Molecular Determinants of Function in Kir2.x channels

描述（由申请人提供）：向内整流电流（IK1）在心脏细胞的兴奋性特性中起作用。人类通道蛋白（Kir2.1）的其中一个基因（KCNJ2）的功能丧失会导致安徒生综合征，其特征是畸形、瘫痪和心律失常。离子通道的性质取决于它们的三维结构以及它们与辅助蛋白的相互作用。关于与Kir2直接相关的蛋白质的信息很少。X通道，或者这些蛋白质如何改变心脏中的通道功能。靶向、聚集和调节离子通道的支架蛋白（SAP97、Veli和CASK）已被证明直接结合或与Kir2共定位。心脏中的X通道，其含义在很大程度上是未知的。我们的目标是确定参与Kir2调控的蛋白-蛋白相互作用。X通道函数。我们的主要假设是SAP97， Veli和CASK与Kir2相互作用。X通道导致心脏通道生物物理、调控、表达和亚细胞靶向的改变。我们将结合电生理学、表面等离子体共振、生化和分子生物学技术，在HEK293细胞和分离的心肌细胞中进行我们的研究。具体目的：1)研究Kir2生物物理性质变化的分子基础。x通道与心脏PDZ结构域蛋白共表达。2)研究Kir2之间相互作用的影响。x通道和PDZ结构域蛋白对PKA磷酸化通道的影响。3)确定PDZ结构域蛋白在Kir2中的作用。X通道在心脏和非心脏细胞中的表达、定位和功能。这些研究将有助于更好地理解调节经典心脏内向整流电流功能的分子特性。

DESCRIPTION (provided by applicant): The inward rectifier current (IK1) plays a role in the excitability properties of the cardiac cell. Loss of function in one of the genes (KCNJ2) underlying the channel protein (Kir2.1) in humans leads to Andersen's syndrome, characterized by dysmorphic features, paralysis and cardiac arrhythmias. Properties of ion channels depend on their three dimensional structure as well as by their interactions with accessory proteins. There is little information available on proteins that directly associate with Kir2.x channels, or how these proteins modify channel function in the heart. The scaffolding proteins, (SAP97, Veli and CASK) that target, cluster and regulate ion channels, have been shown to directly bind or are co localized with Kir2.x channels in the heart, with implications that are largely unknown. Our objective is to determine the protein-protein interactions involved in the regulation of Kir2.x channel function. Our main hypothesis is that SAP97, Veli and CASK interactions with Kir2.x channels results in modification of channel biophysics, regulation, expression and subcellular targeting in the heart. We will use a combination of approaches including electrophysiological, surface plasmon resonance, biochemical and molecular biological techniques and carry out our studies in HEK293 cells and in isolated cardiac myocytes. Specific Aims: 1) To investigate the molecular basis of changes in the biophysical properties of Kir2.x channels following co-expression with cardiac PDZ domain proteins. 2) To study the impact of interactions between Kir2.x channels and PDZ domain proteins on channel phosphorylation by PKA. 3) To determine the role of PDZ domain proteins on Kir2.x channel expression, localization and function in cardiac and non-cardiac cells. These studies should lead to a better understanding of the molecular properties that modulate the function of the classical cardiac inward rectifier current.