Abstract
Ionotropic GABAA receptors (GABAARs), 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 GABAARs. We used Xenopus laevis oocytes to investigate the 6′ position in the second transmembrane region of GABAARs as a site influencing alcohol inhibition. We asked whether modification of the 6′ 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 6′ position in either α2 or β2 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 6′ position. We verified a role for the 6′ position in previously tested α2β2 as well as more physiologically relevant α2β2γ2s GABAARs. Finally, we built a novel molecular model based on the invertebrate glutamate-gated chloride channel receptor, a GABAAR homolog, revealing that the 6′ 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 6′ positions in both α2 and β2 GABAAR 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.
Footnotes
This work was supported by the National Institutes of Health National Institute of Alcohol Abuse and Alcoholism [Grants T32-AA007471, F32-AA019851, AA06399].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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ABBREVIATIONS:
- GABAAR
- GABAA receptor
- nAChR
- nicotinic acetylcholine receptor
- GlyR
- glycine receptor
- TM
- transmembrane
- DTT
- dithiothreitol
- MTSEA
- 2-aminoethyl methanethiolsulfonate
- MTS
- methanethiosulfonate
- MMTS
- methyl methanethiosulfonate
- PMTS
- propyl methanethiosulfonate
- HMTS
- hexyl methanethiosulfonate
- EtOH
- ethanol
- MeOH
- methanol
- HeOH
- hexanol
- BuOH
- butanol
- EC5
- 5% of maximal response
- GluCl
- invertebrate glutamate-gated chloride channel receptor.
- Received September 21, 2011.
- Accepted November 8, 2011.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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