This Article Full Text Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Safayhi, H. Articles by Striessnig, J. Search for Related Content PubMed PubMed Citation Articles by Safayhi, H. Articles by Striessnig, J. Pubmed/NCBI databases Compound via MeSH Substance via MeSH

L-Type Calcium Channels in Insulin-Secreting Cells: Biochemical Characterization and Phosphorylation in RINm5F Cells

Pharmazeutisches Institut, Lehrstuhl Pharmakologie (H.S., U.K., A.B., M.R., H.P.T.A.) Universität Tübingen D-72076 Tübingen, Germany
Max Delbrück Centrum für Molekulare Medizin (H.H., I.M.) Cardiology D-13125 Berlin, Germany
Pfizer (M.K.A.) Central Research Division Groton, Connecticut 06340
Institut für Biochemische Pharmakologie (J.S., M.F., T.N.C.) Universität Innsbruck A-6020 Innsbruck, Austria

Opening of dihydropyridine-sensitive voltage-dependent L-type Ca²⁺-channels (LTCCs) represents the final common pathway for insulin secretion in pancreatic ß-cells and related cell lines. In insulin-secreting cells their exact subunit composition is unknown. We therefore investigated the subunit structure of (+)-[³H]isradipine-labeled LTCCs in insulin-secreting RINm5F cells. Using subunit-specific antibodies we demonstrate that 1C subunits (199 kDa, short form) contribute only a minor portion of the total 1 immunoreactivity in membranes and partially purified Ca²⁺-channel preparations. However, 1C forms a major constituent of (+)-[³H]isradipine-labeled LTCCs as 54% of solubilized (+)-[³H]isradipine-binding activity was specifically immunoprecipitated by 1C antibodies. Phosphorylation of immunopurified 1C with cAMP-dependent protein kinase revealed the existence of an additional 240-kDa species (long form), that remained undetected in Western blots. Fifty seven percent of labeled LTCCs were immunoprecipitated by an anti-ß-antibody directed against all known ß-subunits. Isoform-specific antibodies revealed that these mainly corresponded to ß1b- and ß3-subunits. We found ß2- and ß4-subunits to be major constituents of cardiac and brain L-type channels, respectively, but not part of L-type channels in RINm5F cells. We conclude that 1C is a major constituent of dihydropyridine-labeled LTCCs in RINm5F cells, its long form serving as a substrate for cAMP-dependent protein kinase. ß1b- and ß3-Subunits were also found to associate with L-type channels in these cells. These isoforms may therefore represent biochemical targets for the modulation of LTCC activity in RINm5F cells.

This article has been cited by other articles:

				M. D. Nitert, C. L F Nagorny, A. Wendt, L. Eliasson, and H. Mulder CaV1.2 rather than CaV1.3 is coupled to glucose-stimulated insulin secretion in INS-1 832/13 cells J. Mol. Endocrinol., July 1, 2008; 41(1): 1 - 11. [Abstract] [Full Text] [PDF]

				N. Baroukh, M. A. Ravier, M. K. Loder, E. V. Hill, A. Bounacer, R. Scharfmann, G. A. Rutter, and E. Van Obberghen MicroRNA-124a Regulates Foxa2 Expression and Intracellular Signaling in Pancreatic beta-Cell Lines J. Biol. Chem., July 6, 2007; 282(27): 19575 - 19588. [Abstract] [Full Text] [PDF]

				V. Navarro-Tableros, T. Fiordelisio, A. Hernandez-Cruz, and M. Hiriart Physiological development of insulin secretion, calcium channels, and GLUT2 expression of pancreatic rat beta-cells Am J Physiol Endocrinol Metab, April 1, 2007; 292(4): E1018 - E1029. [Abstract] [Full Text] [PDF]

				S.-N. Yang and P.-O. Berggren The Role of Voltage-Gated Calcium Channels in Pancreatic {beta}-Cell Physiology and Pathophysiology Endocr. Rev., October 1, 2006; 27(6): 621 - 676. [Abstract] [Full Text] [PDF]

				G. A. Rutter and E. V. Hill Insulin Vesicle Release: Walk, Kiss, Pause ... Then Run Physiology, June 1, 2006; 21(3): 189 - 196. [Abstract] [Full Text] [PDF]

				S. Vignali, V. Leiss, R. Karl, F. Hofmann, and A. Welling Characterization of voltage-dependent sodium and calcium channels in mouse pancreatic A- and B-cells J. Physiol., May 1, 2006; 572(3): 691 - 706. [Abstract] [Full Text] [PDF]

				S. Seino and T. Shibasaki PKA-Dependent and PKA-Independent Pathways for cAMP-Regulated Exocytosis Physiol Rev, October 1, 2005; 85(4): 1303 - 1342. [Abstract] [Full Text] [PDF]

				J.-C. Hoda, F. Zaghetto, A. Koschak, and J. Striessnig Congenital Stationary Night Blindness Type 2 Mutations S229P, G369D, L1068P, and W1440X Alter Channel Gating or Functional Expression of Cav1.4 L-type Ca2+ Channels J. Neurosci., January 5, 2005; 25(1): 252 - 259. [Abstract] [Full Text] [PDF]

				S.-N. Yang and P.-O. Berggren {beta}-Cell CaV channel regulation in physiology and pathophysiology Am J Physiol Endocrinol Metab, January 1, 2005; 288(1): E16 - E28. [Abstract] [Full Text] [PDF]

				M. Grunnet and W. A. Kaufmann Coassembly of Big Conductance Ca2+-activated K+ Channels and L-type Voltage-gated Ca2+ Channels in Rat Brain J. Biol. Chem., August 27, 2004; 279(35): 36445 - 36453. [Abstract] [Full Text] [PDF]

