Alcohol, GABA and hormones: Physiology of subunit change

描述（由申请人提供）：我们报道了一种新的GABA-A受体（GABAR）异构体，α 4- β 2- δ，对低浓度但不高浓度的乙醇有反应。当gaba调节类固醇THP在边缘区域（如海马体）正常低表达受体的水平显著增加时，这种效应在重组受体和天然受体中均可见。本研究将研究这种效应的可能机制，通过在HEK-293细胞中的瞬时表达来检测乙醇对单通道特性和失活动力学的剂量依赖性影响。因为在激动剂的饱和浓度下，乙醇会增加电流振幅，我们的假设是乙醇会增加α - 4- β - δ GABAR的门控效力，正如类固醇对该受体的作用所显示的那样。因此，位点定向诱变将用于在HEK-293细胞中表达的GABAR中创建组成活性通道，以区分对结合和门控的影响。此外，TM2残基270处丝氨酸到色氨酸的突变和TM3残基A到S的突变将被用来测试高剂量乙醇作用的结合腔中残基是否介导低剂量乙醇对α - 4- β - δ GABAR的影响。其他实验室报告了高浓度乙醇对含有δ的GABAR有效的影响。因此，我们也假设，在30 mM乙醇存在下，长时间（2-5分钟）预暴露于1-3 mM乙醇可以加速脱敏率。由于α - 4- β - δ GABAR仅存在于突触外，低剂量乙醇对齿状回颗粒细胞记录的紧张电流的影响将被测试，齿状回颗粒细胞通常表达高水平的这些受体。α -4和δ亚基的反义敲除以验证α -4 - β - δ GABAR在乙醇介导作用中的作用。这些研究的结果可能为低剂量乙醇作用GABAR的潜在机制提供见解。

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.