Rapid kinetics of single InsP3R channel regulation

描述（由申请方提供）：本提案的目的是研究天然内质网（ER）膜中肌醇1，4，5-三磷酸（InsP 3）受体（InsP 3R）Ca 2+释放通道对其配体浓度快速生理变化的动态响应。 通过单个InsP 3R通道的Ca 2+释放是复杂的细胞内Ca 2+信号的构建块，该信号控制从细胞凋亡和分泌到免疫应答和记忆的许多生理过程。 理解不同的Ca 2+信号传导现象，包括量子Ca 2+释放，频率调制的Ca 2+振荡，传播Ca 2+波已严重阻碍了缺乏见解的动态配体调控的单InsP 3R通道。我们发现，在培养的Sf 9细胞的分离的细胞核中的InsP 3R通道可以在核膜片钳实验中定期检测到，具有高度令人联想到哺乳动物InsP 3R的特性和调节。此外，现在有可能以高成功率获得所有，特别是胞质侧外构型的切除的核膜补片。 随着这些进展，单InsP 3R通道活性的时间过程可以监测，第一次，在胞质或内腔侧的配体浓度的快速（毫秒）变化。 将在生理条件下表征在Ca 2+诱导的Ca 2+释放事件期间InsP 3R通道活性的动力学。 将研究有助于量子Ca 2+释放的内源性InsP 3R通道行为，包括时间依赖性失活、异质性InsP 3敏感性和ER Ca 2+浓度（Ca 2+）ER对InsP 3R通道活性的调节。 (Ca2+）还将检查InsP 3R通道电导性质、门控行为和活性持续时间的ER调节。这些研究代表了在通道在体内经历的各种动态生理条件下对单个InsP 3R通道动力学的首次系统研究。它们将为将来从分子到细胞水平的Ca 2+信号传导的数学建模提供急需的关于单个InsP 3R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.