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

描述：心脏肌浆网钙的释放 被钙激活。这有可能成为一种内在的 自我再生过程，但它是良好控制和分级相对于 激活钙的水平。调查员提出， 单个RyR信道适配可以用作负反馈机制 这抵消或稳定了 钙诱导的钙释放机制。该项目将使用单一渠道 解决两个目标的方法。第一个将检验这样的假设 单个RyR通道的适应确实有助于稳定CICR过程 在内心深处。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 心脏RyR通道重构为平面双层膜的动力学。这些 数据将被用来区分停用和较慢的监管 机制，如适应和/或失活。这些产品的特性 较慢的机制将被分析并用于测试几个现有的模型 对RyR的适应。 第二个目标将检验短暂触发钙信号较少的假设 持续时间大于1毫秒就足以激活RyR通道，并且 单个RyR通道的激活足以产生钙火花。 笼中钙的光解将被用来产生短暂的触发钙刺激 以双层形式重建的单个心脏RyR通道的邻近区域 定义有效触发Ca信号的动力学极限。要定义 钙火花的基础，钙在双层膜中通过RyR通道转运 将使用共聚焦显微镜进行成像。如何单RyR通道选通 支配火花的空间和时间特征将被定义。 基于这些数据，一种去卷积钙火花样荧光的模型 发信号并准确预测基础通道选通行为将 将在细胞中开发和测试。在最后一系列研究中， 调查员将尝试定义有效触发器的属性 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.