Elementary Events of Intracellular Calcium Signaling

描述（由申请人提供）：Ca2+离子从细胞外液和内质网（ER）储存库进入细胞质，被用作几乎所有细胞类型的信号传导机制，以调节各种功能，如电兴奋性、分泌、增殖和细胞死亡。改进的光学技术现在可以可视化Ca2+信号事件的层次结构，包括单通道Ca2+可渗透通道的打开（“基本”事件），集群通道的协调打开（“基本”事件）和传播Ca2+波。通过单个和集群通道产生的局部游离[Ca2+]升高具有自主的信号功能，其活性可能进一步通过Ca2+扩散和Ca2+诱导的Ca2+释放来协调传播全局细胞Ca2+波。因此，基本和基本事件形成了复杂时空Ca2+信号的分层构建块，允许细胞功能的分级和选择性调节。因此，阐明它们的产生、相互作用和功能后果对于理解无处不在的Ca2+信使通路的生理功能及其在疾病中的作用至关重要。我们的总体目标是阐明，在单通道水平上，细胞如何产生Ca2+信号的层次结构，以及Ca2+信号的中断如何参与疾病的发病机制。我们专注于普遍存在的肌醇三磷酸第二信使途径产生的生理Ca2+信号，以及与阿尔茨海默病有关的淀粉样蛋白低聚物形成的致病性Ca2+渗透孔。通过利用新的生物光子工具，现在可以通过单个通道进行钙通量的光学成像，并以纳米精度定位和跟踪通道蛋白，我们的目标是：(i)进一步完善光学和分析技术，同时监测完整细胞的质膜和内质网中数百个单独通道的Ca2+通量。（ii）阐明个体IP3受体（IP3R）在释放位点的活性如何被精心安排以产生基本的Ca2+泡。（iii）利用超分辨率成像技术确定IP3R的纳米级空间分布，以及这如何影响它们的功能。（iv）同时监测单个淀粉样蛋白孔的Ca2+通量和肽化学计量，研究膜掺入、通道门控和离子渗透的基本机制。

DESCRIPTION (provided by applicant): The entry of Ca2+ ions into the cytosol from the extracellular fluid and from endoplasmic reticulum (ER) stores is used as a signaling mechanism by virtually all cell types to regulate functions as diverse as electrical excitability, secretion, proliferation and cell death. Improved optical technology now enables visualization of a hierarchy of Ca2+ signaling events, ranging from openings of single-channel Ca2+-permeable channels ('fundamental' events), concerted openings of clustered channels ('elementary' events) and propagating Ca2+ waves. The localized free [Ca2+] elevations arising through individual and clustered channels serve autonomous signaling functions, and their activity may further be coordinated through Ca2+ diffusion and Ca2+-induced Ca2+ release to propagate global cellular Ca2+ waves. Fundamental and elementary events thus form hierarchical building blocks underlying the complex spatiotemporal Ca2+ signals that permit graded and selective regulation of cell functions. Elucidation of their generation, interaction and functional consequences is, therefore, pivotal to understand the physiological functioning of the ubiquitous Ca2+ messenger pathway and its involvement in disease. Our overall goal is to elucidate, at the single-channel level, how cells generate the hierarchy of Ca2+ signals and how disruptions in Ca2+ signaling may be involved in disease pathogenesis. We focus on physiological Ca2+ signals generated by the ubiquitous inositol trisphosphate second messenger pathway, and on the pathogenic Ca2+-permeable pores formed by amyloid oligomers implicated in Alzheimer's disease. By utilizing novel biophotonic tools that now enable the optical imaging of calcium flux through individual channels and the localization and tracking of channel proteins with nanometer precision we aim to: (i) Further refine optical and analytical techniques for simultaneously monitoring Ca2+ flux through hundreds of individual channels in the plasma membrane and ER of intact cells. (ii) Elucidate how the activity of individual IP3 receptors (IP3R) at a release site is orchestrated to generate elementary Ca2+ puffs. (iii) Employ superresolution imaging techniques to determine the nanoscopic spatial distribution of IP3R, and how this impacts their functioning. (iv) Simultaneously monitor Ca2+ flux and peptide stoichiometry of individual amyloid pores to study fundamental mechanisms of membrane incorporation, channel gating and ion permeation.