Regulation of GABAA Receptor Cell Surface Expression

GABAA 受体细胞表面表达的调节

• 批准号: 7057836
• 负责人: NANCY J LEIDENHEIMER
• 金额: $ 25.17万
• 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 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7057836
• 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(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)的快速抑制效应。体内存在超过17种不同的受体亚基和多种受体亚型。GABAA受体受多种蛋白磷酸化的调节。这种调控是复杂的，涉及多种亚基和多个第二信使系统途径。我们最近发现，钙磷脂依赖的蛋白激酶(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作用尚不清楚。最近证实，GABAA受体经历依赖于笼蛋白的结构性内吞作用。我们推测，该受体还经历了PKC调节的依赖于笼蛋白的内吞作用。此外，我们假设受体上的二亮氨酸和酪氨酸AP2适配素识别基序在结构性和PKC调节的内吞作用中发挥选择性作用，并参与决定受体内吞后对循环途径和溶酶体途径的分选。最后，我们假设PKC连接的代谢性谷氨酸受体mGluR115的激活可能代表了神经元中发生PKC依赖的GABAA受体内吞作用的机制。本研究的具体目的是：1)确定GABAA受体上的AP2适配素识别基序是否在依赖于笼蛋白的GABAA受体内吞作用中起作用；2)确定PKC激活是否促进依赖于笼蛋白的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中进行。细胞表面受体将通过使用间接免疫荧光和荧光蛋白标签的共聚焦显微镜进行可视化。使用胰凝乳酶消化/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的表达。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。这些研究将有助于了解GABAA受体的功能和神经元的可塑性。GABAA受体在焦虑、睡眠和癫痫障碍中的作用突显了了解该受体的细胞和分子机制的重要性。