Regulation of GABAA Receptor Cell Surface Expression

GABAA 受体细胞表面表达的调节

• 批准号: 6543956
• 负责人: NANCY J LEIDENHEIMER
• 金额: $ 26.9万
• 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/6543956
• 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受体是一种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17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磷酸化调节。这种调节是复杂的，涉及多种亚基和多个第二信使系统通路。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GABAA受体的功能。 PKC对GABAA受体细胞表面表达的调控仍有待研究。最近已经确定GABAA受体经历网格蛋白依赖性组成性内吞作用。我们推测该受体也经历PKC调节的网格蛋白依赖性内吞作用。此外，我们假设受体上的双亮氨酸和酪氨酸AP 2适应素识别基序在组成型与PKC调节的内吞作用中起选择性作用，并且还参与确定受体的内吞后分选以再循环与溶酶体途径。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PKC依赖性GABAA受体内吞作用在神经元中发生的机制。该提议的具体目的是：1）确定GABAA受体上存在的AP 2适应素识别基序是否在网格蛋白依赖性组成型GABAA受体内吞作用中起作用; 2）确定PKC活化是否促进网格蛋白依赖性GABAA受体内吞作用; 3）研究神经元中GABAA受体细胞表面表达的mGluR 1/5受体调节。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拟定实验。使用间接免疫荧光和荧光蛋白标签，通过共聚焦显微镜观察细胞表面受体。还将使用胰凝乳蛋白酶消化/免疫印迹技术进行补充生化实验，以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物化学研究提供功能相关性。这些研究将有助于理解GABAA受体的功能和神经元的可塑性。GABAA受体参与焦虑，睡眠和癫痫发作疾病强调了了解这种受体的细胞和分子机制的重要性。