REGULATION OF SINGLE CALCIUM RELEASE CHANNELS IN HEART

在心脏，细胞内[Ca2+]的快速和短暂的变化是细胞功能的基础。通过ryanodine受体（RyR）通道从肌浆网快速释放Ca2+控制收缩性。然而，在离体心脏细胞中，RyR通道并不是唯一的细胞内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的IP3Rs。IP3Rs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。在本提案中，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通道对快速校准的Ca2+和IP3刺激的反应动力学。闪光光解会产生快速配体刺激。磷酸化影响两种通道差异调节的可能性也将被研究。总体目标是在单通道水平上确定心脏RyR和IP3R功能的关键决定因素。这是理解心脏局部细胞内Ca2+信号复杂性的重要一步（即定义/对比RyR和IP3R调控机制）。目的1：确定离体心室肌细胞IP3R通道的定位、鉴定和单通道功能。目标2：定义“开启”和“关闭”单个RyR和IP3R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IP3R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.