Electric Studies of Excitation, Secretion & Contraction

描述（由申请人提供）：钙信号和胞吐调节是所有动物细胞生理学的核心问题。该资助旨在通过电兴奋性和非电兴奋性哺乳动物细胞系（PC12嗜铬细胞瘤细胞、tsA上皮细胞和胰管上皮细胞）的生物物理实验，定量了解这种信号。一些实验将使用啮齿动物的原代染色质细胞。两个长期的假设指导了这项工作：Ca2+清除和胞吐调节在不同的细胞中采取不同的形式，并调整到每个细胞的生理作用；并且一些胞内细胞器对细胞Ca2+动力学有重要贡献。本研究的目的是：(1)验证分泌颗粒积累和释放Ca2+在生理反应中对细胞Ca2+信号传导有重要贡献的假设。(2)测量受体引起的肌醇1,4,5，三磷酸（IP3）升高的幅度，并验证IP3信号通过IP3被ip35 -磷酸酶快速代谢，随后Ca2+快速再摄取到内质网Ca2+储存而终止的假设。(3)验证细胞骨架轨迹和快速细胞骨架重塑参与了分泌颗粒从储备池向分泌能力池动员的假设。这项工作需要一系列的生物物理技术，包括：离子电流的膜片钳；胞吐的安培和电容测量；基因靶向探针、指标和细胞蛋白的转染；比率光度法和荧光共振能量转移（FRET）指示剂；视频荧光成像；全内反射显微镜；共焦显微镜;定量动力学建模。

DESCRIPTION (provided by applicant): Calcium signaling and the regulation of exocytosis are central issues in the physiology of all animal cells. This grant seeks quantitative understanding of such signaling through biophysical experiments in electrically excitable and non-excitable mammalian cell lines: PC12 pheochromocytoma cells, tsA epithelial cells, and pancreatic duct epithelial cells. A few experiments will use rodent primary chromaffin cells. Two long-term hypotheses guide this work: That Ca2+ clearance and the regulation of exocytosis take different forms in different cells and are tuned to the physiological role of each cell; and that several intracellular organelles make significant contributions to cellular Ca2+ dynamics. The aims in this grant period are: (1) To test the hypothesis that accumulation and release of Ca2+ by secretory granules can make significant contributions to cellular Ca2+ signaling during physiological responses. (2) To measure the amplitude of receptor evoked inositol 1,4,5, trisphosphate (IP3) elevations and to test the hypothesis that Ca2+ signaling via IP3 is terminated by rapid metabolism of IP3 by IP3 5- phosphatase followed by rapid reuptake of Ca2+ into the endoplasmic reticulum Ca2+ stores. And (3) To test the hypothesis that cytoskeletal tracks and fast cytoskeletal remodeling participate in the mobilization of secretory granules from reserve pools into secretion-competent pools. The work requires a range of biophysical techniques including: patch clamp of ion currents; amperometric and capacitance measurements of exocytosis; transfection of genetically targeted probes, indicators, and cellular proteins; ratiometric photometry and fluorescence resonance energy transfer (FRET) of indicators; video fluorescence imaging; total internal reflection microscopy (TIRF); confocal microscopy; quantitative kinetic modeling.