Molecular Components of A-type K+ Channels

描述(申请人提供)：快速瞬时A-型钾电流(IA)，如阈值以下激活神经元的体树突状A-型钾电流(ISA)和心肌细胞(ITO)的快速瞬时外向钾电流(ITO)对于大脑和心脏的正常功能是必不可少的。在病理生理条件下(例如，心脏缺血和脑中风发生期间)，这些电流的异常导致与这些疾病条件相关的异常。该项目致力于研究产生这些电流的离子通道的分子性质。它试图建立这些通道的分子组成，并阐明所识别的成分的生理意义。在阐明介导ISA和Ito的通道的分子组成方面取得了进展，并鉴定了两个关键组分，即Kv4成孔亚基和KCHIP相关蛋白。然而，在许多神经元中，ISA通道的动力学速度快于由Kv4和KChIP蛋白组成的通道。最近有证据表明，大脑中存在编码一个因子(称为KAF)的转录本，它可能是一个新的相关亚基，它加速了Kv4通道的动力学。此外，利用生化方法鉴定了一种新的Kv4通道相关蛋白(DPPX)，并获得了该蛋白与KAF活性有关的证据。本项目的目的是验证这一假设，即DPPX是许多神经元中Kv4通道的重要组成部分，并对天然A型K+通道的特性和多样性做出贡献。目的1研究DPPX在异源表达系统中对Kv4通道功能的影响。目的2利用原位杂交和免疫组织化学方法研究DPPX蛋白在脑内表达的地点和时间，以及它们与Kv4通道其他已知成分的关系。目的3利用基因打靶和反义技术，更直接地研究DPPX蛋白在神经元中的生理意义。

DESCRIPTION (provided by applicant): Fast transient A-type K+ currents (IA), such as the subthreshold-activating somato-dendritic A-type K+ currents in neurons (ISA) and the fast transient outward K+ current in cardiac ventricular myocytes (Ito) are essential for the proper functioning of the brain and the heart. During pathophysiological conditions (e.g. ischemia in the heart and during the occurrence of stroke in brain) abnormalities in these currents contribute to the abnormalities associated with these disease conditions. This project addresses the molecular nature of the ion channels responsible for the generation of these currents. It seeks to establish the molecular composition of these channels and to elucidate the physiological significance of the identified components. Progress has been made in elucidating the molecular composition of the channels mediating the ISA and the Ito, and two key components, Kv4 pore-forming subunits and KCHIP associated proteins, have been identified. However, the kinetics of ISA channels in many neurons is faster than that of channels composed of Kv4 and KCHIP proteins. Evidence has been recently obtained for the presence in brain mRNA of transcripts encoding a factor (termed KAF), probably a novel associated subunit, which accelerates the kinetics of Kv4 channels. Moreover, a novel Kv4 channel associated protein (DPPX) has been identified utilizing biochemical methods, and evidence that this protein is responsible for KAF activity has been obtained. The goal of this project is to test the hypothesis that DPPX is an important component of Kv4 channels in many neurons and contributes to the properties and diversity of native A-type K+ channels. Aim 1 will investigate the effects of DPPX on Kv4 channel function in heterologous expression systems. Aim 2 will investigate where and when DPPX proteins are expressed in brain and their relationship to the other known components of Kv4 channels utilizing in-situ hybridization and immunohistochemistry. Aim 3 will investigate more directly the physiological significance of DPPX proteins in neurons utilizing gene targeting and antisense technology.