MUSCARINIC GATED ATRIAL K+ CHANNEL

描述：副交感神经修饰的分子机制 包括毒蕈碱门控心房肌的激活 钾离子通道GIRK-1 激活途径似乎 涉及G蛋白与离子通道亚基的直接相互作用。 这种相互作用的位置和化学计量仍然未知。 最近的证据表明，GIRK-1亚基与另一个亚基共组装， 内向整流钾通道亚单位，称为CIR。 氨基酸 GIRK的C-末端的缺失使该分子对G不敏感 蛋白质活化 本提案中实验的目标是 确定GIRK蛋白中影响G蛋白活化偶联的位点 蛋白质的通道，而不影响通道组装。 信道 亚基将用表位标记以允许特异性抗体结合 并允许识别。 将进行实验以使 这些通道被预测参与蛋白质-蛋白质 交互. 将进行诱变实验以确定 通道是否正常工作，如果不正常，将确定是否 突变体通道被合成并靶向质膜， 使用表位标记的亚基。 其次，将确定是否 这些对GIRK通道功能至关重要的区域也是 G蛋白结合位点。 第三个策略是尝试 通过创建嵌合构建体的非功能性突变GIRK-1通道 含有已知结合来自其他系统的G蛋白的结构元件。 这些调查将提供证据，以帮助确定结构 参与G蛋白和这类重要的 内向整流钾通道 从更广泛的意义上说， 在这些类型的实验中可实现的分辨率测量可提供 对我们理解G蛋白如何与 其他类型的蛋白质，他们是已知的耦合。 分子 关于G蛋白相互作用和这类离子的信息 通道对于理解兴奋性的分子基础很重要， 蜂窝信令和信息传递。

DESCRIPTION: A molecular mechanism for parasympathetic nervous modification of cellular excitability involves activation of a muscarinic-gated atrial potassium channel known as GIRK-1. The activation pathway appears to involve a direct interaction of G proteins with the ion channel subunit. The location and the stoichiometry of this interaction remains unknown. Recent evidence proposes that the GIRK-1 subunit coassembles with another inwardly rectifying potassium channel subunit known as CIR. Amino acid deletions in the C-terminus of GIRK render the molecule insensitive to G protein activation. The goal of the experiments in this proposal is to identify the sites in the GIRK protein which affect activation coupling of G proteins to the channel without affecting channel assembly. Channel subunits will be tagged with an epitope to allow specific antibody binding and to permit identification. Experiments will be done to mutate regions of the channel that are predicted to be involved in protein-protein interactions. Mutagenesis experiments will be done to determine whether the channels are functional or not and, if not, it will be determined whether the mutant channels are synthesized and targeted to the plasma membrane by using the epitope tagged subunits. Secondly, it will be determined whether these regions that are critical to the function of the GIRK channel are also G protein binding sites. The third strategy will be to try and revert nonfunctional mutant GIRK-1 channels by creating a chimeric construct containing structural elements known to bind G proteins from other systems. These investigations will provide evidence to help identify the structural elements involved in coupling between G proteins and this important class of inwardly rectifying potassium channels. In a broader sense, the high resolution measurements achievable in these types of experiments may provide important insight into our understanding of how G proteins interact with other types of proteins to which they are known to couple. Molecular information about G protein interactions and about this class of ion channels is important for understanding the molecular basis of excitability, cellular signalling and information transfer.