REGULATION OF INWARD RECTIFIER POTASSIUM CHANNELS

长期目标是了解 内向整流钾通道（IRKs）。IRK在兴奋性 神经和肌肉等组织，我们特别强调心脏 肌肉. IRKs调节静息膜电位， 心脏复极期。因此，它们的功能是巨大的。 在评价和发现心脏抗心律失常药物中的重要性， 尤其是III类抗真菌药。三个主要的假设进行审查： 首先，是否特异性天然存在的细胞质多胺（PAs） 调节心肌细胞中的IRK功能;第二， IRK的拓扑模型是正确的;第三，人类心脏是否 IRK、hIRK是I-Kr的主要成分，I-K的快速部分， 整流钾电流。具体目标是：1） 定位Mg 2+和PA的C-末端结合位点， 赋予内向矫正的独特属性; 2）比较 PA阻滞剂有效效价和时间依赖性 IRK的“内在”门控; 3）测试PA水平与 心肌细胞中IRK活性; 4）IRK的特异性拓扑模型 使用糖基化位点插入诱变;和5）鉴定 hIRK上III类抗肿瘤多非利特的结合位点， 克隆该通道的辅助亚基修饰剂。目标1和2以及 第一部分目的5，研究设计采用诱变方法， 异源表达和电生理学。为 目的2用化学方法阻断PA的代谢， 检测IRK功能时心肌细胞水平 电生理学对于目标4， 在杆状病毒-Sf 9系统中过表达，免疫纯化并检测 目前预测为细胞外位点的N-糖基化 拓扑模型使用膜片钳进行功能测试 同步关于目标5的第二部分， 我们克隆的修饰子亚基将被测试它们的能力， 使hIRK更接近模拟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.