Rapid kinetics of single InsP3R channel regulation

描述（由申请人提供）：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（InsP3）受体（InsP3R） Ca2+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InsP3R通道释放Ca2+是复杂的细胞内Ca2+信号的基石，这些信号控制着许多生理过程，从凋亡和分泌到免疫反应和记忆。了解多种Ca2+信号现象，包括量子Ca2+释放，Ca2+振荡的频率调制和Ca2+波的传播，由于缺乏对单个InsP3R通道的动态配体调节的了解，严重阻碍了Ca2+信号的传播。我们在核膜片钳实验中发现，在培养的Sf9细胞的离体细胞核中可以定期检测到InsP3R通道，其特性和调控与哺乳动物的InsP3R非常相似。此外，现在有可能以高成功率获得所有切除的核膜斑块，特别是细胞质侧出构型。随着这些进展，单个InsP3R通道活性的时间过程首次可以在细胞质或管腔侧配体浓度快速（msec）变化期间进行监测。在Ca2+诱导的Ca2+释放事件中，InsP3R通道活性的动力学将在生理条件下进行表征。将研究有助于量子Ca2+释放的内在InsP3R通道行为，包括时间依赖性失活，异质InsP3敏感性以及ER Ca2+浓度（Ca2+）ER对InsP3R通道活性的调节。（Ca2+）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。他们将为未来从分子到细胞水平的Ca2+信号的数学建模提供关于单个InsP3R通道配体调控的急需的动力学信息。

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.