GIRK CHANNEL INTERACTION SITES ON G PROTEINS

G蛋白门控向内整流K+(GIRK)通道是第一个由G蛋白的β-伽马亚基直接调节的效应器的例子。然而，对GIRK通道调节重要的Gbeta-Gamma上的元素知之甚少。我们以前曾使用嵌合策略来识别通道上的重要元件，这些元件对Gbeta-Gamma(他等人)的调节很重要。艾尔1999年)。在这里，我们提供的初步数据表明，在非洲爪哇卵母细胞中，Gbeta1-beta4都可以激活GIRK通道，具有相似的效果。然而，Gbeta5无法在相同条件下激活这些通道。在表达Gbeta5的卵母细胞中进行的Western印迹分析表明，该蛋白得到了表达。因此，缺乏表达不可能是缺乏通道激活的原因。序列比对表明，Gbeta1-beta4与其他Gbeta1亚基的同源性约为90%，而Gbeta5与其他Gbeta亚基的同源性仅为51%。基于这些发现，我们建议使用Gbeta1和Gbeta5之间的嵌合策略来确定导致这两个亚基的通道激活差异的区域。一旦这些区域被确定，我们将使用突变来确定参与Gbeta1和通道相互作用的特定残基。这些研究将有助于我们更好地了解G蛋白与GIRK通道的相互作用。这些研究还将帮助我们更好地了解G蛋白介导的信号转导，这具有重要的生理意义。

G protein-gated inwardly rectifying K+ (GIRK) channels were the first example of an effector that is directly regulated by the beta-gamma subunits of G proteins. However, relatively little is known about the elements on the Gbeta-gamma that are important for GIRK channel regulation. We have previously used a chimeric strategy to identify important elements on the channel that are important for regulation by Gbeta-gamma (He et. al. 1999). Here, we present preliminary data indicating that in Xenopus oocytes, Gbeta1-beta4 can all activate the GIRK channels with similar efficacy. However, the Gbeta5 is incapable of activating these channels under identical conditions. Western blot analysis in oocytes expressing Gbeta5 showed that the protein is expressed. Therefore lack of expression can not be responsible for lack of channel activation. Sequence alignment showed that Gbeta1-beta4 display approximately 90% identity, whereas Gbeta5 shows only 51 % identity with other Gbeta subunits. Based on these findings, we propose to use a chimeric strategy between Gbeta1 and Gbeta5 to identify the regions responsible for the difference in channel activation by the two subunits. Once these regions are identified, we will use mutagenesis to identify specific residues involved in the interaction of Gbeta1 and the channel. These studies will help us better understand the interaction of G proteins with GIRK channels. The proposed studies will also help us achieve a better understanding of G protein mediate signaling, which is of major physiological importance.