				I. Leclerc, W. W. Woltersdorf, G. da Silva Xavier, R. L. Rowe, S. E. Cross, G. S. Korbutt, R. V. Rajotte, R. Smith, and G. A. Rutter Metformin, but not leptin, regulates AMP-activated protein kinase in pancreatic islets: impact on glucose-stimulated insulin secretion Am J Physiol Endocrinol Metab, June 1, 2004; 286(6): E1023 - E1031. [Abstract] [Full Text] [PDF]

				K. J. Mitchell, T. Tsuboi, and G. A. Rutter Role for Plasma Membrane-Related Ca2+-ATPase-1 (ATP2C1) in Pancreatic {beta}-Cell Ca2+ Homeostasis Revealed by RNA Silencing Diabetes, February 1, 2004; 53(2): 393 - 400. [Abstract] [Full Text]

				T. Tsuboi, G. da Silva Xavier, I. Leclerc, and G. A. Rutter 5'-AMP-activated Protein Kinase Controls Insulin-containing Secretory Vesicle Dynamics J. Biol. Chem., December 26, 2003; 278(52): 52042 - 52051. [Abstract] [Full Text] [PDF]

				A. Koschak, D. Reimer, D. Walter, J.-C. Hoda, T. Heinzle, M. Grabner, and J. Striessnig Cav1.4{alpha}1 Subunits Can Form Slowly Inactivating Dihydropyridine-Sensitive L-Type Ca2+ Channels Lacking Ca2+-Dependent Inactivation J. Neurosci., July 9, 2003; 23(14): 6041 - 6049. [Abstract] [Full Text] [PDF]

				G. Liu, N. Dilmac, N. Hilliard, and G. H. Hockerman Cav1.3 Is Preferentially Coupled to Glucose-Stimulated Insulin Secretion in the Pancreatic beta -Cell Line INS-1 J. Pharmacol. Exp. Ther., April 1, 2003; 305(1): 271 - 278. [Abstract] [Full Text]

				K. J. Mitchell, F. A. Lai, and G. A. Rutter Ryanodine Receptor Type I and Nicotinic Acid Adenine Dinucleotide Phosphate Receptors Mediate Ca2+ Release from Insulin-containing Vesicles in Living Pancreatic beta -Cells (MIN6) J. Biol. Chem., March 21, 2003; 278(13): 11057 - 11064. [Abstract] [Full Text] [PDF]

				P. Pinton, T. Tsuboi, E. K. Ainscow, T. Pozzan, R. Rizzuto, and G. A. Rutter Dynamics of Glucose-induced Membrane Recruitment of Protein Kinase C beta II in Living Pancreatic Islet beta -Cells J. Biol. Chem., September 27, 2002; 277(40): 37702 - 37710. [Abstract] [Full Text] [PDF]

				C. Andreolas, G. da Silva Xavier, F. Diraison, C. Zhao, A. Varadi, F. Lopez-Casillas, P. Ferre, F. Foufelle, and G. A. Rutter Stimulation of Acetyl-CoA Carboxylase Gene Expression by Glucose Requires Insulin Release and Sterol Regulatory Element Binding Protein 1c in Pancreatic MIN6 {beta}-Cells Diabetes, August 1, 2002; 51(8): 2536 - 2545. [Abstract] [Full Text] [PDF]

				A. Pereverzev, M. Mikhna, R. Vajna, C. Gissel, M. Henry, M. Weiergraber, J. Hescheler, N. Smyth, and T. Schneider Disturbances in Glucose-Tolerance, Insulin-Release, and Stress-Induced Hyperglycemia upon Disruption of the Cav2.3 ({alpha}1E) Subunit of Voltage-Gated Ca2+ Channels Mol. Endocrinol., April 1, 2002; 16(4): 884 - 895. [Abstract] [Full Text] [PDF]

				A. Varadi, E. K. Ainscow, V. J. Allan, and G. A. Rutter Involvement of conventional kinesin in glucose-stimulated secretory granule movements and exocytosis in clonal pancreatic {beta}-cells J. Cell Sci., January 11, 2002; 115(21): 4177 - 4189. [Abstract] [Full Text] [PDF]

				H. Grabsch, A. Pereverzev, M. Weiergräber, M. Schramm, M. Henry, R. Vajna, R. E. Beattie, S. G. Volsen, U. Klöckner, J. Hescheler, et al. Immunohistochemical Detection of {alpha}1E Voltage-gated Ca2+ Channel Isoforms in Cerebellum, INS-1 Cells, and Neuroendocrine Cells of the Digestive System J. Histochem. Cytochem., August 1, 1999; 47(8): 981 - 994. [Abstract] [Full Text]

				L. B. Lester, L. K. Langeberg, and J. D. Scott Anchoring of protein kinase A facilitates hormone-mediated insulin secretion PNAS, December 23, 1997; 94(26): 14942 - 14947. [Abstract] [Full Text] [PDF]

				A. Koschak, D. Reimer, I. Huber, M. Grabner, H. Glossmann, J. Engel, and J. Striessnig alpha 1D (Cav1.3) Subunits Can Form L-type Ca2+ Channels Activating at Negative Voltages J. Biol. Chem., June 15, 2001; 276(25): 22100 - 22106. [Abstract] [Full Text] [PDF]

				K. J. Mitchell, P. Pinton, A. Varadi, C. Tacchetti, E. K. Ainscow, T. Pozzan, R. Rizzuto, and G. A. Rutter Dense core secretory vesicles revealed as a dynamic Ca2+ store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera J. Cell Biol., October 1, 2001; 155(1): 41 - 52. [Abstract] [Full Text] [PDF]