REGULATION OF SINGLE CALCIUM RELEASE CHANNELS IN HEART

在心脏中，细胞内[Ca 2 +]的快速和短暂变化是细胞功能的基础。 肌浆网通过Ryanodine受体（RyR）通道快速释放Ca 2+控制收缩性。 然而，RyR通道并不是分离的心脏细胞中唯一的细胞内Ca 2+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的IP 3R。 IP 3R是与RyR通道同源的细胞内Ca 2+释放通道。 RyR和IP 3R具有相似的渗透特性，但受Ca 2+和IP 3的差异调节。 RyR和IP 3R介导的细胞内Ca 2+信号通路必须在同一细胞中同时起作用。 这些通路如何同时工作并保持各自的信号保真度尚不清楚。在这个提议中，心脏RyR和IP 3R Ca 2+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IP 3R通道响应快速校准的Ca 2+和IP 3刺激的动力学。 快速配体刺激将通过闪光光解产生。 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IP 3R功能的关键决定因素。 这代表了理解心脏中局部细胞内Ca 2+信号传导的复杂性的重要一步（即定义/对比RyR和IP 3R调节机制）。 目的1：确定分离的心室肌细胞IP 3R通道的定位、鉴定和单通道功能。 目标2：定义“打开”和“关闭”单个RyR和IP 3R释放通道的机制的特性和动力学。 目的3：确定PKA和PKC依赖性磷酸化如何影响单个RyR和IP 3R通道的功能。

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.