Alcohol, GABA and hormones: Physiology of subunit change

描述（由申请人提供）：我们已经报道了一种新的GABA-A受体（GABAR）亚型α 4-β 2-δ对低浓度乙醇有反应，但对高浓度乙醇无反应。这种效应在重组和天然受体中都可以看到，当这种正常低表达的受体水平在边缘系统区域如海马中显著增加时，从GABA调节类固醇THP中撤出。该提案将研究这种效应的可能机制，使用HEK-293细胞中的瞬时表达来检查乙醇对单通道特性和失活动力学的剂量依赖性效应。因为乙醇在激动剂的饱和浓度下增加电流幅度，所以我们假设乙醇起增加α 4-β-δ GABAR的门控功效的作用，如已经显示的类固醇对该受体的作用。因此，定点诱变将用于在HEK-293细胞中表达的GABAR中产生组成型活性通道，以区分对结合和门控的影响。此外，将使用TM 2中第270位残基处的丝氨酸至色氨酸突变和TM 3中的A至S突变来检测结合腔中鉴定为高剂量乙醇作用的残基是否介导低剂量乙醇对α 4-β-δ GABAR的作用。其他实验室已经报道了更高浓度的乙醇的效果，这对含δ的GABAR有效。因此，我们还假设，在30 mM乙醇存在下，延长（2-5 min）预暴露于1-3 mM乙醇可加速脱敏速率。由于α 4-β-δ GABAR仅在突触外，因此将测试低剂量乙醇对从齿状回颗粒细胞记录的强直电流的影响，齿状回颗粒细胞通常表达高水平的这些受体。α-4和δ亚基的反义敲低以验证α 4-β-δ GABAR在介导乙醇作用中的作用。这些研究的结果可能为低剂量乙醇对GABAR的影响提供潜在机制。

DESCRIPTION (provided by applicant): We have reported that a novel GABA-A receptor (GABAR) isoform, alpha4-beta2-delta, is responsive to low, but not high, concentrations of ethanol. This effect was seen both in recombinant and native receptors following withdrawal from the GABA-modulatory steroid THP, when levels of this normally underexpressed receptor are markedly increased in limbic areas such as hippocampus. This proposal will investigate possible mechanisms for this effect, using transient expression in HEK-293 cells to examine dose-dependent effects of ethanol on single channel properties and deactivation kinetics. Because ethanol increases current amplitude at saturating concentrations of agonist, it is our hypothesis that ethanol is acting to increase gating efficacy of alpha4-beta-delta GABAR, as has been shown for steroid effects at this receptor. Therefore, site directed mutagenesis will be used to create constitutively active channels in GABAR expressed in HEK-293 cells to distinguish between effects on binding and gating. In addition, serine to tryptophan mutations at residue 270 in TM2 and A to S mutations in TM3 will be used to test whether residues in the binding cavity identified for effects of high dose ethanol acts to mediate the effects of low dose ethanol on alpha4-beta-delta GABAR. Other labs have reported effects of higher concentrations of ethanol which are effective at delta-containing GABAR. Therefore, it is also our hypothesis that the prolonged (2-5 min) pre-exposure to 1-3 mM ethanol can accelerate desensitization rate in the presence of 30 mM ethanol. Because alpha4-beta-delta GABAR are exclusively extrasynaptic, effects of low dose ethanol will be tested on tonic current recorded from dentate gyrus granule cells, which normally express high levels of these receptors. Antisense knock-down of alpha-4 and delta subunits to verify the role of alpha4-beta-delta GABAR in mediating effects of ethanol. The results from these studies may provide insight into potential mechanisms for effects of low dose ethanol at GABAR.