Rapid kinetics of single InsP3R channel regulation

单 InsP3R 通道调节的快速动力学

• 批准号: 7255811
• 负责人: Don-On Daniel Mak
• 金额: $ 25.4万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7255811
• 关键词: Apoptosis; Behavior; Binding Sites; Cell Nucleus; Cells; Classification; Complex; Condition; Endoplasmic Reticulum; Event; Frequencies; Future; Generations; Immune response; Individual; Inositol 1,4,5-Trisphosphate; Investigation; Kinetics; Ligand Binding; Ligands; Mediating; Membrane; Memory; Molecular; Monitor; Nuclear; Nuclear Envelope; Physiological; Physiological Processes; Probability; Property; Rate; Refractory; Regulation; Research Personnel; Side; Signal Transduction; Time; base; experience; in vivo; insight; mathematical model; millisecond; patch clamp; programs; receptor; research study; response; success

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.

描述（由申请人提供）：本提案的目的是研究其天然内质网（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的快速生理变化的动态响应。 单个 InsP3R 通道释放的 Ca2+ 是复杂细胞内 Ca2+ 信号的组成部分，控制着从细胞凋亡和分泌到免疫反应和记忆等众多生理过程。 由于缺乏对单个 InsP3R 通道的动态配体调节的深入了解，理解各种 Ca2+ 信号传导现象（包括量子 Ca2+ 释放、Ca2+ 振荡的频率调制和传播 Ca2+ 波）受到严重阻碍。我们发现，在核膜片钳实验中可以定期检测到培养的 Sf9 细胞分离核中的 InsP3R 通道，其特性和调节与哺乳动物 InsP3R 高度相似。此外，现在可以以高成功率获得所有形态的切除核膜斑块，尤其是细胞质侧朝外的形态。 凭借这些进展，首次可以在细胞质或腔侧配体浓度快速（毫秒）变化期间监测单个 InsP3R 通道活性的时间过程。 Ca2+ 诱导的 Ca2+ 释放事件期间 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 敏感性以及通过 ER Ca2+ 浓度 (Ca2+)ER 对 InsP3R 通道活性的调节。 还将检查 (Ca2+)ER 对 InsP3R 通道电导特性、门控行为和活动持续时间的调节。这些研究代表了在通道体内经历的各种动态生理条件下单个 InsP3R 通道动力学的首次系统研究。他们将为未来从分子到细胞水平的 Ca2+ 信号传导数学建模提供急需的有关单个 InsP3R 通道的配体调节的动力学信息。