Regulation of Kv4 channels by Ca2+ binding proteins

描述（由申请人提供）：心脏瞬时外向电流对于动作电位期间细胞膜的复极化非常重要。因此，调节这种电流的因素可以对兴奋性和神经元生成产生深远的影响。该电流的分子组分是Kv4亚基。早期的工作表明存在辅助亚基，调节Kv4通道的表达水平和动力学。最近描述的KChIP蛋白实现了这样的作用。我们鉴定了另一个远亲蛋白，frequenin（属于同一家族的Ca2+结合蛋白），在人类心脏中表达，它作为一个额外的Kv4辅助蛋白。Frequenin与Kv4蛋白共免疫沉淀和共定位，增加Kv4膜运输并减缓Kv4失活过程。虽然最初描述为主要在神经元中表达，但我们发现在小鼠心脏中的表达水平很高。我们假设KChIP 2（心脏表达的原型KChIP）和frequenin是心脏Ito表达和功能的重要调节因子。在多学科的方法中，我们将在三个具体目标中研究这一假设。首先，我们将研究KChIP2和frequenin对Ito（和其他膜电流）在小鼠心肌细胞中的作用，通过检查Ito的Ca2+敏感性，以及旨在破坏这些蛋白质的功能和表达的干预的影响。我们的初步数据表明，frequenin和KChIPs可能相互作用。这将在特定目标中使用免疫共沉淀和电生理学测定进行研究。在具体目标3中，我们将研究一种拟议的机制，通过这种机制，frequenin影响Kv4电流，假设其对Kv4运输的部分影响是由PI4K β（磷酸肌醇途径的关键调节因子）的相互作用和刺激介导的。我们将使用生物化学，贩运，分子生物学和电生理学检测来研究这一点。这些实验的完成将是非常宝贵的，在确定一个新的辅助蛋白的功能的一个重要的心脏复极化钾电流的作用。所获得的信息在未来可能是非常宝贵的合理设计的新的抗肿瘤药物。

DESCRIPTION (provided by applicant): The cardiac transient outward current is important for repolarizing the cell membrane during the action potential. As such, factors that modulate this current can have profound effects on excitability and arrhythmogenesis. The molecular components of this current are Kv4 subunits. Early work suggested the existence of accessory subunits that modulate the expression levels and kinetics of Kv4 channels. The recently described KChIP proteins fulfill such a role. We identified another distantly related protein, frequenin (belonging to the same family of Ca2+binding proteins), expressed in human heart, which acts as an additional Kv4 accessory protein. Frequenin co-immunoprecipitates and co-localizes with Kv4 proteins, increases Kv4 membrane trafficking and slows the Kv4 inactivation process. Although initially described as expressed mainly in neurons, we find high frequenin expression levels in mouse heart. We hypothesize that KChIP2 (the prototype KChIP expressed in heart) and frequenin, are important regulators of cardiac Ito expression and function. In a multi-disciplinary approach, we will investigate this hypothesis in three Specific Aims. First, we will examine the role of KChIP2 and frequenin on Ito (and other membrane currents) in mouse cardiac myocytes by examining the Ca2+ sensitivity of Ito, as well as the effects of intervention designed to disrupt the function and expression of these proteins. Our preliminary data suggest that frequenin and KChIPs may interact. This will be investigated in Specific Aim, using co-immunoprecipitation and electrophysiological assays. In Specific Aim 3, we will investigate a proposed mechanism by which frequenin affects Kv4 currents, with the hypothesis that part of its effect on Kv4 trafficking is mediated by an interaction and stimulation of PI4Kbeta, a key regulator of the phosphoinositol pathway. We will investigate this using biochemical, trafficking, molecular biology and electrophysiological assays. The completion of these experiments will be invaluable in identifying the role of a novel accessory protein on the function of an important cardiac repolarizing K+ current. The information gained may in future be invaluable for the rational design of novel anti-arrhythmic agents.