Specific coupling of L-type voltage-gated calcium channels to signaling events in pancreatic β Cells

Marcy L Guerra, Purdue University

Abstract

Influx of calcium ions through L-type voltage-gated calcium channels (VGCCs) stimulates insulin secretion and ERK1/2 phosphorylation in pancreatic β cells. The L-type channels, Cav1.2 and Cav1.3, are both present in β cells and insulin-secreting cell lines, but their specific roles in regulating insulin secretion and ERK phosphorylation are not clear. The α1 pore-forming subunits of Cav1.2 and Cav1.3 are composed of four highly conserved hexahelical transmembrane domains, however, the intracellular loop connecting domains II and III (II-III loop) share only about 43% amino acid identity. The II-III loops of several VGCCS are known to interact with proteins involved in signaling thereby coupling calcium influx to cellular responses. I identified proteins that specifically associated with the II-III loop of either Cav1.2 or Cav1.3 in INS-1 cells. The II-III loop of Cav1.2 immunoprecipitated the proteins Rab-3 interacting molecule (Rim) 2, Piccolo, and IQGAP1. Kir6.2, the pore-forming subunit of the KATP channel, was the only protein identified that immunoprecipitated with the II-III loop of Cav1.3. We have previously shown that Cav1.2 and Cav1.3 reside in lipid rafts and that overexpressing the II-III loop of either channel specifically shifts the corresponding channel out of rafts. The immunoprecipitated proteins were also found to reside in lipid rafts where they most likely act as anchors to localize Cav1.2 and Cav1.3 to rafts through interactions with the II-III loop regions. I investigated the functional consequence of overexpressing the II-III loops of Cav1.2 and Cav1.3 by assessing sulfonylurea-stimulated insulin secretion. I determined that the sulfonylureas tolbutamide and gliclazide elicit insulin secretion by different mechanisms of action. Tolbuatmide’s response was dependent upon release of calcium from intracellular stores while gliclazide’s was not. In INS-1cells overexpressing the II-III loop of Cav1.2 (Cav1.2/II-III loop cells), the tolbutamide response occurred independently of release of calcium from intracellular stores and was mediated completely by influx of calcium through P/Q-type channels, but gliclazide was significantly less effective at stimulating secretion compared to responses observed in the WT INS-1 or Cav1.3/II-III loop cells. Sulfonylurea-stimulated secretion in the INS-1 cells overexpressing the IIIII loop of Cav1.3 (Cav1.3/II-III loop cells) was unaltered. The differences observed with the sulfonylurea response in the Cav1.2/II-III loop cells were not associated with changes in electrical activity but were correlated with reduced intracellular calcium levels. I also investigated the ability of glucose and GLP-1 to stimulate ERK1/2 phosphorylation. In Cav1.2/II-III loop cells ERK1/2 phosphorylation occurred on a slower time scale and reduced amplitude in response to glucose and potentiation by GLP-1. Reduced ERK1/2 activation in the Cav1.2/II-III loop cells correlated with a reduced rise in intracellular calcium in response to glucose and GLP-1. GLP-1 alone did not significantly stimulate ERK1/2 phosphorylation in the Cav1.2/II-III loop cells which could be explained by reduced cAMP accumulation in response to GLP-1. The deficits observed in the Ca v1.2/II-III loop cells were not demonstrated in Cav1.3/II-III loop cells. The reduced ability of glucose and/or GLP-1 could also have resulted from disrupting the IQGAP1-Cav1.2 interaction since IQGAP1 has been shown to be a scaffold for the ERK1/2 pathway. Our observations that impairments in sulfonylurea-stimulated insulin secretion and ERK1/2 phosphorylation coincides with the specific displacement of Cav1.2 from lipid rafts, but not Cav1.3, suggests that the II-III loop domains couple the channels to distinct signaling pathways that are dependent upon specific protein-protein interactions. Deficits associated with type II diabetes may result from the uncoupling of calcium influx through L-type channels from critical signaling pathways.

Degree

Ph.D.

Advisors

Hockerman, Purdue University.

Subject Area

Pharmacology

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