Ryanodine Receptor Calcium Fluxes

描述(申请人提供)：钙火花成像为研究完整肌肉细胞中肌浆网(SR)的钙释放提供了一种手段。要获取有关Ryanodine受体(RyR)SR钙释放通道调节和功能的最大信息，需要准确地量化火花背后的钙释放通量。这种量化已经被证明是困难的，因为火花被成像的细胞环境的复杂性质，以及在一个完整的细胞内操纵单个独立因素的困难。我们的目标是实施一种新的、自下而上的方法来解决这个问题。一种光学系统，针对完整细胞中的火花成像进行了优化，将用于在平面脂质双层的受控和相对简单的条件下，通过单个RyR通道获得已知钙通量的简单钙染料信号。对细胞因素影响的直接实验评估结果，如钙的扩散和缓冲液的结合，将与基本的钙双层信号结合起来，形成更复杂的火花波形。为了实现这一方法，我们开发了一个光学双层系统，能够同时测量钙染料荧光和单RyR通道电流。已经设计了用于在双层中固定RyR通道以连续记录与单通道电流相关的光信号的方法。此外，RyR通道电流仅取决于施加的跨双分子层电压(已知)以及顺式和反式溶液中的离子浓度(已知)的电压阶跃方案已被开发、实施和测试。 拟议的研究还将产生新的见解。不仅可以获得火花背后的钙通量的细节，还可以通过直接测量来剖析单个细胞因素如何影响火花。此外，火花波形的属性和RyR通道功能的特定方面之间的关系将被定性和定量地定义。我们的方法是对现有建模-仿真方法的实验补充，并将提供模型参数和结论的独立验证。我们将对许多必须用当前方法估计的因素进行实证测量。

DESCRIPTION (provided by applicant): Imaging of calcium sparks has provided a means for investigating calcium release from sarcoplasmic reticulum (SR) stores in intact muscle cells. Extraction of maximal information about the regulation and function of ryanodine receptor (RyR) SR calcium release channels requires that the calcium release flux underlying a spark be accurately quantified. Such quantification has proven difficult due to the complex nature of the cellular environment where sparks are imaged, and to the difficulty of manipulating single independent factors within an intact cell. Our goal is to implement a new, bottom-up approach to this problem. An optical system, optimized for imaging sparks in intact cells, will be used to obtain simple calcium dye signals to known calcium fluxes through single RyR channels under the controlled and relatively simple conditions of a planar lipid bilayer. The results of direct experimental evaluations of influences by cellular factors, such as calcium diffusion and binding by buffers, will be combined with the basic calcium bilayer signal to build up to the more complex waveform of a spark. To implement this approach we have developed an optical bilayer system capable of simultaneous measurement of calcium dye fluorescence and single RyR channel currents. Methods for immobilizing RyR channels in bilayers for continuous recording of optical signals related to single channel currents have been devised. In addition, a voltage-step protocol where RyR channel currents depend only on the imposed trans-bilayer voltage (which is known) and the ion concentrations in the cis and trans solutions (which are known) has been developed, implemented and tested. New insights will also be generated by the proposed studies. Not only will the details of the calcium flux underlying a spark be obtained, but also how individual cellular factors influence a spark will be dissected with direct measurements. In addition, the relationships between properties of the spark waveform and specific aspects of RyR channel function will be defined both qualitatively and quantitatively. Our approach is an experimental complement to current methods for modeling-simulation and will provide independent verification of model parameters and conclusions. We will measure empirically many of the factors that must be estimated using current methods.