Gregory H. Hockerman - US grants

calcium channel; phenylalkylamine; molecular site; striated muscles; dihydropyridines; complementary DNA; gene deletion mutation; mutant; electrophysiology; calcium channel blockers; thiazide; nucleic acid hybridization; transfection; site directed mutagenesis; voltage /patch clamp; molecular cloning; L cell;

calcium channel; phenylalkylamine; molecular site; striated muscles; dihydropyridines; complementary DNA; gene deletion mutation; mutant; electrophysiology; calcium channel blockers; thiazide; nucleic acid hybridization; transfection; site directed mutagenesis; voltage /patch clamp; molecular cloning; L cell;

DESCRIPTION (provided by applicant): In type II diabetes, the insulin secreting beta cells of pancreatic islets fail to secrete insulin in sufficient quantities to maintain normal blood glucose levels. The resulting hyperglycemia can lead to many serious complications. Therefore, understanding the mechanisms that mediate insulin secretion could lead to new therapies to prevent the onset and complications of Type II diabetes. Two sub-classes of L-type calcium channels, Cav1.2 and Cav1.3 are expressed in pancreatic beta cells. We have developed a "knock in" method to introduce Cav1.2 and Cav1.3 mutant channels that are insensitive to the dihydropyridine (DHP) class of L-type channel blockers into the insulinoma cell line INS-1. In this system, the endogenous L-type channels can be "shut off" with DHP drugs, thus pharmacologically isolating either Cav1.2 of Cav1.3 channels. Using this system, we have shown that Cav1.3 but not Cav1.2 channels can mediate glucose-stimulated insulin secretion. Insulin secretion is potentiated by the hormone GLP-1, by its binding to the GLP-1 receptor. We have identified a short peptide, derived from the GLP-1 receptor primary amino acid sequence, that can mimic some, but not all of the actions of GLP-1. We hypothesize that in the context of the receptor this peptide comprises an autoactivation domain that is unmasked upon ligand binding. In Aim 1 of this project, we will characterize both the activity of this peptide as a small molecule agonist, and its contribution to GLP-1 receptor activation in the context of the GLP-1 receptor structure. In Aim 2, we will further examine the mechanisms that couple L-type calcium channels to insulin secretion and beta cell proliferation, and how they are modulated by GLP-1 receptor activation. Although most of this work will be done in the INS-1 cell model, viral vectors have been developed to introduce mutant channels and channel fragments into rat primary beta cells. This proposal will utilize techniques such as patch clamp whole-cell electrophysiology, Fluorescence Lifetime Imaging, Total Internal Reflection Fluorescence Microscopy, insulin secretion assays, immunoprecipitation assays, and western blot assays. This proposal is consistent with the PI's long term goal of understanding L-type calcium channel modulation and cellular function. PUBLIC HEALTH RELEVANCE: This project will explore a set of novel molecules that stimulate beta cells of the pancreas to secret insulin in the presence of glucose, and may also promote the proliferation of beta cells. It will also examine the mechanisms by which the influx of Ca2+ into beta cells through specific types of Ca2+ channels regulates insulin secretion. The results of these studies may provide information for the development of new drugs to treat type 2 diabetes.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

With this award from the Chemistry Research Instrumentation and Facilities: Instrument Development (CRIF:ID) programs, Prof. Garth Simpson, Gregory Hockerman and Chengde Mao of Purdue University will develop a new kind of scanning probe microscope, based upon the dielectrophoretic force. The microscope that they will develop will allow the team to probe the surfaces of nanoscopic and biological samples without contact of the probe tip with the sample. The graduate student researchers working on this project will collaborate with the Jonathon Amy Facility for Chemical Instrumentation on the Purdue University campus. The project also benefits from collaborations with Veeco instruments, a manufacturer of scanning probe microscopes. Prof. Simpson and his colleagues will use the microscope developed in this award as a topic for outreach activities with the wider community, partnering with a number of on-campus programs, including the Bindley Biosciences Program in Purdue's Discovery Park.

New kinds of microscopy techniques open up brand new avenues of research. The instrument developed with this award will enable a new kind of force microscopy, which, unlike other kinds of force microscopies, does not require contact between probe and sample. Technology like that developed in the present award will aid in future advances in chemistry, biotechnology and materials science. In addition, the young scientists working on this project will receive a practical education on bringing new scientific devices from the bench-top to the marketplace.

Summary- Electrical excitability and Ca2+ influx are key features of neurons, endocrine cells, and cardiovascular muscle. However, excessive Ca2+ influx can sensitize these cells to cytotoxic agents, and activate pathological cellular events. Ca influx via Cav1.3 L-type Ca channels is hypothesized to play a key 2+ 2+ role in the death of dopaminergic neurons in Parkinson's Disease, and in cytokine-mediated cell death in pancreatic beta cells during the onset of Type 1 diabetes. Therefore, the development of Cav1.3 inhibitors as potential treatments for Parkinson's disease and diabetes is being pursued. Current drugs that inhibit Cav1.3 also block the closely related channel Cav1.2. Selective Cav1.3 inhibitors would be desirable since they could potentially protect neurons and endocrine cells without inducing hypotension. We've found that the 60 amino acid extracellular IIIS5-3P loops of these channels confer distinct pharmacological properties, and shown that the isolated Cav1.2 IIIS5-3P loop can bind the toxin calcicludine using biolayer interferometry (BLI). In Aim 1, we'll screen a 2.5 x 10 membered, DNA-coded chemical library for binding to the IIIS5-3P loop of Cav1.2 or 8 Cav1.3., confirm binding using BLI, and assess the hits for the ability to selectively inhibit Cav1.2 or Cav1.3 activity using whole-cell electrophysiology. We've also found that the intracellular II-III loop of Cav1.3 selectively enhances inactivation of Cav1.3 but not that of Cav1.2 or a C-terminal truncated splice variant of Cav1.3. In Aim 2, we'll define the minimal peptide sequence within the Cav1.3 II-III loop that confers this activity, and define the role of auxiliary beta subunits and the C-terminal tail of Cav1.3 in the mechanism of action. Completion of these aims will provide structural templates for the development of Cav1.2-selective inhibitors, and two mechanistically distinct classes of Cav1.3-selective inhibitors.