Rapid kinetics of single InsP3R channel regulation

描述(申请人提供)：这项建议的目的是研究其天然内质网(ER)膜上的肌醇1，4，5-三磷酸(InsP3)受体(InsP3R)钙释放通道对其配体浓度快速生理变化的动态反应。单个InsP3R通道释放的Ca~(2+)是复杂的细胞内Ca~(2+)信号的组成部分，这些信号控制着许多生理过程，从细胞凋亡和分泌到免疫反应和记忆。由于缺乏对单个InsP3R通道的动态配基调控的深入了解，对包括量子钙释放、钙振荡的频率调制和钙波传播在内的各种钙信号现象的理解受到了严重的阻碍。我们发现，在核膜片钳实验中，可以定期在培养的Sf9细胞的分离细胞核中检测到InsP3R通道，其性质和调节方式非常类似于哺乳动物的InsP3R。此外，现在有可能获得全部切除的核膜补片，特别是细胞质侧面朝外的结构，成功率很高。有了这些进展，可以第一次监测单个InsP3R通道活动的时间进程，在细胞质或管腔侧的配体浓度快速(毫秒)变化期间。在生理条件下，InsP3R通道活性在钙离子诱导的钙释放事件中的动力学特征将被描述。我们将研究导致量子钙释放的内在InsP3R通道行为，包括依赖时间的失活、异质性InsP3敏感性以及内质网钙离子浓度(Ca~(2+))ER对InsP3R通道活性的调节。(Ca~(2+))ER对InsP3R通道电导特性、门控行为和活动持续时间的调节也将被研究。这些研究首次系统地研究了单个InsP3R通道在体内经历的各种动态生理条件下的动力学。它们将为未来从分子到细胞水平的钙信号的数学建模提供急需的关于单个InsP3R通道的配体调节的动力学信息。

DESCRIPTION (provided by applicant): The objective of this proposal is to study the dynamic responses of the inositol 1,4,5-triphosphate (InsP3) receptor (InsP3R) Ca2+-release channel in its native endoplasmic reticulum (ER) membrane to rapid physiological changes in the concentrations of its ligands. Ca2+ release by individual InsP3R channels is the building block of complex intracellular Ca2+ signals that control numerous physiological processes, from apoptosis and secretion to immune responses and memory. Understanding diverse Ca2+ signaling phenomena including quantal Ca2+ release, frequency modulation of Ca2+ oscillations, and propagating Ca2+ waves has been severely hampered by the lack of insights into dynamic ligand regulation of single InsP3R channels. We discovered that InsP3R channels in isolated nuclei of cultured Sf9 cells can be detected regularly in nuclear patch clamp experiments, with properties and regulation that are highly reminiscent of mammalian InsP3R. Furthermore, it is now possible to obtain, with high success rates, excised nuclear membrane patches in all, especially the cytoplasmic-side-out, configurations. With these advances, the time course of single InsP3R channel activity can be monitored, for the first time, during rapid (msec) changes in ligand concentrations on the cytoplasmic or lumenal side. The kinetics of InsP3R channel activity during Ca2+-induced Ca2+ release events will be characterized under physiological conditions. Instrinsic InsP3R channel behaviors that can contribute to quantal Ca2+ release, including time-dependent inactivation, heterogenous InsP3 sensitivity, and regulation of InsP3R channel activity by ER Ca2+ concentration (Ca2+)ER, will be investigated. (Ca2+)ER regulation of InsP3R channel conductance properties, gating behaviors and activity durations will also be examined. These studies represent the first systematic investigation of single InsP3R channel kinetics under various dynamic physiological conditions the channels experience in vivo. They will provide much-needed kinetic information on ligand regulation of single InsP3R channel for future mathematical modeling of Ca2+ signaling, from the molecular to cellular levels.