Ryanodine Receptor Calcium Fluxes

描述（由申请人提供）：钙火花成像提供了一种研究完整肌细胞中肌浆网（SR）储存的钙释放的方法。提取最大信息的调控和功能的Ryanodine受体（RyR）SR钙释放通道需要的钙释放流量下的火花被准确地量化。由于火花被成像的细胞环境的复杂性质，以及在完整细胞内操纵单个独立因子的困难，这种量化已被证明是困难的。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。一个光学系统，优化成像火花在完整的细胞，将被用来获得简单的钙染料信号，已知的钙通量通过单一的RyR通道下的控制和相对简单的条件下的平面脂质双层。对细胞因素（如钙扩散和缓冲剂结合）影响的直接实验评估结果将与基本钙双层信号结合，以形成更复杂的火花波形。为了实现这种方法，我们已经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。已经设计了用于将RyR通道固定在双层中以连续记录与单通道电流相关的光信号的方法。此外，电压阶跃协议，其中RyR通道电流仅取决于所施加的反式双层电压（这是已知的）和离子浓度的顺式和反式解决方案（这是已知的）已经开发，实施和测试。 拟议的研究也将产生新的见解。不仅将获得火花背后的钙流量的细节，而且还将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通道功能的具体方面之间的关系将定性和定量定义。我们的方法是一个实验的补充，目前的建模仿真方法，并将提供独立的验证模型参数和结论。我们将根据经验测量许多必须使用当前方法估计的因素。

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.