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SECOND MESSENGER REGULATION OF CALCIUM ION CHANNELS

钙离子通道的第二信使调节

基本信息

批准号：
2210009
负责人：
THOMAS V MCDONALD
金额：
$ 9.04万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1990
资助国家：
美国
起止时间：
1990-04-01 至 1995-03-31
项目状态：
已结题

项目摘要

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.

该拟议计划的目标是全面培训申请人，专门从事心脏病学的医生，成为一名独立的医生具有现代细胞经验和技能的研究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。实验室培训将包括接触现代细胞生物学的技术和概念框架膜片钳和闪光光解技术的大量使用菲利斯·加德纳博士的赞助。第一阶段的研究将涉及钙通道调节的分子机制研究两个系统中的细胞内第二信使；有丝分裂原和表面抗体激活 T 淋巴细胞通道，以及内皮素诱导的通道平滑肌细胞中的通道。 T 淋巴细胞的全细胞钳研究其中细胞通过光解敏感的第二-进行内部透析将执行信使（“笼式”-肌醇1,4,5-三磷酸）。这闪光光解后立即激活 Ca2+ 电流将调查笼式化合物。膜片钳实验将进行表征电生理特性、动力学和单通道和平滑肌 Ca2+ 通道的药理学全细胞电流测量。在第二阶段进一步研究这些 Ca2+ 通道的细胞内调节因子（即 GTP 结合蛋白、蛋白激酶的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使用特定毒素、激活剂、抑制剂和“笼子”进行测量- 计划使用 Ca2+ 螯合剂。通过阐明Ca2+的机制 T 细胞激活和平滑肌反应中的通道调节内皮素对钙的一般理解的贡献将实现体内平衡、细胞生长和血管收缩。