REGULATION OF INWARD RECTIFIER POTASSIUM CHANNELS

我们的长期目标是了解 内向整流K通道(IRK)。在《易激动》中，反感很重要 组织，如神经和肌肉，我们特别强调心脏 肌肉。图中IRKS调节静息膜电位和终末 心脏复极时相。因此，它们的作用是巨大的 在评价和发现心脏抗心律失常药物方面的重要性 尤其是三类抗心律失常药。我们考察了三个主要假设： 第一，特定的天然产生的细胞质多胺(PAS) 调节心肌细胞中的irk功能；第二，是否存在 IRKS的拓扑模型是正确的；第三，人类心脏 IRK，hIRK是I-Kr的主要成分，是I-K延迟的快速部分 心肌细胞的整流钾电流。具体目标是：1) 将一起结合的镁离子和PAS的C末端结合位点本地化 赋予内向整流的独特性质；2)比较 PA阻滞剂的有效价态和时间依赖性 IRKS的“内在”门控；3)检测PA水平与 IRK在心肌细胞中的活性；4)IRK的EST特异性拓扑模型 使用糖基化位点插入突变；以及5)鉴定 抗心律失常药物多非利特在hIRK和HIRK上的结合位点 克隆此通道的辅助子单位修饰符。对于目标1和目标2以及 目标5的第一部分，研究设计采用了诱变的方法， 异源表达和电生理的迭代方式。为 目的2化学干扰PA代谢用于控制PA 检测IRK功能时的心肌细胞水平 电生理学的。对于Aim 4，一种基因工程的irk蛋白是 在杆状病毒-Sf9系统中过表达，免疫纯化并检测 目前预测的胞外位点的N-糖基化 拓扑模型。使用膜片钳进行功能测试 同时。对于目标5的第二部分，各种候选人 我们已经克隆的修饰亚基将被测试它们的能力 使hirk更接近于i-kr。

The long-term objectives are to understand the function and structure of inward rectifier K channels (IRKs). IRKs are important in excitable tissues such as nerve and muscle and our particular emphasis is on heart muscle. Here IRKs regulate resting membrane potential and the terminal phase of cardiac repolarization. Consequently their function is of great importance in evaluation and discovery of cardiac antiarrhythmic drugs, especially Class III antiarrhythmics. Three major hypotheses are examined: first whether specific naturally occurring cytoplasmic polyamines (PAs) regulate IRK function in cardiomyocytes; second whether the present topological model of IRKs is correct; and third whether a human cardiac IRK, hIRK is a major component of I-Kr, the rapid part of I-K the delayed rectifier K+ current of cardiomyocytes. The specific aims are to: 1) localize C-terminus binding sites for both Mg2+ and PAs which together confer the unique property of inward rectification; 2) compare the effective valence and time-dependence of PA block with so-called "intrinsic" gating of IRKs; 3) test the relationship between PA levels and IRK activity in cardiomyocytes; 4) est specific topological models of IRKs using glycosylation site insertion mutagenesis; and 5) identify the binding site for the Class Ill antiarrhythmic dofetilide, on hIRK and clone accessory subunit modifiers of this channel. For Aims 1 and 2 and the first part of Aim 5, the research design uses methods of mutagenesis, heterologous expression and electrophysiology in an iterative manner. For Aim 2 chemical interruption of PA metabolism is used to manipulate PA levels of cardiomyocytes while testing IRK function electrophysiologically. For Aim 4 a genetically engineered IRK protein is overexpressed in a baculovirus-Sf9 system, immunopurified and tested for N-glycosylation at sites predicted to be extracellular by present topological models. Functional tests using patch clamp are performed simultaneously. For the second part of Aim 5, a variety of candidate modifier subunits that we have cloned will be tested for their ability to make hIRK more closely mimic I-Kr.