MOLECULAR MECHANISM OF CARDIAC K+ CHANNEL INACTIVATION

心脏K通道失活的分子机制

• 批准号: 2332422
• 负责人: LESLIE K SPRUNGER
• 金额: $ 8.74万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-02-01 至 1999-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2332422
• 关键词: Xenopus; Xenopus oocyte; action potentials; animal genetic material tag; antiarrhythmic agent; complementary DNA; drug screening /evaluation; electrophysiology; gel electrophoresis; genetic library; heart cell; heart pharmacology; human genetic material tag; molecular cloning; nucleic acid sequence; potassium channel; protein sequence; protein structure function; site directed mutagenesis; voltage /patch clamp

The delayed reciter K channel (lKr) in the heart plays an essential role in the membrane repolarization during the plateau phase of the cardiac action potential (AP). The objective of this proposal is to characterize the structure of the human IKr channel and to identify regions of the channel that are responsible for the properties of rapid inactivation and rectification. We propose that the human IKr channel will show enough sequence homology to the RK family of delayed rectifier K channels in rat heart and brain that cDNAs from members of this family can be used to screen a human heart cDNA library for IKr. We will test this hypothesis by cloning cDNAs that hybridize to the rat heart delayed rectifier K channel and expressing them in Xenopus oocytes. The rapid inactivation and rectification properties of this channel are primary determinants of the amount of current which can flow through the channel, and hence, the degree to which this current affects the shape and duration of the cardiac AP. We also propose that inactivation and rectification can be selectively altered by certain class III antiarrhythmic drugs. This hypothesis will be tested by examining the biophysical properties of the clone channels using two-electrode voltage clamp and macro-patch clamp techniques to determine which clones possess physiological and pharmacological properties that correspond to IKr. Clones that produce IKr channels will be sequenced and subjected to site-directed mutagenesis experiments to identify critical regions of the protein that are involved in rapid inactivation and rectification. Normal and altered clones will be characterized in the presence and absence of specific antiarrhythmic agents to determine their effect on inactivation and rectification. Information from these experiments may be useful in the design of new antiarrhythmic drugs with greater efficacy and improved safe.

心脏中延迟的朗诵k通道（LKR）起着至关重要的作用 在心脏高原阶段的膜复极中 动作电位（AP）。 该提议的目的是表征 人IKR渠道的结构，并确定 负责快速失活的特性的通道和 纠正。 我们建议人类IKR频道足够显示 与大鼠延迟整流器K通道的RK家族的序列同源性 这个家庭成员的cdnas的心脏和大脑可用于 筛选IKR的人心脏cDNA库。 我们将检验这个假设 通过克隆与大鼠心脏延迟整流器k杂交的cDNA 通道并在爪蟾卵母细胞中表达它们。 快速失活 该通道的纠正特性是 可以流过通道的电流量，因此 该电流影响的程度 心AP。 我们还建议灭活和纠正可以是 通过某些III类抗心律失常药物有选择地改变。 这 假设将通过检查的生物物理特性来检验 使用两电极电压夹和宏观点夹的克隆通道 确定哪些克隆具有生理和 与IKR相对应的药理特性。 产生的克隆 IKR通道将进行测序并进行现场定向诱变 实验以鉴定涉及蛋白质的关键区域 在快速失活和纠正中。 正常和改变的克隆将 在存在和不存在特定抗心律失常的情况下进行表征 确定其对失活和恢复的影响的代理。 这些实验的信息可能在新的设计中有用 具有更大疗效并改善安全性的抗心律失常药物。