The long term goal of this research is understanding the molecular mechanisms by which the cytosolic Ca concentration Ca regulates insulin secretion. Understanding this process may hold the key to unraveling the beta cell defects in type II diabetes where alterations in glucose sensitivity and defective dynamics of insulin release are major abnormalities. We will use a variety of biochemical and biophysical techniques to characterize how Ca currents in a glucose-responsive beta cell line (HIT cells) are regulated to alter insulin release. A more sensitive fluroescent probe, fura 2 will be used to further test the hypothesis that a rise in Ca is the primary intracellular signal which triggers insulin secretion and establish if there are differences in Ca signaling between normal and transformed beta cells. In permeabilized HIT cells, the precise role of the myosin light chain kinase, a Ca- calmodulin-dependent enzyme, in Ca signal transduction will be established by introducing synthetic peptides which bind to the calmodulin binding site of the enzyme and inhibit enzymatic activity. We will determine the effects of these inhibitors and control truncated peptides which are inactive on insulin release and phosphorylation reactions evoked by increasing concentrations of Ca. We will establish if the potentiation of insulin secretion by insulin secretagogues which activate cAMP, protein kinase C or Ca-CaM kinases are brought about by increasing the rate or phosphorylation state of certain specific substrate proteins. To understand the involvement of a novel low threshold Ca channel in insulin secretion we will examine how insulin secretagogues alter single and whole cell Ca currents measured by the patch clamp technique. The functional significance of the voltage-dependent Ca channel will be further evaluated by establishing if the flux of Ca ions through these channels is altered by phosphorylation of Ca channels identified by the use of photoaffinity probes, (3H dihydropyroidines or phenylalkylamines which bind to the channel in a specific manner. These studies should establish the molecular, electrical, and cellular mechanisms by which physiologic and pharmacologic insulin secretagogues activate voltage-dependent Ca channels and allow Ca to enter the beta cell and trigger the secretory process
