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.
| Premack, B A; McDonald, T V; Gardner, P (1994) Activation of Ca2+ current in Jurkat T cells following the depletion of Ca2+ stores by microsomal Ca(2+)-ATPase inhibitors. J Immunol 152:5226-40 |
| Kindman, L A; Kim, S; McDonald, T V et al. (1994) Characterization of a novel intracellular sphingolipid-gated Ca(2+)-permeable channel from rat basophilic leukemia cells. J Biol Chem 269:13088-91 |
| McDonald, T V; Premack, B A; Gardner, P (1993) Flash photolysis of caged inositol 1,4,5-trisphosphate activates plasma membrane calcium current in human T cells. J Biol Chem 268:3889-96 |
| Gollapudi, S; McDonald, T; Gardner, P et al. (1992) Abnormal chloride conductance in multidrug resistant HL60/AR cells. Cancer Lett 66:83-9 |
