Electric Studies of Excitation, Secretion & Contraction

描述（由申请人提供）：钙信号传导和胞吐调节是所有动物细胞生理学的中心问题。这项资助旨在通过电兴奋和非兴奋哺乳动物细胞系的生物物理实验定量了解这种信号传导：PC 12嗜铬细胞瘤细胞，tsA上皮细胞和胰管上皮细胞。一些实验将使用啮齿动物的原代嗜铬细胞。两个长期的假设指导这项工作：Ca 2+清除和胞吐的调节在不同的细胞中采取不同的形式，并根据每个细胞的生理作用进行调整;以及几个细胞内细胞器对细胞Ca 2+动力学做出重要贡献。本研究的主要目的是：（1）验证分泌颗粒对Ca ~（2+）的积累和释放在生理反应中对细胞Ca ~（2+）信号转导的重要作用。(2)测定受体诱发的1，4，5，三磷酸肌醇（IP 3）升高的幅度，并检验通过IP 3的Ca 2+信号传导是通过IP 3 5-磷酸酶快速代谢IP 3，然后快速重摄取Ca 2+进入内质网Ca 2+库而终止的假设。（3）验证细胞骨架的快速重塑和细胞骨架轨迹参与了分泌颗粒从储备池向分泌池的动员。这项工作需要一系列生物物理技术，包括：离子电流的膜片钳;胞吐的电流和电容测量;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）的指标;视频荧光成像;全内反射显微镜（TIRF）;共聚焦显微镜;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.