CONTROL OF CALCIUM RELEASE IN CARDIAC MYOCYTES

心肌细胞钙释放的控制

• 批准号: 2415634
• 负责人: JAMES SK SHAM
• 金额: $ 11.74万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2415634
• 关键词: action potentials; biological signal transduction; calcium channel; chickens; guinea pigs; hamsters; heart cell; heart contraction; laboratory rat; muscle cells; receptor coupling; ryanodine; sarcoplasmic reticulum; sodium channel; species difference; stoichiometry; voltage /patch clamp

In cardiac myocytes, there are multiple Ca2+ entry pathways which may participate in triggering Ca2+ release from the sarcoplasmic reticulum (SR). Since the spatial distribution, kinetics of Ca2+ transport, and the micro-environment for various Ca2+ transporters are not the same, the efficacies and/or the mechanisms of these Ca2+ entry pathways in activating Ca2+ release can be very different. Our objective is to investigate the cellular mechanisms of controlling Ca2+ release by L-type Ca2+ channel, T-type Ca2+ channel, Na+-Ca2+ exchanger, and Na+ channel. Based on previous studies and our preliminary data, we proposed the following hypotheses. First, the characteristics Ca2+ release induced by Ca2+ current (I[ca]) is determined by the functional coupling of L-type Ca2+ channels and Ca2+ release channels (ryanodine receptors) in the junctional SR, and the uncoupling of these channels in the corbular SR. Second, Na+-Ca2+ exchanger, T-type Ca2+ channels, and Na+ channel activate Ca2+ release via mechanisms/pathways different from I[ca]-induced Ca2+ release, and their efficacies varies among animal species depend on the availability, distribution, and activity of the ion channel or transporter. To test the first hypothesis, we will (1) determine whether Ca2+ channels and ryanodine receptors are functionally coupled, and if so, whether such coupling is essential for the I[Ca]-induced fast Ca2+ release, and (2) determine whether I[Ca] can activate regenerative (partial or complete) Ca2+ release, and if so, whether such regenerative Ca2+ release involves corbular SR. To test the second hypothesis, we will (1) determine whether the Na+-Ca2+ exchanger mediated Ca2+ release is similar to the I[ca]-induced-Ca2+ release or the Ca2+-overload induced spontaneous oscillation, (2) examine the Na+-Ca2+ exchange mediated Ca2+ release and the possible Ca2+ release activated indirectly by Na+ influx and accumulation in animal species with higher exchanger activities, and (3) determine whether the transient Ca2+ entry via the T-type Ca2+ channel activates Ca2+ release, and if so, what is the difference between this T- type Ca2+ channel activated Ca2+ release and those activated by L-type Ca2+ channel and Na+-Ca2+ exchanger. In the proposed experiments, a combination of patch-clamp and Indo-1 fluorescence techniques will be applied to single isolated ventricular myocytes to monitor simultaneously the trans-membrane currents, and intracellular Ca2+ concentration. This project will provide important information on the control mechanisms of cardiac excitation-contraction coupling, and improve our understanding on Ca2+ signalling and processing in cardiac myocytes.

在心肌细胞中，存在多种 Ca2+ 进入途径，这些途径可能 参与触发肌浆网 Ca2+ 释放 （SR）。由于 Ca2+ 传输的空间分布、动力学以及 各种Ca2+转运蛋白的微环境不一样， 这些 Ca2+ 进入途径的功效和/或机制 激活 Ca2+ 释放可能会有很大不同。我们的目标是 研究L-型控制Ca2+释放的细胞机制 Ca2+通道、T型Ca2+通道、Na+-Ca2+交换器、Na+通道。 根据之前的研究和我们的初步数据，我们提出了 以下假设。 一、Ca2+释放的特点 Ca2+ 电流 (I[ca]) 由 L 型的功能耦合决定 Ca2+ 通道和 Ca2+ 释放通道（兰尼碱受体） 连接 SR，以及球状 SR 中这些通道的解偶联。 二、Na+-Ca2+交换器、T型Ca2+通道、Na+通道激活 Ca2+ 通过与 I[ca] 诱导的 Ca2+ 不同的机制/途径释放 释放，其功效因动物种类而异，取决于 离子通道的可用性、分布和活性或 运输者。为了检验第一个假设，我们将 (1) 确定是否 Ca2+ 通道和兰尼碱受体在功能上耦合，如果是这样， 这种耦合对于 I[Ca] 诱导的快速 Ca2+ 是否是必需的 释放，（2）确定I[Ca]是否可以激活再生 （部分或全部）Ca2+释放，如果是，是否具有再生性 Ca2+ 释放涉及球粒 SR。 为了检验第二个假设，我们将 (1)确定Na+-Ca2+交换器介导的Ca2+释放是否是 类似于 I[ca] 诱导的 Ca2+ 释放或 Ca2+ 超载诱导 自发振荡，(2) 检查 Na+-Ca2+ 交换介导的 Ca2+ 释放和可能的 Ca2+ 释放由 Na+ 流入间接激活 和在具有较高交换活性的动物物种中的积累，以及 (3)判断瞬时Ca2+是否通过T型Ca2+通道进入 激活Ca2+释放，如果是的话，这个T-有什么区别 型Ca2+通道激活的Ca2+释放和L型激活的Ca2+释放 Ca2+ 通道和 Na+-Ca2+ 交换器。在提议的实验中， 膜片钳和 Indo-1 荧光技术的结合 应用于单个分离的心室肌细胞以同时监测 跨膜电流和细胞内 Ca2+ 浓度。这 项目将提供有关控制机制的重要信息 心脏兴奋-收缩耦合，并提高我们对心脏兴奋-收缩耦合的理解 心肌细胞中的 Ca2+ 信号传导和处理。