ELEMENTARY EVENTS OF INTRACELLULAR CALCIUM SIGNALING

几乎所有类型的细胞都将细胞内储存的钙释放到细胞质中，作为一种信号机制来调节分泌、收缩、增殖和细胞死亡等多种功能。改进的成像技术显示，钙释放通过涉及肌醇三磷酸和良胺受体/通道的途径不连续发生，作为“初级”钙释放事件。这些短暂的，局部的，亚细胞的游离[Ca2+]升高出现在内质网内形成离散功能释放位点的通道簇中。单个位点可以产生涉及单个或多个通道的自主事件，其活动可能通过钙扩散和钙诱导的钙释放来协调传播全局细胞钙波。因此，基本事件构成了复杂时空钙信号的基本组成部分，这些信号允许细胞功能的分级和选择性调节。因此，了解它们的产生、相互作用和功能后果对于理解无处不在的钙信使通路的生理和病理功能至关重要。我们的具体目标是确定(i)基本钙事件产生的机制，(h)允许钙波起始和传播的事件之间的协调，以及（iii）局部钙微域对效应系统特异性激活的原理。我们将使用爪蟾卵母细胞和心肌细胞作为模型系统，分别研究肌醇三磷酸受体和红嘌呤受体的信号传导。此外，使用培养细胞系将允许调查由其他受体同种异构体产生的事件。我们的实验方法包括高分辨率的1光子和2光子共聚焦显微镜（小于0.5微米和1毫秒），以成像完整细胞中由三磷酸肌醇和Ca2+光释放引起的亚细胞钙事件。这些光学技术提供了一个独特的机会来可视化活细胞中的单通道活性，我们的总体目标是阐明单个钙释放通道如何促进细胞钙反应。

The liberation of calcium from in intracellular stores into the cytosol is used as a signaling mechanism by virtually all cell types to regulate functions as diverse as secretion, contraction, proliferation and cell death. Improved imaging technology has revealed that calcium liberation through pathways involving both inositol trisphosphate and ryanodine receptor/channels occurs discontinuously, as 'elementary' calcium release events. These transient, localized, subcellular free [Ca2+] elevations arise at clusters of channels that form discrete functional release sites within the endoplasmic reticulum. Individual sites can generate autonomous events involving single or multiple channels, and their activity may be coordinated by calcium diffusion and calcium-induced calcium release to propagate global cellular calcium waves. Elementary events thus form the basic building blocks underlying the complex spatiotemporal calcium signals that permit graded and selective regulation of cell functions. An understanding of their generation, interaction and functional consequences is, therefore, pivotal to understand the physiological and pathological functioning of the ubiquitous calcium messenger pathway. Our specific aims are to determine, (i) the mechanisms underlying the generation of elementary calcium events, (h) the coordination between events allowing the initiation and propagation of calcium waves, and (iii) the principles underlying specific activation of effector systems by local calcium microdomains. We will use Xenopus oocytes and cardiac myocytes as model systems to study signaling by inositol trisphosphate- and ryanodine-receptors, respectively. Furthermore, use of cultured cell lines will allow investigation of events generated by other receptor isoforms. Our experimental methodology involves 1- and 2-photon confocal microscopy with high (less than 0.5 mum and 1 ms) resolution to image subcellular calcium events in intact cells evoked by photorelease of inositol trisphosphate and Ca2+. These optical techniques provide a unique opportunity to visualize single channel activity in living cells, and our overall goal is to elucidate how individual calcium release channels contribute to cellular calcium responses.