G PROTEIN COUPLED ION CHANNELS IN HEART RATE REGULATION

心脏病仍然是全球范围内发病率和死亡率的主要原因。 美国老年人口。心率下降和发病率 心律失常随着年龄的增长而增加。然而，心率的分子基础 控制权仍不明朗。超极化激活电流I(F) 和T型钙电流I(Ca-T)是两个重要的因素 舒张期去极化，它设定心脏的频率和节律 起搏器。斑马鱼(心脏发育的一种新模型)的突变 导致基础心率减慢的基因已经被证明已经改变 I(F)。G蛋白偶联介导的心率神经调控 感受器。交感神经(通过β-肾上腺素能受体传递信号 GS)和副交感神经(通过M2-M受体传递信号 百日咳毒素敏感的GI/GO对心脏产生相反的作用 功能。我们已经培育出了三种基因敲除的细胞系和动物 Gi/Goα亚基在心脏α(I2)α(I3)和 Alpha(0)。我们建议使用这些敲除来理解信号 转导途径及其调控的分子基础 两个离子通道，并利用这一知识来理解 老化的心脏。结合我最近获得的经验 在电生理学和生物物理学方面有额外的经验 分子生物学，我将在这个奖项期间获得，我将是 在独特的位置研究G蛋白调节的机制 从细胞电生理事件到它们的起搏活动 功能后果。 这项提议的具体目标是：目标1：确定离子 肾上腺素能和胆碱能调控舒张期去极化的基础 在起搏器活动中。目标2：确定 GI/GO异三聚体蛋白介导I(F)的胆碱能调节 和I(Ca-T)，以及调控是直接补丁限制还是 通过一条间接途径。目标3：确定信号 介导离子通道调控的信号转导通路：3A。 确定一氧化氮是否介导肾上腺素能和胆碱能 离子通道和心率的调节。3B。以确定G- 蛋白质信号是由β-伽马或α亚基介导的。目标4：实现 确定阿尔法亚单位基因失活对底物的影响 心率与年龄的关系，并将这些变化与G- 使用WT和基因敲除小鼠的蛋白质水平。 这些实验的结果将增加我们对正常 信号转导途径与G蛋白改变的影响 心率调节随年龄增长的表达。

Heart disease remains a major cause of morbidity and mortality in the U.S. older population. Heart rate declines and the incidence arrhythmias increase with age. Yet, the molecular basis for heart rate control remains unclear. The hyperpolarization activated current I(f) and T-type calcium current I(Ca-T) are two important contributors to the diastolic depolarization which sets the rate and rhythm of the cardiac pacemaking. In zebrafish (a ne model of cardiac development) mutations which result in slow basal heart rate have been shown to have altered I(f). The neural control of heart rate is mediated by G-protein coupled receptors. Sympathetic (via beta-adrenergic receptors signaling through Gs) and parasympathetic (via m2-muscarinic receptors that signal through pertussis toxin sensitive Gi/Go) produce opposing effects on cardiac function. We have produced knockout cell lines and animals of the three Gi/Go alpha subunits expressed in heart alpha(i2) alpha(i3) and alpha(0). We propose to use these knockouts to understand the signal transduction pathways and the molecular basis of the control of these two ion channels and to use that knowledge to understand the effects in the aging heart. Combining the experience that I have recently obtained in electrophysiology and biophysics with the additional experience in molecular biology that I would gain during this award period, I will be in an unique position to study the mechanism of G-protein regulation of pacemaking activity from cellular electrophysiological events to their functional consequences. The specific aims for this proposal are: Aim 1: To determine the ionic basis for adrenergic and cholinergic control of diastolic depolarization in pacemaker activity. Aim 2: To determine which Gi/Go heterotrimeric protein mediates the cholinergic regulation of I(f) and I(Ca-T), and whether the regulation is direct patch limited or through an indirect pathway. Aim 3: To determine the signal transduction pathway mediating control of ion channels: 3A. To determine whether nitric oxide mediates adrenergic and cholinergic regulation of ion channels and heart rate. 3B. To determine if the G- protein signal is mediated by beta-gamma or alpha subunits. Aim 4: To determine the effect of the alpha subunit gene inactivations on basal heart rate with aging and correlate these changes with changes in G- protein levels using WT and knockout mice. Results from these experiments will add to our knowledge on the normal signal transduction pathways and the impact of altered G protein expression on heart rate regulation with aging.