Role for Plasma Membrane-Related Ca2+-ATPase-1 (ATP2C1) in Pancreatic β-Cell Ca2+ Homeostasis Revealed by RNA Silencing

Abstract

Changes in intracellular Ca2+ 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 Ca2+-ATPase, plasma membrane-related Ca2+-ATPase-1 (PMR1; human orthologue ATP2C1) in intracellular Ca2+ homeostasis in pancreatic islet β-cells. Endogenous PMR1 mRNA and protein were detected in both isolated rat islets and β-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 Ca2+ uptake into the endoplasmic reticulum and secretory vesicles by ∼20%, as assessed using organelle-targeted aequorins in permeabilized INS1 cells. In intact cells, PMR1 depletion markedly enhanced flux though L-type Ca2+ channels and augmented glucose-stimulated, but not basal, insulin secretion. Whereas average cytosolic [Ca2+] increases in response to 30.0 mmol/l glucose were unaffected by PMR1 depletion, [Ca2+] 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 Ca2+-ATPases, in the control of β-cell Ca2+ homeostasis and insulin secretion.