The Physician Scientist Award will enable the applicant to develop his longstanding interest in the mechanisms of biological processes. During the tenure of this award, he will mature as an independent investigator with expertise in biochemistry and membrane electrophysiology. This will be accomplished by active participation in graduate level courses, seminars, and workshops, and regular meetings with an advisory committee composed of cell biologists, electrophysiologists and cardiologists. An intensive laboratory experience under the direction of the sponsors Dr. Lubert Stryer and Dr. Phyllis Gardner will provide experimental training. Neuroendocrine signal transduction is often mediated by a rise in cytosolic free calcium. This occurs through the Inositol 1,4,5 trisphosphate (InsP3)-gated release of calcium from the endoplasmic reticulum and by InsP3-gated entry of calcium from the extracellular space. Amplitude modulated hormonal stimuli lead to frequency-modulated cytosolic calcium transients. Central to the generation of the calcium transient is the regulation of calcium entry by the InsP3-gated calcium channel. To further characterize this important family of calcium channels, a comparative study of InsP3-gated calcium channels from the endoplasmic reticulum of the rat basophilic leukemia (RBL) cell and the Jurkat human leukemia cell lines is proposed. The electrophysiologic and regulatory properties of the InsP3-gated calcium channel of the endoplasmic reticulum will be studied in saponin-permeabilized cells using a fluorescent indicator assay for calcium flux. To better understand the regulation of the InsP3-gated calcium channel, the channel will be purified by detergent and lipid solubilization, followed by affinity and gel filtration chromatography. It will be functionally reconstituted into calcium-loaded phospholipid vesicles. In this environment, the effects of putative modulators of InsP3-gated calcium channel activity will be investigated. Further understanding of channel inactivation will be gained by investigating InsP3 binding and channel gating in this system. During the second phase of this award, training in cellular electrophysiology will be obtained by performing a comparative patch-clamp investigation of the properties of the plasma membrane InsP3-gated calcium channel of RBL and Jurkat cells. This data will be related to the findings of the endoplasmic reticulum InsP3-gated calcium channel. By understanding the similarities and differences of these two classes of channels, further insight into the mechanism of hormonally mediated calcium transients will be gained.
