Cav1.2-mediated Calcium-induced Calcium Release and Regulation of cAMP Dynamics and ERK Activation in Pancreatic Beta-cells

Evan Pratt, Purdue University

Abstract

L-type voltage-gated Ca2+ channels (L-VGCC) are required for pancreatic β-cell function and regulation of pancreatic β-cell mass. Although the two L-VGCCs expressed in pancreatic β-cells, Ca v1.2 and Cav1.3, are structurally very similar, there is evidence that the channels are functionally distinct. One the most divergent regions between the amino acid sequences of Cav1.2 and Ca v1.3 is the II-III intracellular loop. This region of Cav1.2 is required for interactions with SNARE and scaffolding proteins and coupling of Cav1.2-mediated Ca2+ influx with downstream signaling events. We previously demonstrated that expression of the Cav1.2 II-III loop in INS-1 cells (Cav1.2/II-III cells) disrupts proper localization of the channel to lipid raft domains and perturbs Cav1.2-mediated signaling. To investigate the role of the L-VGCC Cav1.2 in INS-1 cells, I examined Ca2+ dynamics in response to the sulfonylurea tolbutamide in Cav1.2/II-III cells. Using the cytosolic Ca2+ indicator Fura2, I found that tolbutamide-stimulated Ca2+ dynamics are sensitive to the L-VGCC blocker nicardipine and the ryanodine receptor (RyR) inhibitor ryanodine in control INS-1 cells. Furthermore, I showed that while the Ca2+ response was blunted by the SERCA inhibitor thapsigargin in control INS-1 cells, Cav1.2/II-III cells remained resistant to ER Ca2+ depletion, suggesting that ER Ca 2+ release does not contribute to Ca2+ dynamics in these cells. Indeed, using the ER-targeted Ca2+ sensor D1ER, I found that the tolbutamide-stimulated drop in ER Ca2+ was diminished in Cav1.2/II-III cells. Thus, my results suggest that Cav1.2-mediated Ca2+ influx is normally coupled with RyR-mediated Ca2+ release in INS-1 cells, and this is disrupted in Cav1.2/II-III cells. Glucose-stimulated cAMP accumulation (GS-cAMP) and ERK phosphorylation (GSEP) are driven by L-VGCC-mediated Ca2+ influx; however, whether Ca2+-induced Ca2+ release (CICR) is required to amplify both processes is unclear. Using fluorescence resonance energy transfer (FRET)-based sensors of cAMP levels and ERK activity, I found that GS-cAMP and GSEP were diminished in Cav1.2/II-III cells as well as in control INS-1 cells treated with ryanodine. Furthermore, I showed that the Epac1 inhibitor CE3F4 abolished GSEP, suggesting that Epac1 provides a link between GS-cAMP and GSEP in INS-1 cells. Taken together, Cav1.2-mediated CICR is required for efficient cAMP generation and Epac1-mediated ERK activation in response to glucose. cAMP-mediated activation Epac has been shown to enhance glucose-stimulated insulin secretion (GSIS) by increasing intracellular Ca2+ levels. Using Fura2 Ca2+ imaging, I found that an Epac-selective cAMP agonist (ESCA) potentiated Ca2+ dynamics and insulin secretion stimulated with tolbutamide and dose-dependently elevated intracellular Ca 2+ levels independently. Furthermore, the effects of ESCA were abolished by the TRP channel blocker 2-APB. I investigated whether 2-APB was selective for TRP channels in INS-1 cells and found that 2-APB inhibited the second phase of Ca2+ dynamics stimulated with carbachol; however, it did not inhibit SOCE in these cells. Therefore, we propose that the source of extracellular Ca2+ that is stimulated by ESCA is a TRPC channel. cAMP levels are tightly controlled by phosphodiesterase (PDE) activity. Earlier reports have suggested that the Ca2+-calmodulin regulated subtype PDE1 as well as PDE3 and PDE4 compartmentalize cAMP signaling in pancreatic β-cells; however, these studies have yielded conflicting results. I evaluated the role of PDE1, PDE3 and PDE4 in INS-1 cells and human pancreatic β-cells using subtype-selective inhibitors. Using a FRET-based sensor of cAMP, I found that PDE1 is the primary subtype in INS-1 cells, whereas PDE3 appears to be required in human pancreatic β-cells. Furthermore, measurements of GSIS revealed that PDE1 regulates cAMP-dependent insulin secretion in both cell types. I also demonstrated that PDE1 inhibition can rescue INS-1 cells from lipotoxicity. Collectivity, this work highlights the interplay of Ca2+ signaling and cAMP signaling in pancreatic β-cells, and this has drastic effects on pancreatic β-cell proliferation and survival.

Degree

Ph.D.

Advisors

Hockerman, Purdue University.

Subject Area

Cellular biology|Pharmacology

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