Regulation of the surface expression of 伪4尾2未 GABA_A receptors by high efficacy states

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• 作者：Aarti Kuver ; Hui Shen ; Sheryl S. Smith ; ^{Sheryl.smith@downstate.edu}
• 关键词：伪4F ; 伪4FLAG (3xFLAG tag) ; BDNF ; Brain-derived neurotrophic factor ; DMSO ; dimethyl sulfoxide ; FLAG ; DYKDDDDK epitope ; GABAR ; GABAA receptor ; GBX ; Gaboxadol ; 4 ; 5 ; 6 ; 7-tetrahydroisoxazolo(5 ; 4-c)pyridin-3-ol ; THIP ; HEK-293 ; human embryonic kidney cell-293 ; P
• 刊名：Brain Research
• 出版年：2012
• 期刊代码：8_00068993
• 类别：neu
• 出版时间：29 June, 2012
• 卷：1463
• 期：Complete
• 页码：1-20
• 文件大小：1997 K

摘要

伪4尾未 GABA_A receptors (GABARs) have low CNS expression, but their expression is increased by 48 h exposure to the neurosteroid THP (3伪-OH-5伪[尾]-pregnan-20-one). THP also increases the efficacy of 未-containing GABARs acutely, where GABA is a partial agonist. Thus, we examined effects of THP (100 nM) and full GABA agonists at 伪4尾2未 (gaboxadol, 10 渭M, and 尾-alanine, 10 渭M-1 mM), on surface expression of 伪4尾2未. To this end, we used an 伪4 construct tagged with a 3XFLAG (F) epitope or measured expression of native 伪4 and 未. HEK-293 cells or cultured hippocampal neurons were transfected with 伪4F尾2未 and treated 24 h later with GABA agonists, THP, GABA plus THP or vehicle (0.01%DMSO) for 0.5 h-48 h. Immunocytochemistry was performed under both non-permeabilized and permeabilized conditions to detect surface and intracellular labeling, respectively, using confocal microscopy. The high efficacy agonists and GABA (1 or 10 渭M) plus THP increased 伪4尾2未 surface expression up to 3-fold after 48 h, an effect first seen by 0.5 h. This effect was not dependent upon the polarity of GABAergic current, although expression was increased by KCC2. Intracellular labeling was decreased while functional expression was confirmed by whole cell patch clamp recordings of responses to GABA agonists. GABA plus THP treatment did not alter the rate of receptor removal from the surface membrane, suggesting that THP-induced 伪4尾2未 expression is likely via receptor insertion. Surface expression of 伪4尾2未 was decreased by rottlerin (10 渭M), suggesting a role for PKC-未. These results suggest that trafficking of 伪4尾2未 GABARs is regulated by high efficacy states.