The long-range goal of this research is to determine at the molecular level how extracellular signals regulate voltage-dependent Ca2+ channels (VDCCs) and the free cytosolic calcium level ([Ca2+]i) in the beta cell to control insulin secretion. In the preceding grant period we have shown that these Ca2+ channels play a central role in controlling the [Ca2+]i, the major """"""""second messenger"""""""" that signals insulin release. We have recently cloned a major beta cell VDCC subunit, the alpha(1) subunit, from a hamster (HIT cell) cDNA library. In the proposed studies we will express the cloned HIT cell alpha(1) subunit to establish that it is capable of forming a pore and passing Ca2+ current. In order to study the structure/function relationships of the Ca2+ channel subunits we will complete the cloning of a second subunit, the cardiac-like alpha(1) subunit, we have identified in the HIT cells and rat islets. We will also clone the beta subunit, raise antibodies to unique sequences of these subunits, and determine in which tissues and cells they are found. By expressing the subunits alone or together in stable transfected cell lines we will determine if these subunits interact to control Ca2+ current. Mutational studies will be performed to establish which of the cytoplasmic regions of the alpha(1) subunits interact with the beta subunit and which amino acids regulate the gating of the channel by specific protein kinases A phosphorylations. The significance of carboxy terminus of the subunits will be established by deletion analysis and the expression of hybrid genes. We will also determine if the vasopressin receptor, or other receptors, communicate with the VDCCs through a phosphoinositol signal, inositol 1,3,4,5-tetrakisphosphate. These investigations should provide a framework for beta cell Ca2+ channel function that will be extremely useful in defining the abnormalities in signal transduction that are a hallmark of non-insulin dependent diabetes. By understanding at the molecular level how these channels are regulated, it may be possible to target therapeutic strategies at the Ca2+ channels to increase insulin secretion in diabetic individuals.
