Effects Of Bilayer Composition And Ethanol On The GABA(A

双层组合物和乙醇对 GABA(A) 的影响

• 批准号: 6680131
• 负责人: DRAKE C MITCHELL
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6680131
• 关键词: G protein; GABA receptor; Sf9 cell line; biological signal transduction; ethanol; fluorescence spectrometry; inhibitor /antagonist; intermolecular interaction; lipid bilayer membrane; liposomes; pharmacology; posttranslational modifications; protein isoforms; protein purification; protein structure function; receptor binding; recombinant proteins; serotonin receptor; stimulant /agonist; tissue /cell culture; G protein; GABA receptor; Sf9 cell line; biological signal transduction; ethanol; fluorescence spectrometry; inhibitor /antagonist; intermolecular interaction; lipid bilayer membrane; liposomes; pharmacology; posttranslational modifications; protein isoforms; protein purification; protein structure function; receptor binding; recombinant proteins; serotonin receptor; stimulant /agonist; tissue /cell culture

The GABA(A) receptor family of ligand-gated ion channels consists of heteroligomeric complexes of five subunits from six different classes with each class consisting of several isoforms, however, only ten subunit combinations have been reported. GABA(A) receptors contain binding sites for GABA, barbituates, benzodiazepines, and neurosteroids. The goal of this project is to determine the effects of membrane composition on the function of the GABA(A) receptor, particularly with respect to the changes in membrane composition commonly associated with chronic exposure to ethanol. GABA(A) receptors are among the most sensitive neuronal signaling systems to ethanol and clearly play a role in the neural adaptation which underlies ethanol dependence. Many behavioral effects of ethanol are induced by GABA(A) receptor agonists, and behavioral effects of acute ethanol are sharply reduced by GABA(A) receptor antagonists. In addition, its sensitivity to barbituates and benzodiazapenes means that compromised GABA(A) receptor function is a hallmark of people who simultaneously abuse drugs and alcohol. Changes in GABA(A) receptor function are associated with ethanol tolerance and dependence and may be due to the altered neuronal membrane composition associated with chronic ethanol exposure. GABA(A) receptor function is altered by membrane phospholipid composition because purification and reconstitution of fully active receptor depends on the inclusion of native brain lipids. A second indication of functional interaction with lipids is the highly selective effect of free fatty acids. Two highly polyunsaturated fatty acids, docosahexaenoic acid and arachidonic acid, increase the GABA-induced peak chloride ion current when applied at concentrations below 1 micromolar. At higher concentrations, both fatty acids gradually suppressed the peak amplitude. Interestingly, two other polyunsaturated fatty acids, docosapentaenoic acid and docosatetraenoic acid, had no effect at any concentration. Currently we are investigating the effects of membrane cholesterol content on ligand binding and the interaction of ligands with apparently overlapping binding sites. This work has emphasized assays of ligand binding saturation and competition using radio-labeled compounds and filter binding assays. Initial results indicate that both enrichment and depletion of membrane cholesterol reduce the affinity of several important ligands. This would suggest that the native membrane has an optimal cholesterol content with respect to GABA(A) receptor function. This hypothesis will be tested using purified receptor and membranes consisting of defined phospholipids and variable levels of cholesterol. Purification of the receptor free of native lipid has been accomplished on a limited scale. Our purification method involves solubilization of synaptosomal membranes in detergent and purification via affinity chromatography using a high affinity ligand linked to sepharose. Initially, the optimal ligand for this process, Ro 7-1986, was only available in limited quantity as a gift from a pharmaceutical company. Thus, the scale of the purification procedure was quite limited. Recently we were able to obtain a large quantity of Ro 7-1986 via contract with an organic synthesis laboratory. It is anticipated that this will allow the purification procedure to be scaled up to produce quantities of purified receptor suitable for reconstitution into phospholipid bilayers of defined composition. This will make it possible to conduct detailed investigations of receptor interaction with the lipid bilayer, and interaction with ethanol under controlled and defined conditions. A distinct advantage of working with the receptor in recombinant membranes is the ability to directly monitor, in real time, all aspects of receptor activity; agonist/antagonist binding, activation, and passage of chloride ions. This will be accomplished via a variety of non-invasive spectroscopic techniques. The binding of fluorescent ligands will be monitored via stopped-flow. Both fluorescence and CD assays of agonist binding will be conducted with a recently acquired stopped flow instrument that is capable of simultaneously collecting CD and fluorescence as well as fluorescence polarization stopped flow data. Both the fluorescence and CD techniques have the added advantage that they require very little sample in order to make accurate measurements.

