Calcium dynamics in secretory granule-containing cells

说明(申请人提供)：钙离子是调节许多重要过程的第二信使；因此，激发、终止和塑造钙离子信号的细胞机制吸引了密切的研究。胞浆、胞外间隙和内质网(ER)之间的Ca~(2+)通量是对Ca~(2+)动力学研究最深入的组成部分。然而，其他细胞器，如分泌颗粒(SG)，也可能起作用。人们很早就知道SG能维持远高于胞浆中的钙离子水平。最近的工作证明了功能释放通道的存在，并强调了钙信号的高度局部化作用，进一步证明了SG是细胞内钙信号机制的重要组成部分。一个令人困惑的因素是，SG运输质子和Ca~(2+)；这通过影响Ca~(2+)报告器和改变SG的Ca~(2+)缓冲而使Ca~(2+)测量变得复杂。这项研究将定量地剖析含SG的细胞内的整体细胞钙转运，表征SG的钙缓冲能力，并测量SG对细胞刺激的反应，同时测量pH对报告和SG缓冲区的影响。为了实现这些目标，将在完整的神经内分泌细胞和分离的SG中使用使用化学探针和遗传编码报告的钙和pH光度法。这一结果将有助于更好地了解神经内分泌细胞以及其他临床相关细胞(包括胰腺β细胞和肥大细胞)中的钙动态变化。

DESCRIPTION (provided by applicant): Ca2+ is a second messenger regulating many vital processes; therefore, cellular mechanisms for inciting, terminating, and shaping Ca2+ signals attract intense research. Ca2+ fluxes between the cytosol and both the extracellular space and the endoplasmic reticulum (ER) are the best-understood components of Ca2+ dynamics. However, other organelles, such as secretory granules (SG), may also contribute. It has long been known that SG maintain Ca2+ levels well above those of the cytosol. Recent work demonstrating the presence of functional release channels and emphasizing the highly localized action of Ca2+ signals further argues that SG represent important components of the cellular Ca2+ signaling ma- chinery. A confounding factor has been that SG transport protons as well as Ca2+; this complicates Ca2+ measurements by affecting Ca2+ reporters and altering SG Ca2+ buffering. The research in this proposal will quantitatively dissect global cellular Ca2+ transport in SG-containing cells, characterize the Ca2+ buffering capacity of SG, and measure the response of SG Ca2+ to cell stimuli while measuring pH effects on reporters and SG buffers. Ca2+ and pH photometry using both chemical probes and genetically-encoded reporters will be used in both intact neuroendocrine cells and isolated SG to achieve these goals. The results will lead to better understanding of Ca2+ dynamics in neuroendocrine cells, as well as in other clinically relevant cells in which SG Ca2+ is potentially important, including pancreatic beta-cells and mast cells.