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THE ROLE OF N265 OF THE GABAA RECEPTOR β2 SUBUNIT IN THE ACTIONS OF ALCOHOLS AND ANESTHETICS
Author(s) -
McCracken Mandy L.,
Borghese Cecilia M.,
Adron Harris R.
Publication year - 2008
Publication title -
alcoholism: clinical and experimental research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.267
H-Index - 153
eISSN - 1530-0277
pISSN - 0145-6008
DOI - 10.1111/j.1530-0277.2008.00689_15.x
Subject(s) - gabaa receptor , cysteine , chemistry , protein subunit , long term potentiation , receptor , anesthetic , voltage clamp , biochemistry , serine , gaba receptor , gabaa rho receptor , biophysics , biology , medicine , anesthesia , enzyme , membrane potential , gene
Alcohols and volatile anesthetics enhance the function of γ‐aminobutyric acid type A (GABAA) receptors containing α, β, and γ subunits, and the presence of an alcohol and volatile anesthetic binding site has been identified in the α1, α2, and β1 subunits. However, the specific role of the β2 subunit has not been elucidated. The present study examined whether amino acid residue Asparagine‐265 (N265), which is homologous to the implicated residue in the α subunits, contributes to an alcohol and volatile anesthetic binding site in the GABAA receptor β2 subunit. Cysteine was substituted at the candidate residue, and cRNAs encoding wild‐type α1β2γ2S or mutant α1β2(N265C)γ2S receptors were injected into Xenopus laevis oocytes. Using two‐electrode voltage clamp electrophysiology, we asked whether the cysteine mutation affected receptor sensitivity to alcohols, and if octyl methanethiosulfonate (OMTS), an alcohol and anesthetic analog, reacted with the substituted cysteine and irreversibly altered GABA‐induced currents. Further, the potentiation of GABA‐induced currents by alcohols and anesthetics was measured before and after the application of OMTS, which has been shown to convalently bind to cysteine residues located at alcohol and anesthetic binding sites. We found that potentiation of GABA‐induced currents by butanol or octanol was significantly reduced in mutant 1β2(N265C)γ2S receptors compared to wild‐type receptors, and submaximal GABA‐induced currents of mutant receptors were irreversibly potentiated following the application of OMTS, presumably due to the covalent reaction between OMTS and the thiol group in the substituted cysteine. Moreover, the potentiation of GABA‐induced currents by butanol or isoflurane was abolished in mutant receptors following the application of OMTS, suggesting that an alcohol and volatile anesthetic binding site at position 265 of the β2 subunit was irreversibly occupied by OMTS and consequently prevented alcohols and anesthetics from binding and producing their effects. Taken together, our findings provide evidence that N265 may contribute to an alcohol and anesthetic binding site, and ultimately we propose that the GABAA receptor β2 subunit may be important for the actions of these compounds.