Calcium dynamics in secretory granule-containing cells

描述（由申请人提供）：Ca2+是调节许多生命过程的第二信使；因此，激发、终止和塑造Ca2+信号的细胞机制吸引了大量的研究。胞浆与细胞外空间和内质网（ER）之间的Ca2+通量是Ca2+动力学中最容易理解的组成部分。然而，其他细胞器，如分泌颗粒（SG），也可能起作用。人们早就知道，SG维持Ca2+水平远高于那些细胞质。最近的研究证明了功能性释放通道的存在，并强调Ca2+信号的高度局部化作用，进一步表明SG是细胞Ca2+信号传导机制的重要组成部分。一个混淆的因素是，SG传输质子和Ca2+；这通过影响Ca2+报告和改变SG Ca2+缓冲使Ca2+测量复杂化。本提案中的研究将定量剖析含SG细胞中的全局细胞Ca2+运输，表征SG的Ca2+缓冲能力，并测量SG Ca2+对细胞刺激的响应，同时测量pH对报告者和SG缓冲液的影响。使用化学探针和基因编码报告器的Ca2+和pH光度法将在完整的神经内分泌细胞和分离的SG中使用，以实现这些目标。该结果将有助于更好地理解神经内分泌细胞中的Ca2+动力学，以及其他临床相关细胞中SG Ca2+的潜在重要性，包括胰腺β细胞和肥大细胞。

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.