MODULATION GIRK CHANNELS--ALPHA SUBUNITS OF G PROTEINS

GIRK的内向整流性G蛋白激活的K+通道 家庭在调节心跳和介导 大脑中许多神经递质的抑制作用。 GIRK的现象学和调控机制 对神经递质知之甚少。 未解决的问题包括： G蛋白和蛋白质之间相互作用特异性的决定因素 GIRK通道;门控、脱敏和抑制机制 通过神经递质调节。 长期目标是了解 的分子机制和生理意义 通过G-α亚基和G蛋白的抑制性GIRK调节- 耦合神经递质 具体目标是：1.了解 膜间相互作用的分子机制 GIRK亚基和G-α蛋白，通过检查 纯化的G蛋白和影响离体细胞中磷酸化的试剂 非洲爪蟾卵母细胞膜，并通过监测蛋白质-蛋白质 通过免疫共沉淀和重叠方法进行相互作用。 2. 为了研究GIRK的调节，通过蛋白磷酸化， 激活G-q的神经递质和GIRK的过程 脱敏，通过检查蛋白激酶抑制剂的作用， 纯化的蛋白激酶在非洲爪蟾卵母细胞，并通过突变推定的 靶GIRK亚基中的磷酸化位点。 3.评价 G-α亚基对GIRK调节的生理意义。 卵母细胞中所描述的调节的存在，以及 他们的分子机制的身份，将在主要确认 通过测试心脏和神经细胞的直接影响， G-α亚基和相关蛋白激酶，并通过消除 通过反义基因敲除的方式来去除信号通路中的蛋白质成分。 4.研究G-ail、G-as和蛋白质磷酸化 干扰信道选通的过程。 门控详情 将通过检查通道部分的相互作用来探索过程 参与与其他通道的门控，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).