ELEMENTARY EVENTS OF INTRACELLULAR CALCIUM SIGNALING

钙从细胞内储存释放到胞质溶胶中被用作几乎所有细胞类型的信号传导机制，以调节如分泌、收缩、增殖和细胞死亡等多种功能。 改进的成像技术已经揭示，通过涉及三磷酸肌醇和兰尼碱受体/通道的途径的钙释放不连续地发生，作为"基本"钙释放事件。 这些短暂的，局部的，亚细胞游离[Ca2 +]升高出现在通道簇，形成离散的功能释放位点内的内质网。 单个位点可以产生涉及单个或多个通道的自主事件，并且它们的活动可以通过钙扩散和钙诱导的钙释放来协调，以传播全局细胞钙波。 因此，基本事件形成了复杂的时空钙信号的基础构件，允许分级和选择性调节细胞功能。 因此，了解它们的产生，相互作用和功能后果是了解无处不在的钙信使途径的生理和病理功能的关键。 我们的具体目标是确定，（i）基本钙事件产生的机制，（h）事件之间的协调，允许钙波的启动和传播，以及（iii）效应系统的局部钙微域的特异性激活的原则。 我们将使用非洲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和ryanodine受体的信号。此外，使用培养的细胞系将允许研究由其他受体同种型产生的事件。 我们的实验方法涉及1-和2-光子共聚焦显微镜与高分辨率（小于0.5妈妈和1毫秒）图像亚细胞钙事件在完整的细胞诱发的三磷酸肌醇和Ca2+的光释放。 这些光学技术提供了一个独特的机会，可视化活细胞中的单通道活动，我们的总体目标是阐明如何个别钙释放通道有助于细胞钙反应。

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.