MUSCARINIC GATED ATRIAL K+ CHANNEL

描述：副交感神经修饰的分子机制 细胞兴奋性的变化涉及到毒碱门控心房的激活 钾通道称为GIRK-1。激活途径似乎是 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同组装 向内整流钾通道亚基，称为CIR。氨基酸 GIRK C末端的缺失使分子对G不敏感 蛋白质激活。这项提案中实验的目标是 确定GIRK蛋白中影响G活化偶联的位点 蛋白质进入通道而不影响通道组装。渠道 将用表位标记亚基，以允许特定抗体结合 并允许进行身份识别。将进行实验以突变基因区域 被预测参与蛋白质-蛋白质的通道 互动。将进行诱变实验，以确定是否存在 通道是否正常工作，如果不起作用，将确定是否 突变通道被合成并通过以下方式靶向质膜 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。第三种策略将是尝试和恢复 通过构建嵌合结构实现无功能突变体GIRK-1通道 含有已知能与其他系统的G蛋白结合的结构元件。 这些调查将提供证据，帮助识别结构 参与G蛋白和这一重要类别之间偶联的元件 向内整流钾通道。在更广泛的意义上，高 在这些类型的实验中可实现的分辨率测量可以提供 对我们理解G蛋白如何与 已知与之偶联的其他类型的蛋白质。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， 蜂窝信令和信息传输。

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.