GABAA receptor subunit expression and sensitivity to ethanol.
Ethanol has been demonstrated to potentiate GABAergic mechanisms in several different cell types and also appears to be active on GABAA receptors in specific brain regions. By expressing brain mRNA and cloned GABA receptor subunits in Xenopus oocytes, we have investigated the requirements for ethanol modulation of the GABAA receptor. Using hybrid arrest techniques, the GABA receptor subunits alpha 1, beta 1, gamma 1, gamma 2S+ gamma 2L, gamma 2L and gamma 3 were individually prevented from expressing. None of these treatments altered potentiation by pentobarbital, the gamma 2S+ gamma 2L and gamma 2L reduced diazepam potentiation, however only the gamma 2L hybridization reduced ethanol sensitivity. By expression of cloned GABA subunits the effects could be reproduced by comparing ethanol sensitivity of alpha 1 beta 1 gamma 2S and alpha 1 beta 1 gamma 2L. The gamma 2L is an alternatively spliced variant of gamma 2 containing an extra eight amino acids bearing a consensus site for phosphorylation by protein kinase C. By using in vitro mutagenesis techniques, alteration of key residues in the phosphorylation site prevented ethanol modulation of the receptors containing mutant gamma 2 subunits. These results suggest that phosphorylation of a site on the gamma 2L subunit can alter the modulatory effects of ethanol on GABAA receptors containing this subunit.
Wafford
K A
KA
Merck, Sharp & Dohme Research Laboratories, Harlow, Essex, U.K.
Burnett
D
D
Harris
R A
RA
Whiting
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D003001:Cloning, Molecular; D004351:Drug Resistance; D000431:Ethanol / Q000633:toxicity*; D005260:Female; D015870:Gene Expression; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009865:Oocytes / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011963:Receptors, GABA-A / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics*; D014981:Xenopus
D012333:RNA, Messenger; D011963:Receptors, GABA-A; D000431:Ethanol; D011493:Protein Kinase C