MODULATION GIRK CHANNELS--ALPHA SUBUNITS OF G PROTEINS

GIRK的内向整流、G蛋白激活的K+通道 家庭在心跳调节中起着重要的作用 大脑中许多神经递质的抑制作用。 GIRK的现象学和调控机制 人们对神经递质知之甚少。未解决的问题包括： G蛋白与G蛋白相互作用的特异性决定因素 GIRK通道；门控、脱敏和抑制机制 神经递质的调节作用。我们的长期目标是理解 其分子机制及其生理学意义 G-α亚基和G蛋白对GIRK的抑制作用 联结的神经递质。具体目标是：1.了解 膜分隔相互作用的分子机制 GIRK亚基和G-α蛋白，通过检测 纯化的G蛋白及其在体外影响磷酸化的因素 非洲爪哇卵母细胞膜上的斑块，并通过监测蛋白质-蛋白质 免疫共沉淀法和重叠方法的相互作用。2. 研究GIRK的调节，通过蛋白质磷酸化，通过 激活G-Q的神经递质与GIRK的过程 脱敏，通过检测蛋白激酶抑制剂和 非洲爪哇卵母细胞中纯化的蛋白激酶及其可能的突变 靶基因GIRK亚基的磷酸化位点(S)。3.评估 G-α亚基调节GIRK的生理意义。 卵母细胞中所描述的调节的存在，以及 它们的分子机制的身份，将在初步确认 心脏和神经细胞的培养通过测试直接作用 G-α亚基和相关蛋白激酶(S)，并通过消除 反义基因敲除信号通路的蛋白质组分。 4.研究G-AIL、G-AS和蛋白质磷酸化 干扰通道选通过程。选通详细信息 我们将通过检查渠道各部分之间的相互作用来探索流程 参与与渠道的其余部分以及与 调节门控(G-α蛋白和蛋白激酶)。

The inwardly rectifying, G protein-activated K+ channels of the GIRK family are important in regulation of heartbeat and mediate the inhibitory effects of many neurotransmitters in the brain. Phenomenology and mechanisms of modulation of GIRK by neurotransmitters are poorly understood. Unresolved problems include: determinants of specificity of interaction between G proteins and the GIRK channels; mechanisms of gating, desensitization, and inhibitory modulation by neurotransmitters. The long term goal is to understand the molecular mechanisms and the physiological significance of the inhibitory GIRK modulation by G-alpha subunits and by G protein- coupled neurotransmitters. The specific aims are: 1. To understand the molecular mechanisms of membrane-delimited interaction between GIRK subunits and the G-alpha proteins, by examining the effects of purified G proteins and agents affecting phosphorylation in excised patches of Xenopus oocyte membrane, and by monitoring protein-protein interactions by coimmunoprecipitation and overlay methodologies. 2. To study modulation of GIRK, via protein phosphorylation, by neurotransmitters that activate G-q, and the process of GIRK desensitization, by examining effects of protein kinase inhibitors and purified protein kinases in Xenopus oocytes, and by mutating putative phosphorylation sites in target GIRK subunit(s). 3. To evaluate the physiological significance of modulations of GIRK by G-alpha subunits. The existence of the modulations described in the oocytes, and the identity of their molecular mechanisms, will be confirmed in primary cultures of cardiac and nerve cells by testing the direct effects of G-alpha subunits and relevant protein kinase(s), and by eliminating the protein components of signaling pathways by antisense knockout. 4. To investigate how G-ail, G-as and protein phosphorylation interfere with the process of channel gating. Details of gating process will be explored by examining interactions of parts of channel involved in gating with the rest of the channel, and with agents that modulate gating (G-alpha proteins and protein kinases).