Regulation of GABAA Receptor Cell Surface Expression

GABAA 受体细胞表面表达的调节

• 批准号: 6660370
• 负责人: NANCY J LEIDENHEIMER
• 金额: $ 29万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-15 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6660370
• 关键词: GABA receptor; biological signal transduction; cell line; cell membrane; clathrin; confocal scanning microscopy; endocytosis; enzyme activity; enzyme mechanism; gamma aminobutyrate; glutamate receptor; immunofluorescence technique; leucine; neural plasticity; neural transmission; neurons; protein kinase C; protein structure function; receptor expression; tyrosine; voltage /patch clamp; western blottings; yeast two hybrid system

DESCRIPTION (provided by applicant): The GABAA receptor is a ligand-gated chloride channel that mediates the fast inhibitory effects of the neurotransmitter GABA (g-aminobutyric acid). Over seventeen different receptor subunits exist and multiple receptor subtypes are present in vivo. The GABAA receptor is regulated by protein phosphorylation through multiple kinases. This regulation is complex, involving a variety of subunits and multiple second messenger system pathways. We have recently shown that activation of calcium phospholipid-dependent protein kinase (PKC) decreases GABAA receptor function via internalization of GABAA receptors. The regulation of GABAA receptor cell surface expression by PKC remains to be characterized. It has recently been established that GABAA receptors undergo clathrin-dependent constitutive endocytosis. We hypothesize that the receptor also undergoes PKC-regulated clathrin-dependent endocytosis. Furthermore, we hypothesize that the dileucine and tyrosine AP2 adaptin recognition motifs on the receptor play selective roles in constitutive vs. PKC-regulated endocytosis and also are involved in determining the post-endocytic sorting of the receptor to recycling vs. lysosomal pathways. Lastly, we hypothesize that activation of the PKC-linked metabotropic glutamate receptors mGluR 115 may represent a mechanism by which PKC-dependent GABAA receptor endocytosis occurs in neurons. The specific aims of this proposal are: 1)To determine if the AP2 adaptin recognition motifs present on the GABAA receptor play a role in clathrin-dependent constitutive GABAA receptor endocytosis; 2) To determine whether PKC activation promotes clathrin-dependent GABAA receptor endocytosis; 3) To investigate mGluR1/5 receptor regulation of GABAA receptor cell surface expression in neurons. The proposed experiments will be performed in HEK 293 cells expressing recombinant receptors and in primary neuronal cultures. Cell surface receptors will be visualized by confocal microscopy using indirect immunofluorescence and fluorescent protein tags. Complementary biochemical experiments using chymotrypsin digest/Immunoblotting blotting techniques will also be used to measure receptor cell surface expression. Experiments using whole-cell patch-clamp recordings will provide a functional correlate for the confocal microscopy and biochemical studies. The proposed studies will aid in the understanding of GABAA receptor function and neuronal plasticity. The involvement of GABAA receptors in anxiety, sleep and seizure disorders underscores the importance of understanding the cellular and molecular mechanisms of this receptor.

描述（由申请人提供）：GABAA受体是配体门控氯离子通道，介导神经递质GABA（g-氨基丁酸）的快速抑制作用。体内存在超过十七种不同的受体亚基并且存在多种受体亚型。 GABAA 受体通过多种激酶受蛋白质磷酸化的调节。这种调节很复杂，涉及多种亚基和多个第二信使系统途径。我们最近发现，钙磷脂依赖性蛋白激酶（PKC）的激活通过 GABAA 受体的内化降低了 GABAA 受体的功能。 PKC 对 GABAA 受体细胞表面表达的调节仍有待表征。最近已确定 GABAA 受体经历网格蛋白依赖性组成型内吞作用。我们假设该受体也经历 PKC 调节的网格蛋白依赖性内吞作用。此外，我们假设受体上的二亮氨酸和酪氨酸 AP2 适应素识别基序在组成型内吞作用和 PKC 调节的内吞作用中发挥选择性作用，并且还参与确定受体的内吞后分选以再循环和溶酶体途径。最后，我们假设 PKC 连接的代谢型谷氨酸受体 mGluR 115 的激活可能代表了神经元中 PKC 依赖性 GABAA 受体内吞作用发生的机制。该提案的具体目的是： 1）确定GABAA受体上存在的AP2适应素识别基序是否在网格蛋白依赖性组成型GABAA受体内吞作用中发挥作用； 2) 确定PKC激活是否促进网格蛋白依赖性GABAA受体内吞作用； 3) 研究mGluR1/5受体对神经元GABAA受体细胞表面表达的调节。拟议的实验将在表达重组受体的 HEK 293 细胞和原代神经元培养物中进行。细胞表面受体将通过共聚焦显微镜使用间接免疫荧光和荧光蛋白标签进行可视化。使用胰凝乳蛋白酶消化/免疫印迹印迹技术的补充生化实验也将用于测量受体细胞表面表达。使用全细胞膜片钳记录的实验将为共聚焦显微镜和生化研究提供功能关联。拟议的研究将有助于理解 GABAA 受体功能和神经元可塑性。 GABAA 受体与焦虑、睡眠和癫痫疾病的关系强调了了解该受体的细胞和分子机制的重要性。