The overall goals of this project are to clone, sequence and characterize key beta-cell genes which participate in insulin secretion. While the functional importance of genes which regulate intracellular concentrations of Ca2+ and K+ in the generation of the insulin secretory response has been clearly demonstrated, little is known about the molecular structure of the isoforms of these proteins which are expressed in the beta-cell. We will therefore clone and characterize b-cell K+ channels which appear to play an important role in the generation and regulation of the insulin secretory signal. These include the ATP-sensitive K+ channel and several types of voltage-dependent K+ channels. We have recently cloned and functionally characterized a voltage-dependent K+ channel expressed in human insulinoma and human islet cells, and have begun to characterize other K+ channels expressed in insulin-secreting cells. Our experimental approach will involve a number of strategies including expression cloning, PCR based amplification and low stringency cross-hybridization of cDNA libraries. We will also clone and characterize beta-cell Ca2+ channels including voltage dependent Ca2+ channels (VDCC) and inositol trisphosphate receptors. Our preliminary studies have identified an isoform of the VDCC which is expressed in beta-cells and brain. This cloning will be completed and we will attempt to clone the additional forms of the beta-cell VDCCs suggested by electrophysiologic studies. A complementary mechanism for the regulation of intracellular Ca2+ is through its mobilization from sequestered sites in the endoplasmic reticulum. This process is mediated through binding of IP3 to specific receptors. We have utilized the recent cloning of the rat brain IP3 receptor to identify a related isoform that is expressed in rat islets. Completion of the cloning will allow detailed comparison of its structure with similar proteins such as the neuronal IP3 and ryanodine receptors. The biophysical and pharmacological properties of the cloned channel genes and their role in insulin secretion in heterologous systems will be studied in collaboration with Projects 2 and 3. We anticipate that these studies will provide a better understanding of the structure and function of proteins involved in the regulation of insulin secretion at the molecular level and facilitate studies which aim to clarify the alterations which are present in diabetes. In addition we hope to define the minimal system which enables the beta cell to couple secretory stimuli to electrical excitability.
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