MODULATION OF CARDIAC L TYPE CALCIUM CHANNELS

心脏 L 型钙通道的调节

基本信息

批准号：
6430017
负责人：
ROBERT L ROSENBERG
金额：
$ 23.61万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-02-20 至 2001-11-30
项目状态：
已结题

项目摘要

L-type Ca channels serve many vital functions in the heart. They sustain the plateau of the action potential and help set action potential duration and refractoriness. The influx of Ca2+ through L-type Ca channels triggers Ca2+ release from the sarcoplasmic reticulum, establishing the link between electrical activity at the cell surface and the contraction of cardiac myoctes. L-type Ca channels in nodal cells contribute to pacemaker activity and to the speed of conduction in those cells. Their activity may also play an important role in the generation of very slow conduction in ischemic myocardium, and especially at the border between normal and ischemic regions. In order to understand the basis of electrical re-entry that can occur at the ischemic border zone to trigger dangerous ventricular arrhythmias, it is crucial to understand factors that modulate Ca channels. The long-term goal of this project is to characterize the molecular mechanisms responsible for the modulation of cardiac L-type Ca channels, with special attention to the conditions that exist at the ischemic border zone. Because ischemic myocardium has a depolarized resting potential, high intracellular Ca2+, and low border zone. Because ischemic myocardium has a depolarized resting potential, high intracellular Ca2+, and low intracellular and extracellular pH, and because cardiac ischemia produces large beta-adrenergic stimulation, Specific Aim 1 focuses on the regulation of L-type Ca channels by protein kinase A under depolarized, Elevated Ca2+, and acidic pH conditions. We hypothesize that PKA stimulation sustains the activity of Ca channels the might otherwise become inactivated, leading to a abnormal slow conduction. Because beta- adrenergic release of norepinephrine is accompanied by a release of ATP, Specific Aim 2 focuses on some new observations of the effects of extracellular ATP on L-type Ca channels. Lipid metabolism is affected by ischemia, so Specific Aim 3 focuses on the regulation of L-type Ca channels by lysolipids and metabolites of arachidonic acid. Finally, Specific Aim 4 focuses on the regulation of Ca channels by nitric oxide and S-nitrosothiols. The experimental approach is to characterize the regulation of single L-type Ca channels incorporated from cardiac sarcolemma into planar lipid bilayers. We will record single channel activity of the reconstituted Ca channels, and evaluate channel conductance and unitary gating events. We control the extracellular and intracellular ionic conditions on both sides of the channels, the activity of endogenous regulatory enzymes like kinases and phosphatases, and the membrane environment. Thus, we will create conditions that mimic the ischemic border zone, and make a detailed characterization of the modulation of Ca channels there.

L 型 Ca 通道在心脏中发挥许多重要功能。他们维持动作电位的稳定状态并帮助设定动作电位持续时间和不应期。 Ca2+ 通过 L 型 Ca 通道流入触发肌浆网释放 Ca2+，建立细胞表面的电活动与收缩之间的联系心肌细胞。结节细胞中的 L 型 Ca 通道有助于起搏器活动以及这些细胞的传导速度。他们的活动也可能在非常缓慢的产生中发挥重要作用缺血心肌中的传导，特别是在心肌之间的边界处正常区域和缺血区域。为了了解基础电折返可发生在缺血边界区以触发危险的室性心律失常，了解因素至关重要调节 Ca 通道。该项目的长期目标是表征分子负责调节心脏 L 型 Ca 通道的机制，特别注意缺血边界存在的情况区。由于缺血心肌具有去极化静息电位，细胞内 Ca2+ 高，边界区低。因为心肌缺血具有去极化静息电位、高细胞内 Ca2+ 和低细胞内和细胞外的 pH 值，并且由于心脏缺血产生大量 β 肾上腺素能刺激，具体目标 1 侧重于去极化下蛋白激酶 A 对 L 型 Ca 通道的调节， Ca2+ 升高和 pH 值呈酸性条件。我们假设 PKA 刺激维持 Ca 通道的活动，否则可能变得失活，导致异常缓慢的传导。因为贝塔- 去甲肾上腺素的肾上腺素释放伴随着 ATP 的释放，具体目标 2 侧重于对影响的一些新观察 L 型 Ca 通道上的细胞外 ATP。脂质代谢受以下因素影响缺血，所以Specific Aim 3重点关注L型Ca的调节溶血脂和花生四烯酸代谢物的通道。最后，具体目标 4 侧重于一氧化氮对 Ca 通道的调节和S-亚硝基硫醇。实验方法是表征单一的调节 L 型 Ca 通道从心脏肌膜整合到平面脂质中双层。我们将记录重组 Ca 的单通道活动通道，并评估通道电导和单一门控事件。我们控制两侧细胞外和细胞内的离子条件通道的内源性调节酶的活性，例如激酶和磷酸酶以及膜环境。这样，我们将创造模拟缺血边界区的条件，并做出详细的 Ca 通道调制的表征。