PHOSPORYLATION EFFECT ON GABAA RECEPTOR FUNCTION

GABA受体是一种配体门控氯离子通道，介导GABA受体的代谢。 神经递质GABA（γ-氨基丁酸）的抑制作用 酸）。 除了与GABA结合外，该受体还受几种 治疗剂和内源性神经类固醇， 不同的变构结合位点 超过15种不同的受体 亚基已被克隆和测序， 存在于体内。 有证据表明，GABA α受体是受调节的， 通过蛋白质磷酸化，这种调节是复杂的， 涉及多个第二信使系统通路。 激酶和 伴随这种调节的磷酸酶没有得到很好的表征。 本提案的假设是：1）多种激酶， 磷酸酶参与蛋白质的磷酸化/去磷酸化， 受体，2）受体的亚基组成决定了受体如何与受体结合。 受体受磷酸化调节，3）磷酸化改变 受体复合物的变构调节相互作用。 测试 这些假设，蛋白激酶和磷酸酶将被显微注射 转化到表达重组野生型或 诱变的人GABA受体亚单位cDNA。 这些影响 酶对GABA受体门控氯电流和变构 通过苯二氮卓类、巴比妥类和 将使用双电极电压钳测量神经类固醇 法 因此，本研究采取了还原论的策略 研究GABA受体在一定条件下的磷酸化/去磷酸化 其中确定了受体的组成， 可用于鉴定受体上的磷酸化位点。 这些 研究将有助于了解GABA受体在脑中的功能 神经元信号传导的总体方案。 GABA的参与 受体在过度兴奋状态如焦虑症和癫痫， 强调了理解GABA受体功能的重要性。

The GABA receptor is a ligand-gated chloride channel which mediates the inhibitory effects of the neurotransmitter GABA (gamma-aminobutyric acid). In addition to binding GABA, the receptor is modulated by several therapeutic agents and endogenous neurosteroids which act through distinct, allosteric binding sites. Over fifteen different receptor subunits have been cloned and sequenced and multiple receptor subtypes exist in vivo. There is evidence that the GABAa receptor is regulated by protein phosphorylation and that is this regulation is complex and involves multiple second messenger system pathways. The kinases and phosphatases which accompany this regulation are not well-characterized. The hypotheses of the present proposal are: 1) multiple kinases and phosphatases participate in the phosphorylation/dephosphorylation of the receptor, 2) the subunit composition of the receptor determines how the receptor is regulated by phosphorylation, and 3) phosphorylation alters the allosteric modulatory interactions of the receptor complex. To test these hypotheses, protein kinases and phosphatases will be microinjected into Xenopus oocytes expressing either recombinant wildtype or mutagenized human GABA receptor subunit cDNAs. The effect of these enzymes on both GABA receptor-gated chloride currents and the allosteric modulation of these currents by benzodiazepines, barbiturates and neurosteroids, will be measured using the two-electrode voltage-clamp technique. Thus, the proposed research adopts a reductionistic strategy to study GABA receptor phosphorylation/dephosphorylation under conditions where the receptor composition is defined and site-directed mutagenesis can be used to identify phosphorylation sites on the receptor. These studies will aid in the understanding of GABA receptor function in the overall scheme of neuronal signaling. The involvement of the GABA receptor in hyperexcitable states such as anxiety disorders and epilepsy, underscores the importance of understanding GABA receptor function.