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，α4-β2-Delta，对低浓度的乙醇有反应，但不是高浓度。在停用GABA调节的类固醇THP后，这种效应在重组和天然受体中都可以看到，当这种正常表达不足的受体在边缘区域(如海马区)的水平显著增加时。这项建议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效应。由于乙醇在激动剂的饱和浓度下增加电流幅度，我们的假设是乙醇正在作用于增加α4-β-增量GABAR的门控效率，正如类固醇对该受体的影响所显示的那样。因此，定点突变将被用来在HEK-293细胞中表达的GABAR中创建结构性活性通道，以区分对结合和门控的影响。此外，TM2第270位丝氨酸到色氨酸的突变和TM3的A到S突变将被用来测试结合腔中被识别为高剂量乙醇影响的残基是否介导了低剂量乙醇对α4-β-增量GABAR的影响。其他实验室已经报告了更高浓度的乙醇的影响，这对含有三角洲的GABAR有效。因此，我们的假设也是，在30 mM乙醇存在的情况下，长时间(2-5分钟)预先暴露于1-3 mM乙醇可以加快脱敏速度。由于Alpha4-β-Delta GABAR完全是突触外的，因此将测试低剂量乙醇对齿状回颗粒细胞记录的紧张性电流的影响，齿状回颗粒细胞通常表达高水平的这些受体。反义敲除α-4和Delta亚基以验证Alpha4-β-Delta GABAR在乙醇调节效应中的作用。这些研究的结果可能为低剂量乙醇在GABAR产生影响的潜在机制提供洞察力。

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.