Ryanodine Receptor Calcium Fluxes

Ryanodine 受体钙通量

基本信息

批准号：
7185785
负责人：
JOHN L SUTKO
金额：
$ 33.94万
依托单位：
UNIVERSITY OF NEVADA RENO
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-03-05 至 2010-02-28
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：钙火花的成像提供了一种用于研究完整肌肉细胞中核质网（SR）储存钙释放的方法。提取有关Ryanodine受体（RYR）SR钙释放通道的调节和功能的最大信息，要求精确量化钙释放通量。由于对火花成像的细胞环境的复杂性质以及操纵完整细胞内的单个独立因素的困难，这种定量已被证明很困难。我们的目标是针对此问题实施一种新的自下而上的方法。在完整细胞中针对成像火花进行优化的光学系统将用于在平面脂质双层的受控且相对简单的条件下通过单个RYR通道获得简单的钙染料信号，以通过单RYR通道获得已知的钙通量。通过细胞因子（例如钙扩散和通过缓冲液结合）对影响的直接实验评估的结果将与基本的双层钙信号结合使用，以构建到更为复杂的火花波形。为了实施这种方法，我们开发了一种能够同时测量钙染料荧光和单RYR通道电流的光学双层系统。已经设计了将RYR通道固定在双层中的RYR通道的连续记录与单个通道电流相关的光信号的方法。此外，电压步骤协议，其中RYR通道电流仅取决于施加的跨双层电压（已知），并且已经开发，实现和测试了顺式和反式溶液中的离子浓度（已知）。拟议的研究也将产生新的见解。不仅可以通过直接测量来解剖火花的钙通量细节，而且还将如何影响各个细胞因子。此外，将在定性和定量上定义火花波形的属性与RYR通道函数的特定方面之间的关系。我们的方法是对当前建模模拟方法的实验补充，并将提供模型参数和结论的独立验证。我们将凭经验测量许多必须使用当前方法估算的因素。