Abstract
Alcohols and inhaled anesthetics enhance the function of GABAA receptors containing α, β, and γ subunits. Molecular analysis has focused on the role of the α subunits; however, there is evidence that the β subunits may also be important. The goal of our study was to determine whether Asn265, which is homologous to the site implicated in the α subunit (Ser270), contributes to an alcohol and volatile anesthetic binding site in the GABAA receptor β2 subunit. We substituted cysteine for Asn265 and exposed the mutant to the sulfhydryl-specific reagent octyl methanethiosulfonate (OMTS). We used two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes and found that, after OMTS application, GABA-induced currents were irreversibly potentiated in mutant α1β2(N265C)γ2S receptors [but not α1β2(I264C)γ2S], presumably because of the covalent linking of octanethiol to the thiol group in the substituted cysteine. It is noteworthy that this effect was blocked when OMTS was applied in the presence of octanol. We found that potentiation by butanol, octanol, or isoflurane in the N265C mutant was nearly abolished after the application of OMTS, suggesting that an alcohol and volatile anesthetic binding site at position 265 of the β2 subunit was irreversibly occupied by octanethiol and consequently prevented butanol or isoflurane from binding and producing their effects. OMTS did not affect modulation or direct activation by pentobarbital, but there was a partial reduction of allosteric modulation by flunitrazepam and alphaxalone in mutant α1β2(N265C)γ2S receptors after OMTS was applied. Our findings provide evidence that Asn265 may contribute to an alcohol and anesthetic binding site.
Footnotes
This work was supported by the National Institutes of Health National Institute on Alcohol Abuse and Alcoholism [Grants AA06399, AA13378] and the National Institutes of Health National Institute of General Medical Sciences [Grant GM47818].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.170472.
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ABBREVIATIONS:
- TM
- transmembrane
- OMTS
- octyl methanethiosulfonate
- GLIC
- Gloeobacter violaceus ion channel.
- Received May 20, 2010.
- Accepted September 2, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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