Research Interests: My laboratory is focused on the molecular mechanisms which regulate exocytosis, using two models- the neuroendocrine insulin-secreting pancreatic islet beta cell and the epithelial pancreatic acinar cell which secrete digestive enzymes. We are mainly interested in SNARE proteins, originally described to regulate neurotransmitter release, but subsequently found to be highly conserved in neuroendocrine and non-excitable secretory cells to regulate secretion. We were the first to identify the combinations of SNARE proteins which mediate the distinct exocytic events in the acinar cell (apical and basolateral exocytosis, homotypic granule fusion) and the pancreatic islet beta cell. In the islet beta cell, we proceeded to demonstrate that these SNARE proteins could independently bind and regulate potassium channels which control membrane excitability, and thereby regulate the fine sequence of ionic and exocytic events leading to secretion. We are currently examining the precise functional interacting domains between these SNARE proteins and ion channels (K+ and Ca2+) in the hope that we may identify specific drug targets to treat diseases (diabetes) which may have as their basis a dysregulation of exocytosis or ion channels. We are also exploring the specific molecules which interact with SNARE proteins to mediate the priming of insulin granules which could amplify insulin secretion in the sluggish diabetic islet beta cells. We have now generated or acquired transgenic mouse models with altered expression of SNARE and associated proteins to unequivocally elucidate their function in these tissues. Insights from my research are therefore of direct impact to normal secretory biology and pathobiology, specifically in understanding (and rescuing) the dysregulated insulin secretion in diabetes and pathologic membrane fusion in pancreatitis.
My lab has in place molecular biological techniques to examine structure-function, viral gene transfer into primary cells in culture and in vivo into organs (pancreas), biochemical protein binding assays, cell biology methods (confocal microscopy, islet secretion assays), and importantly, single cell analyses by patch-clamp electrophysiology (including capacitance measurements) to directly study ion channel function, and state-of-the art fluorescent imaging techniques (TIRFM, multi-photon, spinning disc confocal, super-resolution imaging) to visualize and track single secretory granule exocytosis and molecular interactions between SNARE and ion channels.
Cell and tissue culture: Pancreatic slice, cardiomyocytes, pancreas cells
Procedures: Adenovirus, electrophysiology, ELISA, glucose clamp, immunohistochemistry, immunocytochemistry, in-vitro electrophysiology, intracellular injection, patch clamp, qRT-PCR, RIA, RT-PCR, signal transduction characterization, siRNA, voltage clamp, western blot.
Amplifier, analytical balances, benchtop centrifuge, blotting apparatus, calcium imaging system, confocal microscope, culture hood, culture incubators, departmental beta and gamma counters, dissecting microscope, electrophysiology rig, EMCCD, fluorescence microscope, gel apparatus, low- and high-speed centrifuge, low and ultralow freezers, micropipette puller, plate reader, real-time/thermocycler, setups for electropherosis, stirrer/hot plate, vibratome, water baths.