STRUCTURE OF GABA-A RECEPTOR ANION-SELECTIVE CHANNEL

GABA-A 受体阴离子选择性通道的结构

• 批准号: 2379673
• 负责人: Myles H. Akabas
• 金额: $ 24.04万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-03-05 至 1998-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2379673
• 关键词: GABA receptor; Xenopus; Xenopus oocyte; animal genetic material tag; anions; biological signal transduction; cysteine; gamma aminobutyrate; gene mutation; ion transport; neural conduction; protein sequence; protein structure function; site directed mutagenesis; sulfhydryl reagents; transfection; voltage /patch clamp

The long term goal of this research is to elucidate the structural basis of ion conduction and gating in the GABAA receptor. The GABAA receptors are the major post synaptic receptors for GABA. The binding of GABA triggers the opening of an anion-selective channel, which is the basis of GABA's role as the major inhibitory neurotransmitter in the brain. The GABAergic system is essential for normal brain function and has been implicated in the treatment and etiology of epilepsy and anxiety. The GABAA receptor is the target of two classes of neuropsychiatric drugs, the benzodiazepines and the barbiturates. Although the pharmacological, electrophysiological, and molecular biological properties of the GABAA receptors have been extensively studied, the structures of these receptors are not well- determined beyond the sequences of their subunits. The subunits all have similar sequences that include four hydrophobic, putative membrane-spanning segments, named M1, M2, M3, and M4. By analogy with the homologous acetylcholine receptor, the subunits likely form pseudosymmetrical, pentameric rings around a central channel. The goal of this project is to identify the residues of these segments that line the GABAA channel lumen. Residues, initially in the alpha1 subunit M1 and M2 segments, will be mutated to cysteine, one at a time. The mutant alpha1 subunit will be coexpressed with the beta1 subunit in Xenopus oocytes. Only mutants which have near-normal function when expressed in oocytes will be studied further. These will be challenged with small, negatively charged, sulfhydryl reagents, including iodoacetate and methanethiosulfonate- ethylsulfonate. These reagents covalently attach a negatively charged group to the sulfhydryl of cysteine. They are highly polar and are much more likely to react with cysteines expressed on the receptor surface, which includes residues lining the channel lumen. If a cysteine faces the channel lumen and reacts with these reagents, the channel conductance should be irreversibly altered. Reagents will be added to the extracellular and intracellular sides of the membrane, and the effects will be monitored by two-electrode voltage clamping or by patch clamping. Reagents will be added in the presence and absence of GABA. By this approach, I will identify the residues that line the anion channel, and determine their secondary structure, the position of the ion selectivity filter, and the position of the gate.

这项研究的长期目标是阐明 GABAA受体中的离子传导和门控。 GABAA受体是 GABA的主要后突触受体。 GABA触发器的结合 阴离子选择通道的开放，这是GABA的基础 作为大脑中主要的抑制性神经递质的作用。 加巴能 系统对于正常的大脑功能至关重要，并且已与 癫痫和焦虑的治疗和病因。 GABAA受体是 两类神经精神药物的靶标，苯二氮卓类药物 和巴比妥 虽然是药理，电生理学，但 GABAA受体的分子生物学特性已经 经过广泛的研究，这些受体的结构不是很好 确定超出其亚基的序列。 亚基都有 类似的序列，包括四个疏水，推定的膜跨度 片段，名为M1，M2，M3和M4。 通过类似于同源 乙酰胆碱受体，亚基可能形成伪对称， 中央通道周围的五聚会环。 这个项目的目标是 确定将GABAA通道管腔排列的这些段的残基。 最初在Alpha1亚基M1和M2段中的残留物将是 一次突变为半胱氨酸，一次。 突变的alpha1亚基将 与爪蟾卵母细胞中的β1亚基共表达。 只有突变体 在卵母细胞中表达时具有接近正常的功能 更远。 这些将受到小额，负电的挑战， 亚硫酰乙酸酯试剂，包括碘乙酸酯和甲烷硫酸盐 - 乙酸乙酯。 这些试剂共价附加带负电荷的 一组到半胱氨酸的硫ry 它们是极性的，很大 更可能与在受体表面上表达的半胱氨酸反应， 其中包括在通道管腔内衬里的残留物。 如果半胱氨酸面对 通道管腔并与这些试剂反应，通道电导 应该不可逆转地改变。 试剂将被添加到 膜的细胞外和细胞内侧，影响将 通过两电极电压夹紧或贴片夹具监测。 在存在和不存在GABA的情况下，将添加试剂。 这样 方法，我将确定在阴离子通道上排列的残留物，以及 确定其二级结构，离子选择性的位置 过滤器和门的位置。