Molecular Determinants of Function in Kir2.x channels

描述（由申请人提供）：内向整流电流（IK 1）在心脏细胞的兴奋性中起作用。人类通道蛋白（Kir2.1）的基因（KCNJ 2）之一的功能缺失导致安德森综合征，其特征在于畸形特征、瘫痪和心律失常。离子通道的性质取决于其三维结构以及与辅助蛋白的相互作用。关于与Kir2.x通道直接相关的蛋白质或这些蛋白质如何改变心脏中的通道功能的信息很少。靶向、聚集和调节离子通道的支架蛋白（SAP 97、Veli和CASK）已被证明直接结合或与心脏中的Kir2.x通道共定位，其含义在很大程度上尚不清楚。我们的目标是确定参与Kir2.x通道功能调节的蛋白质-蛋白质相互作用。我们的主要假设是SAP 97、Veli和CASK与Kir2.x通道的相互作用导致心脏中通道生物物理、调节、表达和亚细胞靶向的改变。我们将使用包括电生理学、表面等离子体共振、生物化学和分子生物学技术在内的方法的组合，并在HEK 293细胞和分离的心肌细胞中进行我们的研究。具体目标：1）研究Kir2.x通道与心脏PDZ结构域蛋白共表达后生物物理性质变化的分子基础。2)研究Kir2.x通道与PDZ结构域蛋白的相互作用对PKA磷酸化通道的影响。3)确定PDZ结构域蛋白对心脏和非心脏细胞中Kir2.x通道表达、定位和功能的作用。这些研究应导致一个更好地了解的分子特性，调节经典的心脏内向整流电流的功能。

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.