Abstract
L-type Ca2+ channels play a key role in the integration of physiological signals regulating insulin secretion that probably requires their localization to specific subdomains of the plasma membrane. We investigated the role of the intracellular II-III loop domains of the L-type channels Cav1.2 and 1.3 in coupling of Ca2+ influx with glucose-stimulated insulin secretion (GSIS) potentiated by the incretin hormone glucagon-like peptide (GLP)-1. In INS-1 cell lines expressing the Cav1.2/II-III or Cav1.3/II-III peptides, GLP-1 potentiation of GSIS was inhibited markedly, coincident with a decrease in GLP-1-stimulated cAMP accumulation and the redistribution of Cav1.2 and Cav1.3 out of lipid rafts. Neither the Cav1.2/II-III nor the Cav1.3/II-III peptide decreased L-type current density compared with untransfected INS-1 cells. GLP-1 potentiation of GSIS was restored by the L-type channel agonist 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester (FPL-64176). In contrast, potentiation of GSIS by 8-bromo-cAMP (8-Br-cAMP) was inhibited in Cav1.2/II-III but not Cav1.3/II-III cells. These differences may involve unique protein-protein interactions because the Cav1.2/II-III peptide, but not the Cav1.3/II-III peptide, immunoprecipitates Rab3-interacting molecule (RIM) 2 from INS-1 cell lysates. RIM2, and its binding partner Piccolo, localize to lipid rafts, and they may serve as anchors for Cav1.2 localization to lipid rafts in INS-1 cells. These findings suggest that the II-III interdomain loops of Cav1.2, and possibly Cav1.3, direct these channels to membrane microdomains in which the proteins that mediate potentiation of GSIS by GLP-1 and 8-Br-cAMP assemble.
Footnotes
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This work was supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant R01-DK064736] (to G.H.H.); a Purdue Research Foundation award (to S.M.P.J. and G.H.H.); and a Showalter Trust Award (to G.H.H. and V.J.W.).
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This work was in partial fulfillment of the requirements for Jacobo SMP (2008) Dichotomy of Cav1.2 and Cav1.3 L-type Ca2+ channel function and modulation in GLP-1-mediated insulin exocytosis and ERK1/2 activation in pancreatic beta cells. Ph.D. thesis, Purdue University, West Lafayette, Indiana.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.109.150672.
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ABBREVIATIONS: GSIS, glucose-stimulated insulin secretion; L-VGCC, L-type voltage-gated Ca2+ channel; 8-Br-cAMP, 8-bromo-cAMP; EPAC2, exchange protein activated by cAMP2; RIM, Rab3-interacting molecule; GLP-1R, glucagon-like peptide-1 receptor; GLP, glucagon-like peptide; PBS, phosphate-buffered saline; KRBH Krebs-Ringer bicarbonate HEPES; BSA, bovine serum albumin; BCA, bicinchoninic acid; GFP, green fluorescent protein; PAGE, polyacrylamide gel electrophoresis; PBST, phosphate-buffered saline containing 0.1% Tween 20; MES, 2-(N-morpholino)ethanesulfonic acid; IBa, barium current; FPL-64176, 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester; mβCD, methyl-β-cyclodextrin.
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- Received January 8, 2009.
- Accepted April 6, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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