Louis Philipson, M.D., Ph.D.

Molecular and Biophysical Aspects of Insulin Secretion; Ion Channels Related to Beta Cell Function; Functional Imaging of Islet Physiology and Exocytosis

Research Summary

Studies on Ion Channels of Insulin Secreting Cells

Our goal is to develop a greater understanding of the relationship of ion channels, regulation of intracellular Ca2+ concentration, and insulin secretion in normal pancreatic ß-cells and how this process may be dysfunctional in diabetes mellitus. The work described here is among the first series of investigations to examine the expression of K+ and Na+ ion channel genes in islet cells. We have also cloned and characterized a new Ca2+ channel alpha1 subunit, which is expressed in both brain and neuroendocrine cells.

Recently we reported the identification of cDNAs encoding delayed rectifier-type K+ channels in human insulin-secreting cells. In addition several other related K+ channel cDNAs were isolated from human genomic and skeletal muscle libraries, some of which have also been shown to be present in normal islets. In collaboration with Dr. D. J. Nelson, our expression studies in Xenopus oocytes and stable cell lines have confirmed the similarity of these currents to those observed in normal cells. We have also shown that these cDNAs encode subunits that can arrange to form active heteromultimers, which also may be an important mechanism in generating the K+ currents present in a variety of tissues, such as brain, heart, and pituitary. Antibodies directed against the channel and a fusion epitope have allowed characterization of the protein in transfected cell lines and transgenic animals. Additional studies have examined the mechanisms of subunit association and inactivation. The expression of one of the human K+ in pancreatic ß-cells of transgenic mice is associated with hyperglycemia, as anticipated. This may provide a model in which to study the role of membrane potential in glucose-induced insulin signalling. Ongoing studies include generation of K+ channel knock-out mice by a dominant-negative approach, examination of the effects of the -subunit, and expression via adenovirus vectors.

Several members of the inward rectifier class have been cloned and expressed. In collaborative studies, we have identified several new cDNAs and their genes encoding new members of this family in insulin secreting cells, including the human ROMK1 ATP-regulated inward rectifier, and GIRK1, a G-protein-linked inward rectifier. An epitope tag has been added to the GIRK1 cDNA, enabling us to study the expression of the protein as we examine its co-expression with G-protein coupled receptors.

Other studies are in progress to examine the role of Na+ and Ca2+ channels in insulin secretion. These studies have shown that a different subset of voltage-dependent Na+ channel gene isoforms (of which five are known) are expressed in islets that are in the adult brain. In a joint project with Prof. R. J. Miller we have cloned and studied the expression of a new Ca2+ channel alpha one subunit alpha 1E. This channel is expressed strongly in multiple nuclei of the brain, in pancreatic ß-cells and neuroendocrine cell lines, and has several splice variants. The intact cDNA is now being examined in several expression systems.

Selected Papers

Tamarina N, Wang Y, Mariotto Y, Bond C, Adelman J, Philipson LH. (2003). Small conductance calcium-activated K+ channels (SK) are expressed in pancreatic islets and regulate glucose responses. Diabetes 52:2000-6.

Bindokas VP, Kuznetsov A, Sreenan S, Polonsky KS, Philipson LH. (2003). Visualizing superoxide production in normal and diabetic rat islets of langerhans. J Biol Chem. 278:9796-9801.

Fridyland LE, Tamarina T, Philipson LH. (2003). Modeling of Ca2+ flux in pancreatic ?-cells: role of plasma membrane and intracellular stores. Am J. Physiol 285:E138-54.

Yaekura K, Julyan R, Wicksteed BL, Hays LB, Alarcon C, Sommers S, Poitout V, Baskin DG, Wang Y, Philipson LH, Rhodes CJ.  (2003). Insulin secretory deficiency and glucose intolerance in rab3a null mice. J Biol Chem. 278:9715-21.

Ueda K, Lipkind G, Kuznetsov A, Philipson LH and Steiner DF. (2003). Mutational analysis of predicted interactions between the catalytic and P domains of prohormone convertase 3 (PC3/PC1). PNAS 100:5622-7.

Fridlyand LE and Philipson LH. (2004). Does the Glucose-dependent Insulin Secretion Mechanism Itself Cause Oxidative Stress in Pancreatic ß-cells? Diabetes, 53(8):1942-8.

Ma L, Bindokas VP, Kuznetsov A, Rhodes C, Hays L, Edwardson JM, Ueda K, Steiner DF and Philipson LH. (2004). Direct Imaging Shows Insulin Granule Exocytosis Occurs by Complete Vesicle Fusion. Proc Natl Acad Sci 101(25):9266-71.

Philipson LH and Roe MW. (2004). Imaging Metabolic and Signaling Targets in the Pancreatic Beta Cell. Current Medicinal Chemistry in press.

Kuznetsov A, Bindokas VP, Marks JD, Philipson LH. (2005). FRET-Based Voltage Probes for Confocal Imaging: Membrane Potential Oscillations Throughout Pancreatic Islets. Am J Physiol Cell Physiol. in press [Epub ahead of print]

Hays LB, Wicksteed B, Wang Y, McCuaig JF, Philipson LH, Edwardson JM and Rhdoes CJ. (2005). Intragranular targeting of syncollin, but not a syncollin-GFP chimera, inhibits regulated insulin exocytsosi in pancreatic beta cells. J. of Endocrinol. In press.

Fridlyand LE, Ma L, Philipson LH. (2005). Adenine Nucleotide Regulation In Pancreatic BetaCells: Modeling of ATP/ADP - Ca2+ Interactions. Am J Physiol Endocrinol Met in press [Epub ahead of print]

Fridlyand LE and Philipson LH. (2005). Reactive Species, Cellular Repair and Risk Factor in the Onset of Type 2 Diabetes Mellitus: Review and Hypothesis. In Press.

Tamarina NA, Kuznetsov A, Fridlyand LE and Philipson LH. (2005). Delayed Rectifie (Kv2.1) Regulation of Pancreatic Beta Cell Calcium Responses to Glucose: Inhibitor Specificity and Modeling. AM. J. Physiol In Press.