Regulation of Kv4 channels by Ca2+ binding proteins

描述(由申请人提供)：心脏瞬间外向电流在动作电位期间对细胞膜的再极化很重要。因此，调节这一电流的因素可以对兴奋性和心律失常的发生产生深远的影响。这种电流的分子成分是Kv4亚基。早期的工作表明，存在调节Kv4通道表达水平和动力学的辅助亚基。最近描述的KChIP蛋白实现了这种作用。我们发现了另一种在人类心脏中表达的远亲蛋白--频率蛋白(同属钙结合蛋白家族)，它是一种额外的Kv4辅助蛋白。Frequenin与Kv4蛋白共沉淀和共定位，增加Kv4膜转运，减缓Kv4失活过程。虽然最初被描述为主要在神经元中表达，但我们发现在小鼠心脏中表达水平很高。我们推测KChIP2(KChIP在心脏中表达的原型)和频率蛋白是心脏Ito表达和功能的重要调节因子。在一个多学科的方法中，我们将从三个具体目标来研究这一假说。首先，我们将通过检测Ito对钙的敏感性，以及旨在干扰这些蛋白功能和表达的干预效果，来研究KChIP2和频率蛋白对Ito(和其他膜电流)的作用。我们的初步数据表明，频率蛋白和KChIP可能存在相互作用。这将通过免疫共沉淀和电生理方法进行有针对性的研究。在具体目标3中，我们将研究频率蛋白影响Kv4电流的机制，假设频率蛋白对Kv4转运的部分影响是通过磷酸肌醇途径的关键调节因子PI4Kβ的相互作用和刺激介导的。我们将使用生化、运输、分子生物学和电生理等方法对此进行研究。这些实验的完成对于确定一种新的辅助蛋白在重要的心脏复极K+电流功能上的作用将是非常有价值的。所获得的信息在未来可能对合理设计新型抗心律失常药物具有无价价值。

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.