Regulation of GABAA Receptor Cell Surface Expression

描述（由申请人提供）：GABAA受体是一种配体门控氯通道，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种不同的受体亚基和多种受体亚型。GABAA受体通过多种激酶受蛋白磷酸化调控。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。我们最近的研究表明，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作用有待进一步研究。最近已经确定GABAA受体经历网状蛋白依赖的组成性内吞作用。我们假设受体也经历pkc调节的网格蛋白依赖的内吞作用。此外，我们假设受体上的二亮氨酸和酪氨酸AP2适应蛋白识别基序在构成型和pkc调节的内吞作用中发挥选择性作用，并且还参与决定受体内吞后对循环和溶酶体途径的分选。最后，我们假设pkc相关的代谢性谷氨酸受体mGluR 115的激活可能代表了pkc依赖性GABAA受体内吞在神经元中发生的机制。本提案的具体目的是：1)确定GABAA受体上存在的AP2适应蛋白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激活是否促进网格蛋白依赖性GABAA受体内吞作用；3)探讨mGluR1/5受体对神经元GABAA受体细胞表面表达的调控作用。该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共聚焦显微镜使用间接免疫荧光和荧光蛋白标签可视化。利用胰凝乳酶消化/免疫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。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受体在焦虑、睡眠和癫痫障碍中的参与强调了理解该受体的细胞和分子机制的重要性。

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.