SECOND-MESSENGER REGULATION OF CALCIUM ION CHANNELS

该计划的目标是全面培训申请人， 一个专门从事心脏病学的医生，成为一个独立的医生， 具有现代细胞学经验和技能的研究人员 电生理学和细胞生物学。 正式培训将包括 参加细胞生物学系的研究生课程， 药理学和神经生物学以及积极参与期刊 俱乐部会议和研讨会。 实验室培训将包括暴露 现代细胞生物学的技术和概念框架， 在此背景下， 菲利斯·加德纳医生的赞助 第一阶段的研究将涉及 研究钙通道调节的分子机制 两个系统中的细胞内第二信使;丝裂原和表面 抗体激活T淋巴细胞通道，内皮素诱导 平滑肌细胞中的通道。 T淋巴细胞的全细胞钳夹研究 其中细胞用光解敏感的第二- 使者（“笼”-肌醇1，4，5-三磷酸）将执行。 的 闪光光解后瞬时激活的钙电流 将对笼中化合物进行研究。 膜片钳实验将在 进行电生理特性、动力学和 单通道平滑肌Ca 2+通道的药理学， 全细胞电流测量。 在第二阶段， 这些Ca 2+通道的细胞内调节剂（即GTP结合蛋白， 蛋白激酶通道磷酸化和钙离子自动调节） 将被执行。 使用全细胞和膜片钳电流的实验 测量特定的毒素，激活剂，抑制剂，和“笼”- 钙离子螯合剂的计划。 通过阐明钙离子的作用机制， T细胞活化和平滑肌对 内皮素对钙的一般理解的贡献 体内平衡、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.