Role for plasma membrane-related Ca2+-ATPase-1 (ATP2C1) in pancreatic beta-cell Ca2+ homeostasis revealed by RNA silencing.
Changes in intracellular Ca(2+) concentration play a key role in the regulation of insulin secretion by glucose and other secretagogues. Here, we explore the importance of the secretory pathway Ca(2+)-ATPase, plasma membrane-related Ca(2+)-ATPase-1 (PMR1; human orthologue ATP2C1) in intracellular Ca(2+) homeostasis in pancreatic islet beta-cells. Endogenous PMR1 mRNA and protein were detected in both isolated rat islets and beta-cell-derived lines (MIN6 and INS1). Subcellular fractionation of the cell lines revealed PMR1 immunoreactivity in both microsomal and dense-core secretory vesicle-enriched fractions. Correspondingly, depletion of cellular PMR1 with small interfering RNAs inhibited Ca(2+) uptake into the endoplasmic reticulum and secretory vesicles by approximately 20%, as assessed using organelle-targeted aequorins in permeabilized INS1 cells. In intact cells, PMR1 depletion markedly enhanced flux though L-type Ca(2+) channels and augmented glucose-stimulated, but not basal, insulin secretion. Whereas average cytosolic [Ca(2+)] increases in response to 30.0 mmol/l glucose were unaffected by PMR1 depletion, [Ca(2+)] oscillation shape, duration, and decay rate in response to glucose plus tetraethylammonium were modified in PMR1-depleted single cells, imaged using fluo-3-acetoxymethylester. PMR1 thus plays an important role, which is at least partially nonoverlapping with that of sarco(endo-)plasmic reticulum Ca(2+)-ATPases, in the control of beta-cell Ca(2+) homeostasis and insulin secretion.
Mitchell
Kathryn J
KJ
Henry Wellcome Laboratories of Integrated Cell Signaling and Department of Biochemistry, School of Medical Sciences, University Walk, University of Bristol, Bristol, U.K.
Tsuboi
Takashi
T
Rutter
Guy A
GA
D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology*; D002478:Cells, Cultured; D020868:Gene Silencing; D006706:Homeostasis; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology / Q000201:enzymology / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000201:enzymology
D018528:ATP-Binding Cassette Transporters; C492023:Atp2c1 protein, rat; D007328:Insulin; D034741:RNA, Small Interfering; C403624:ATP2C1 protein, human; D000252:Calcium-Transporting ATPases; D002118:Calcium