Electric Studies of Excitation, Secretion & Contraction

描述(申请人提供)：钙信号和胞吐的调节是所有动物细胞生理学的中心问题。这项资助旨在通过在电刺激和非刺激的哺乳动物细胞系：PC12嗜铬细胞瘤细胞、TSA上皮细胞和胰管上皮细胞中进行生物物理实验来定量了解这种信号。一些实验将使用啮齿动物的初级嗜铬细胞。两个长期的假设指导了这项工作：钙离子清除和胞吐作用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内钙离子动力学做出了重大贡献。本授权期的目的是：(1)验证分泌颗粒积累和释放钙离子在生理反应中对细胞内钙信号有重要贡献的假说。(2)测定受体引起的1，4，5，三磷酸肌醇(IP3)升高的幅度，并验证通过IP3传递的钙信号是通过IP3被IP3 5-磷酸酶快速代谢，然后迅速重新摄取到内质网钙库中而终止的假说。以及(3)检验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到分泌能力池的动员这一假设。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；胞吐作用的安培和电容测量；基因靶向探针、指示剂和细胞蛋白质的转基因；指示剂的比率光度法和荧光共振能量转移(FRET)；视频荧光成像；全内反射显微镜(TIRF)；共聚焦显微镜；定量动力学模型。

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.