REGULATION OF SINGLE CALCIUM RELEASE CHANNELS IN HEART

在心脏中，细胞内[Ca~(2+)]的快速和瞬时变化是细胞功能的基础。肌浆网通过Ryanodine受体(RyR)通道快速释放钙离子控制收缩。然而，RyR通道并不是存在于分离的心肌细胞内的唯一钙释放通道。像所有哺乳动物细胞一样，心室肌细胞含有大量的IP3R。IP3R是与RyR通道同源的细胞内钙释放通道。RyR和IP3R具有相似的渗透特性，但受钙离子和IP3的调节不同。RyR和IP3R介导的细胞内钙信号通路必须在同一细胞内同时运行。目前尚不清楚这些通路如何同时运行并保持各自的信号保真度。在该方案中，将在平面双层中定义心脏RyR和IP3R钙释放通道的单通道功能。将测量单个RyR和IP3R通道对快速、校准的钙离子和IP3刺激的反应动力学。闪光光解将产生快速的配体刺激。还将研究磷酸化影响这两种类型通道的不同调节的可能性。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。这代表了理解心脏局部细胞内钙信号的复杂性的重要一步(即定义/对比RyR和IP3R调节机制)。目的1：确定IP3R通道在分离的心室肌细胞中的位置、鉴定和单通道功能。目标2：确定“开启”和“关闭”单一RyR和IP3R释放通道的机制的同一性和动力学。目标3：确定依赖于PKA和PKC的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.