配体门控离子通道的GABA（a）受体家族由来自六个不同类别的五个亚基的杂聚集络合物组成，每个类别由几种同工型组成，但是，只有十个亚基组合报道。 GABA（A）受体包含用于GABA，BATBITATES，BINZODIAZEPINE和NEURORSTORIS的结合位点。该项目的目的是确定膜组成对GABA受体功能（a）受体功能的影响，尤其是在通常与慢性暴露于乙醇相关的膜组成的变化方面。 GABA（A）受体是乙醇最敏感的神经元信号传导系统之一，并且在乙醇依赖性的基础的神经适应性中显然发挥作用。 GABA（A）受体激动剂诱导了乙醇的许多行为作用，急性乙醇的行为效应被GABA（A）受体拮抗剂急剧降低。此外，它对巴比西特和苯二氮唑烯的敏感性意味着妥协的GABA（A）受体功能是同时滥用毒品和酒精的人的标志。 GABA（a）受体功能的变化与乙醇的耐受性和依赖性有关，可能是由于与慢性乙醇暴露有关的神经元膜组成改变所致。 GABA（A）受体功能会因膜磷脂组成而改变，因为完全活跃的受体的纯化和重建取决于包含天然脑脂质。功能相互作用与脂质的第二种指示是游离脂肪酸的高度选择性作用。当以低于1微摩尔的浓度施用时，两种高度多不饱和的脂肪酸，二十二碳六烯酸和花生四烯酸会增加GABA诱导的峰值氯离子电流。在较高浓度下，两种脂肪酸都逐渐抑制了峰值振幅。有趣的是，另外两个多不饱和脂肪酸，二十碳烯酸和二十二烯酸，在任何浓度下都没有作用。 目前，我们正在研究膜胆固醇含量对配体结合的影响以及配体与显然重叠结合位点的相互作用。这项工作强调了使用无线电标记化合物和滤波器结合测定法对配体结合饱和度和竞争的测定。最初的结果表明，膜胆固醇的富集和耗竭降低了几种重要配体的亲和力。这表明天然膜相对于GABA（A）受体功能具有最佳的胆固醇含量。该假设将使用由定义的磷脂和可变水平的胆固醇组成的纯化受体和膜进行检验。 没有天然脂质的受体的纯化已经有限地完成。我们的纯化方法涉及使用与Sepharose相关的高亲和力配体通过亲和色谱法溶解洗涤剂中的突触体膜。最初，此过程的最佳配体RO 7-1986仅作为制药公司的礼物以有限的数量提供。因此，纯化程序的规模非常有限。最近，我们能够通过与有机合成实验室的合同获得大量的RO 7-1986。预计这将允许将纯化程序缩放以产生适合重建的纯化受体，以重新结构为定义组成的磷脂双层。这将使对受体相互作用与脂质双层的相互作用进行详细研究，并在受控和确定的条件下与乙醇相互作用。在重组膜中与受体一起工作的一个明显优势是能够实时直接监测受体活性的所有方面。激动剂/拮抗剂结合，氯离子的激活和通过。这将通过多种非侵入性光谱技术来实现。荧光配体的结合将通过停止流量监测。荧光和CD分析的激动剂结合将使用最近获得的停止流量仪器进行，该仪器能够同时收集CD和荧光以及荧光极化停止流量数据。荧光和CD技术都具有额外的优势，即它们几乎不需要样品才能进行准确的测量。