REGULATION OF SINGLE CALCIUM RELEASE CHANNELS IN HEART

心脏单一钙释放通道的调节

• 批准号: 6499053
• 负责人: Michael Fill
• 金额: $ 25.63万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-14 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6499053
• 关键词: animal tissue; calcium channel; calcium flux; cardiac myocytes; chemical kinetics; flash photolysis; heart metabolism; inositol phosphates; phosphorylation; protein kinase A; protein kinase C; protein localization; protein structure function; receptor binding; receptor expression; sarcoplasmic reticulum

In heart, rapid and transient changes in intracellular [Ca2+] are fundamental to cell function. Rapid Ca2+ release from the sarcoplasmic reticulum through ryanodine receptor (RyR) channels controls contractility. The RyR channel, however, is not the only intracellular Ca2+ release channel present in isolated heart cells. Like all mammalian cells, ventricular cardiac myocytes contain a significant number of IP3Rs. The IP3Rs are intracellular Ca2+ release channels that are homologous to the RyR channel. The RyR and IP3R have similar permeation properties but are differentially regulated by Ca2+ and IP3. The RyR and IP3R mediated intracellular Ca2+ signaling pathways must operate simultaneously in the same cell. How these pathways can operate simultaneous and maintain their individual signal fidelity is not yet clear. In this proposal, single channel function of cardiac RyR and IP3R Ca2+ release channels will be defined in planar bilayers. The kinetics of single RyR and IP3R channels as they respond to fast, calibrated Ca2+ and IP3 stimuli will measured. Fast ligand stimuli will be generated by flash photolysis. The possibility that phosphorylation impacts the differential regulation of the two types of channels will also be investigated. The overall goal is to establish key determinants of cardiac RyR and IP3R function at the single channel level. This represents an important step (i.e. defining/contrasting RyR and IP3R regulatory mechanisms) in understanding the complexity of local intracellular Ca2+ signaling in the heart. Aim number1: Determine the location, identify and single channel function of IP3R channels in isolated ventricular myocytes. Aim number 2: Define the identity and kinetics of the mechanisms that "turn-on" and "turn-off" single RyR and IP3R release channels. Aim number 3: Determine how PKA and PKC dependent phosphorylation impacts function of single RyR and IP3R channels.

在心脏中，细胞内 [Ca2+] 的快速且短暂的变化是细胞功能的基础。 Ca2+ 通过兰尼碱受体 (RyR) 通道从肌浆网快速释放，控制收缩力。 然而，RyR 通道并不是离体心脏细胞中唯一存在的细胞内 Ca2+ 释放通道。 与所有哺乳动物细胞一样，心室心肌细胞含有大量 IP3R。 IP3R 是细胞内 Ca2+ 释放通道，与 RyR 通道同源。 RyR 和 IP3R 具有相似的渗透特性，但受 Ca2+ 和 IP3 的调节不同。 RyR 和 IP3R 介导的细胞内 Ca2+ 信号通路必须在同一细胞中同时运行。 这些通路如何同时运行并保持各自的信号保真度尚不清楚。在该提案中，心脏 RyR 和 IP3R Ca2+ 释放通道的单通道功能将在平面双层中定义。 将测量单个 RyR 和 IP3R 通道响应快速、校准的 Ca2+ 和 IP3 刺激时的动力学。 快速光解作用将产生快速配体刺激。 还将研究磷酸化影响两种类型通道的差异调节的可能性。 总体目标是在单通道水平上建立心脏 RyR 和 IP3R 功能的关键决定因素。 这代表了理解心脏局部细胞内 Ca2+ 信号传导复杂性的重要一步（即定义/对比 RyR 和 IP3R 调节机制）。 目标 1：确定离体心室肌细胞中 IP3R 通道的位置、识别和单通道功能。 目标 2：定义“打开”和“关闭”单个 RyR 和 IP3R 释放通道的机制的特性和动力学。 目标 3：确定 PKA 和 PKC 依赖性磷酸化如何影响单个 RyR 和 IP3R 通道的功能。