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，即α4-β2-Delta，它对 乙醇的浓度很低，但不高。这种效应在重组受体和天然受体中都可以看到。 在停用GABA调节类固醇THP后，当这种激素水平正常低表达时 在边缘区域，如海马区，受体显著增加。这项提案将调查 利用HEK-293细胞的瞬时表达来检测这种效应的可能机制 乙醇对单通道特性和失活动力学的剂量依赖效应。因为乙醇 在激动剂饱和浓度下增加电流幅度，我们的假设是乙醇是 作用于增加α4-β-增量GABA的门控效率，正如类固醇效应在 这个感受器。因此，定点突变将被用来创建结构性活性通道。 GABAR在HEK-293细胞中表达，以区分对结合和门控的影响。此外, TM2基因第270位丝氨酸到色氨酸突变和TM3基因A到S突变将用于检测 高剂量乙醇作用下结合腔中的残留物是否参与调节作用 低剂量乙醇对α4-β-增量伽巴的影响。其他实验室已经报告了更高的 乙醇的浓度对含三角洲的伽巴有效。因此，它也是我们的 假设长时间(2-5分钟)预先接触1-3 mM乙醇可以加速脱敏 在30 mM乙醇存在的情况下进行速率测定。因为Alpha4-beta-Delta GaBAR完全是突触外的， 小剂量乙醇对齿状回颗粒细胞记录的紧张性电流的影响 正常情况下，这些受体的表达水平很高。α-4和Delta亚基的反义敲除 验证Alpha4-β-Delta GABAR在乙醇调节作用中的作用。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。

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.