealing that the 6Ј position residue faces the channel pore, and modification of this residue alters volume and polarity of the pore-facing cavity in this region. These results indicate that the 6Ј positions in both ␣2 and ␤2 GABA A R subunits mediate inhibition by short-chain alcohols, which is consistent with the presence of multiple counteracting sites of action for alcohols on ligand-gated ion channels.

Toxicology and the biological role of methanol and ethanol: Current view

“…As an example, methanol acts as a negative allosteric modulator of NMDA and the ability to block NMDA by methanol is very similar to ethanol and butanol 91 . Similar findings can be ascertained for the GABA receptor that has binding sites for low molecular weight alcohols, including methanol and ethanol 92 . From the global point of view, the both methanol and ethanol are able to bind to pentameric ligand-gate ion channels, such as the glycine receptor, nAChR, etc.…”

Barbiturates Bind in the GLIC Ion Channel Pore and Cause Inhibition by Stabilizing a Closed State

“…This site cannot be responsible for the activation of anionic pLGICs, as (i) it blocks the pore and (ii) it has been shown by photoaffinity labeling experiments that in this case the binding site is located at the interface between subunits in the transmembrane domain (34). However, the inhibition potency of this binding site is also likely to be significant for anionic pLGICs, as it has been shown to mediate alcohol inhibition on GABA A receptor (35). The identification of the true binding sites of barbiturates onto GABA A receptors awaits their crystal structure and would be greatly facilitated through the use of molecules such as the ones synthesized for this study thanks to their anomalous signal.…”

Seeking Structural Specificity: Direct Modulation of Pentameric Ligand-Gated Ion Channels by Alcohols and General Anesthetics

“…Indeed, at least one region of the transmembrane domain has been implicated specifically in channel inhibition. Covalent labeling with a small alcohol analog at the pore-facing 69 residue of a2 GABA A receptors produced persistent channel inhibition and increased the net potentiating effects of alcohols (Johnson et al, 2012), consistent with blockage of an independent inhibitory site. Assuming multiple possible binding sites for small amphiphilic agents on these receptors, with different affinities and efficacies of modulation, it remains to be determined which active sites are particularly critical to alcohol and anesthetic action on GABA A receptors, or whether a dynamic balance of multiple sites underlies their clinical phenotype.…”

“…Third, pore-facing residues throughout M2 (Fig. 1F) are implicated in anesthetic inhibition of nicotinic (Ziebell et al, 2004;Nirthanan et al, 2008) and GABA A receptors (Johnson et al, 2012), as well as ELIC (Spurny et al, 2013). Some of these residues also overlap portions of the intersubunit cavity, suggesting these binding regions could be contiguous.…”