SECOND-MESSENGER REGULATION OF CALCIUM ION CHANNELS

这一拟议计划的目标是对申请者进行全面培训， 专攻心脏病学的医生，成为一名独立的 具有现代蜂窝领域经验和技能的调查员 电生理学和细胞生物学。正式的培训将包括 在细胞生物系参加研究生水平的课程， 药理学、神经生物学以及积极参与期刊 俱乐部会议和研讨会。实验室培训将包括接触 现代细胞生物学的技术和概念框架 密集使用膜片钳和闪光光解技术 菲利斯·加德纳博士的赞助。第一阶段的研究将涉及 银杏叶提取物对钙离子通道调控的分子机制研究 有丝分裂原和表面两个系统中的细胞内第二信使 抗体激活的T淋巴细胞通道与内毒素诱导 平滑肌细胞中的通道。T淋巴细胞的全细胞钳制研究 在这种情况下，细胞用光解敏感的秒- 将执行信使(笼子状的-肌醇1，4，5-三磷酸)。这个 闪光光解后钙电流的瞬时激活 笼子里的化合物将被调查。膜片钳实验将 以表征电生理特性、动力学和 单通道和单通道的平滑肌钙通道的药理学研究 全电池电流测量。在第二阶段进一步研究 这些钙通道的细胞内调节因子(即GTP结合蛋白， 由蛋白激酶引起的通道磷酸化和钙离子自动调节) 将会被执行。全细胞膜片钳电流实验 对特定的毒素、激活剂、抑制剂和笼子进行测量- 钙离子的螯合剂正在计划中。通过阐明钙离子的作用机制 T细胞活化和平滑肌反应中的通道调节 内皮素对钙的一般理解作用 将进行动态平衡、细胞生长和血管收缩。

The goal of this proposed program is to fully train the applicant, a physician who has specialized in cardiology, to become an independent investigator with experience and skills in modern cellular electrophysiology and cell biology. The formal training will consists of attending graduate level courses in the departments of Cell Biology, Pharmacology, and Neurobiology as well as active participation in journal club meetings and seminars. The laboratory training will include exposure to the techniques and conceptual framework of modern cell biology with intensive use of patch-clamp and flash-photolysis techniques under the sponsorship of Dr. Phyllis Gardner. Research in Phase I will involve investigation of the molecular mechanisms of calcium channel regulation by intracellular second-messengers in two systems; mitogen and surface antibody activated channels of T-lymphocytes, and Endothelin-induced channels in smooth muscle cells. Whole-cell clamp studies on T-lymphocytes in which cells are internally dialyzed with photolysis-sensitive second- messengers ('caged'-Inositol 1,4,5-trisphosphate) will be performed. The instantaneous activation of Ca2+ current following flash-photolysis of the caged compound will be investigate. Patch-clamp experiments will be performed to characterized the electrophysiologic properties, kinetics, and pharmacology of the smooth muscle Ca2+ channel with single-channel and whole-cell current measurements. In Phase II further studies of intracellular regulators of these Ca2+ channels (i.e. GTP-binding proteins, channel phosphorylation by protein kinases, and calcium ion autoregulation) will be performed. Experiments using whole-cell and patch-clamp current measurements with specific toxins, activators, inhibitors, and 'caged'- chelators of Ca2+ are planned. By elucidating the mechanisms of Ca2+ channel regulation in T-cell activation and smooth muscle response to endothelin contributions to the general understanding of calcium homeostasis, cell growth, and vasoconstriction will be made.