CONTROL OF CA++- INDUCED CA++ RELEASE IN HEART

描述：心脏肌浆网释放钙， 激活Ca。这有可能成为一个内在的 自我再生的过程，但它是很好的控制和分级相对于 激活Ca的水平。研究者提出， 单个RyR信道自适应可以用作负反馈机制 它能抵消或稳定 Ca诱导的Ca释放机制。 本项目将采用单通道 解决两个目标的方法。 第一个实验将检验一个假设， 单个RyR通道的适应确实有助于稳定CICR过程 在心里。 激光闪光光解产生的游离钙的快速变化 笼状化合物将被用来定义激活和失活 重构成平面双层的心脏RyR通道的动力学。 这些 数据将用于区分停用与较慢的监管 机制，如适应和/或失活。 这些的性质 较慢的机制将被分析，并用于测试几个现有的模型 RyR适应 第二个目标将测试短暂触发Ca信号较少的假设 持续时间超过1毫秒足以激活RyR通道， 单个RyR通道的激活足以产生Ca火花。 笼状Ca的光解将用于产生短暂的触发Ca刺激， 单个心脏RyR通道附近在双层中重建， 定义有效触发Ca信号的动力学极限。 来定义 钙火花的基础是钙通过双层RyR通道的转运 将使用共聚焦显微镜成像。 单个RyR通道门控 将定义支配火花的空间和时间特征的参数。 基于这些数据，一个模型，去卷积钙火花样荧光 信号并准确预测潜在的通道门控行为， 在细胞中进行开发和测试。 在最后一系列研究中， 研究者将尝试定义有效触发物的属性 Ca信号。

DESCRIPTION: The release of Ca from the sarcoplasmic reticulum in heart is activated by Ca. This has the potential to be an inherently self-regenerating process, yet it is well controlled and graded relative to the level of activating Ca. It has been proposed by the investigator that single RyR channel adaptation may serve as a negative feedback mechanism that counters or stabilizes the intrinsic positive feedback of the Ca-induced Ca release mechanism. This project will utilize single channel approaches to address two Aims. The first will test the hypothesis that adaptation of single RyR channels does serve to stabilize the CICR process in heart. Rapid changes in free Ca generated by laser flash photolysis of caged compounds will be utilized to define the activation and deactivation kinetics of cardiac RyR channels reconstituted into planar bilayers. These data will be used to differentiate deactivation from slower regulatory mechanisms, such as adaptation and/or inactivation. The properties of these slower mechanisms will be analyzed and used to test several existing models of RyR adaptation. The second Aim will test the hypothesis that brief trigger Ca signals less than 1 msec in duration are adequate to activate the RyR channel and that activation of a single RyR channel is sufficient to produce a Ca spark. Photolysis of caged Ca will be used to generate brief trigger Ca stimuli in the vicinity of single cardiac RyR channels reconstituted in bilayers to define the kinetic limits of an effective trigger Ca signal. To define the basis of the Ca spark, Ca translocation through RyR channels in bilayers will be imaged using a confocal microscope. How single RyR channel gating governs the spatial and temporal features of the spark will be defined. Based on these data, a model that deconvolves Ca spark-like fluorescence signals and accurately predicts the underlying channel gating behavior will be developed and tested in cells. In a final series of studies, the investigator will attempt to define the properties of an effective trigger Ca signal.