Ryanodine Receptor Calcium Fluxes

Ryanodine 受体钙通量

• 批准号: 7017075
• 负责人: JOHN L SUTKO
• 金额: $ 34.96万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-05 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7017075
• 关键词: Rana; animal tissue; bioimaging /biomedical imaging; calcium channel; calcium flux; calcium indicator; cardiac myocytes; confocal scanning microscopy; fluorescent dye /probe; imaging /visualization /scanning; laboratory mouse; laboratory rabbit; lipid bilayer membrane; neuromuscular transmission; sarcoplasmic reticulum; striated muscles

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）：钙火花成像提供了一种研究完整肌肉细胞中肌浆网（SR）储存的钙释放的方法。要提取有关兰尼碱受体 (RyR) SR 钙释放通道的调节和功能的最大信息，需要准确量化火花背后的钙释放通量。由于火花成像的细胞环境的复杂性，以及在完整细胞内操纵单个独立因素的困难，这种量化已被证明是困难的。我们的目标是采用一种新的、自下而上的方法来解决这个问题。针对完整细胞中的火花成像进行了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 通道获得已知钙通量的简单钙染料信号。对细胞因素（例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相结合，以建立更复杂的火花波形。为了实施这种方法，我们开发了一种光学双层系统，能够同时测量钙染料荧光和单 RyR 通道电流。已经设计出将 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，还开发、实施和测试了电压阶跃协议，其中 RyR 通道电流仅取决于施加的跨双层电压（已知）以及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。不仅可以获得火花背后的钙通量的详细信息，而且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，还将定性和定量地定义火花波形的特性与 RyR 通道功能的特定方面之间的关系。我们的方法是对当前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。我们将根据经验测量许多必须使用当前方法估计的因素。