PHOSPORYLATION EFFECT ON GABAA RECEPTOR FUNCTION

GABA受体是一种配体门控的氯离子通道，它介导 神经递质γ-氨基丁酸的抑制作用 酸)。除了与GABA结合外，受体还受几种 治疗药物和内源性神经类固醇通过 不同的变构结合部位。超过15种不同的受体 亚基已被克隆和测序，并有多种受体亚型 存在于活体内。有证据表明GABAA受体是受调控的 通过蛋白质磷酸化，也就是说，这种调节是复杂的 涉及多个第二信使系统通路。这些激动素和 伴随这一调节的磷酸酶还没有得到很好的描述。 本方案的假设是：1)多个激酶和 磷酸酶参与细胞的磷酸化/去磷酸化 受体，2)受体的亚基组成决定如何 受体受磷酸化调控，3)磷酸化改变 受体复合体的变构调节相互作用。为了测试 这些假说、蛋白激酶和磷酸酶将被显微注射 转化非洲爪哇卵母细胞，表达重组野生型或 诱变的人GABA受体亚单位cDNA。这些因素的影响 GABA受体门控氯电流和变构上的酶 苯二氮卓类、巴比妥类和 神经类固醇，将使用双电极电压钳进行测量 技术。因此，拟议的研究采用了一种简约主义策略 条件下GABA受体磷酸化/去磷酸化的研究 确定受体组成的地方和定点突变 可用于识别受体上的磷酸化位点。这些 研究将有助于了解脑内GABA受体的功能。 神经元信号传递的总体方案。GABA的参与 处于过度兴奋状态的受体，如焦虑症和癫痫， 强调了解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.