Alcohol, GABA and hormones: Physiology of subunit change

我们已经报道了一种新的GABA-A受体（GABAR）亚型，α 4-β 2-δ，是响应于 低浓度但不高浓度的乙醇。这种效应在重组和天然受体中都可以看到 在停用GABA调节类固醇THP后， 受体在边缘区域如海马体中显著增加。该提案将调查 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影响。因为乙醇 在激动剂的饱和浓度下增加电流幅度，我们假设乙醇是 作用于增加α 4-β-δ GABAR的门控功效，如已显示的类固醇作用， 这个受体。因此，将使用定点诱变来在细胞中产生组成型活性通道。 GABAR在HEK-293细胞中表达，以区分对结合和门控的影响。此外，本发明还提供了一种方法， 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效应的残留物是否起到介导作用 低剂量乙醇对α 4-β-δ γ-氨基丁酸受体的影响。其他实验室报告说， 乙醇的浓度，这是有效的δ含有GABAR。因此，这也是我们的 假设长时间（2-5分钟）预暴露于1-3 mM乙醇可加速脱敏 在30 mM乙醇的存在下，因为α 4-β-δ GABAR只在突触外， 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， 正常情况下这些受体的表达水平很高。反义敲低α-4和δ亚基， 验证α 4-β-δ GABAR在介导乙醇效应中的作用。这些研究的结果 可能提供洞察低剂量乙醇在GABAR的影响的潜在机制。

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.