Effects of Bilayer Composition and Ethanol on GABA(A) Re

双层组成和乙醇对 GABA(A) Re 的影响

• 批准号: 6983079
• 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/6983079
• 关键词: GABA receptor; Xenopus oocyte; benzodiazepines; chloride channels; chloride ion; cholesterol; dietary lipid; ethanol; gender difference; intermolecular interaction; laboratory rat; lipid bilayer membrane; long chain fatty acid; membrane lipids; membrane structure; mutant; nutrition related tag; protein purification; protein structure function; receptor binding; receptor sensitivity; recombinant proteins; synaptosomes; tissue /cell culture; unsaturated fatty acids; voltage /patch clamp

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 changes in membrane composition commonly associated with chronic ethanol exposure. The GABA(A) receptor is a member of the cys-loop super family of ligand-gated ion channels and conducts chloride ions in response to binding the neurotransmitter gamma-amino butyric acid (GABA). GABA(A) receptors are among the most sensitive neuronal signaling systems to ethanol and play a role in the neural adaptation which underlies ethanol dependence. 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. During the past year we completed two studies of the interaction between the synaptic membrane and the GABA(A) receptor, made significant progress towards purifying the receptor from native tissue in quantities suitable for biophysical measurements, and initiated a study that will allow direct examination of ligand-induced chloride flux. The first completed study was an investigation of the effects of membrane cholesterol content on ligand binding and the interaction of ligands with allosterically linked binding sites. We showed that depletion of membrane cholesterol reduces the affinity of several benzodiazepines (BZs), while enrichment of membrane cholesterol had no effect on BZ binding. The effects of cholesterol on BZ binding were completely reversible, regardless of the order in which membrane cholesterol was manipulated. This strongly suggests that the effect of depleted membrane cholesterol is not due to alteration of a cholesterol-dependent membrane structure such as a lipid raft, but is due to specific receptor- cholesterol interaction. This study is currently under review for publication. The second completed study showed that a diet deficient in long chain n-3 fatty acids compromises BZ binding in rat synaptosomes in a manner that varies with gender. Binding of BZ by endogenous GABA(A) receptor was examined in synaptosomes from the cerebra of seven and twelve week old Sprague-Dawley rats raised on diets that were either deficient or adequate in long chain (LC) n-3 fatty acids. For rats fed the adequate LC n-3 fatty acid (FA) diet, there were no gender-related differences in BZ binding at either seven or twelve weeks of age. Likewise, no differences in synaptosome fatty acid composition were observed between male and female rats on the adequate LC n-3 FA diet. The deficient LC n-3 FA diet produced significant differences in BZ binding between male and female rats. Males on the n-3 FA deficient diet lost DHA and total LC n-3 FA as they aged, while females increased their synaptosomal levels of both DHA and total LC n-3 FA. The divergence in synaptosomal fatty acid composition between n-3 FA deficient male and female rats as both aged was correlated with increasing gender-related differences in GABAA receptor function. This study is currently under review for publication. A new study initiated this year will enable direct, electrophysiological monitoring of GABA(A) chloride flux activity of receptors either expressed in Xenopus oocytes or injected into Xenopus oocytes. We are using the two-electrode voltage clamp technique to monitor ligand-induced chloride currents. Currently this technique is being used to examine the effects of membrane cholesterol on chloride flux by using methyl-beta-cyclodextrin (MBCD) to deplete and enrich oocytes with cholesterol. Preliminary results indicate that the cholesterol content of the outer oocyte membrane can be manipulated with MBCD using the same techniques we successfully applied to brain synaptosomes. The next stage in this study will be to examine the effects of cholesterol on the chloride flux behavior of several single-site mutants of GABA(AAA) where putatively membrane-exposed residues have been altered. In preparation for this work we have acquired the DNA for several single-site GABA(A) mutants.

该项目的目的是确定膜组成对GABA（A）受体功能的影响，尤其是在通常与慢性乙醇暴露相关的膜组成变化方面。 GABA（A）受体是配体门控离子通道的Cys-Loop超级家族的成员，并响应于结合神经递质γ-氨基丁酸（GABA）而进行氯离子。 GABA（A）受体是乙醇最敏感的神经元信号传导系统之一，并在基于乙醇依赖性的神经适应性中发挥作用。 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。 在过去的一年中，我们完成了两项关于突触膜与GABA（a）受体之间相互作用的研究，在净化天然组织的受体中，以适合生物物理测量的量净化受体，并启动了一项研究，该研究将允许直接检查配体诱导的氯化物的氯化物。首次完成的研究是对膜胆固醇含量对配体结合的影响以及配体与变构连接结合位点的相互作用的作用。我们表明膜胆固醇的耗竭降低了几种苯二氮卓类（BZ）的亲和力，而富集膜胆固醇对BZ结合没有影响。胆固醇对Bz结合的影响是完全可逆的，无论操纵膜胆固醇的顺序如何。这强烈表明，耗尽的膜胆固醇的作用不是由于胆固醇依赖性的膜结构（例如脂质筏）的改变，而是由于特定的受体胆固醇相互作用所致。目前正在审查这项研究以进行发表。 第二项完成的研究表明，长链N-3脂肪酸缺乏饮食会以随性别变化的方式损害了大鼠突触体的BZ结合。内源性GABA（A）受体的BZ在脑的突触体中检查了七个和十二周大的Sprague-Dawley大鼠的突触体，该大鼠是在长链（LC）N-3脂肪酸中不足或足够的饮食中饲养的。对于喂养足够的LC N-3脂肪酸（FA）饮食的大鼠，在七个或十二周龄时，BZ结合的BZ结合均无差异。同样，在足够的LC N-3 FA饮食上，男性和雌性大鼠之间也没有观察到突触体脂肪酸组成的差异。不足的LC N-3 FA饮食在男性和雌性大鼠之间的BZ结合产生了显着差异。 N-3 FA缺乏饮食的雄性年龄损失了DHA和总LC N-3 FA，而女性则增加了DHA和总LC N-3 FA的突触体水平。 N-3 FA缺乏的雄性和雌性大鼠之间突触体脂肪酸组成的差异与GABAA受体功能的性别相关差异的增加相关。目前正在审查这项研究以进行发表。 今年启动的一项新研究将实现直接的电生理监测，对在Xenopus卵母细胞中表达或注射到爪蟾卵母细胞中的受体的GABA（A）氯化物通量活性。我们正在使用两电极电压夹技术来监测配体诱导的氯离子电流。目前，该技术用于检查膜胆固醇对氯化物通量的影响，使用甲基-Beta-Cyclodextrin（MBCD）耗尽并富集胆固醇的卵母细胞。初步结果表明，外卵母细胞膜的胆固醇含量可以使用MBCD操作，使用我们成功应用于脑突触体的相同技术。这项研究的下一个阶段将是检查胆固醇对GABA（AAA）几个单位突变体的氯化物通量行为的影响，在这些突变体已更改了膜暴露的残基。为了准备这项工作，我们为几个单位GABA（A）突变体获得了DNA。