Dynamic imaging of endoplasmic reticulum Ca2+ concentration in insulin-secreting MIN6 cells using recombinant targeted cameleons: Roles of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)-2 and ryanodine receptors

Varadi, Aniko; Rutter, Guy A.

Abstract

The endoplasmic reticulum (ER) plays a pivotal role in the regulation of cytosolic Ca2+ concentrations ([Ca2+]cyt) and hence in insulin secretion from pancreatic β-cells. However, the molecular mechanisms involved in both the uptake and release of Ca2+ from the ER are only partially defined in these cells, and the presence and regulation of ER ryanodine receptors are a matter of particular controversy. To monitor Ca2+ fluxes across the ER membrane in single live MIN6 β-cells, we have imaged changes in the ER intraluminal free Ca2+ concentration ([Ca2+]ER) using ER-targeted cameleons. Resting [Ca2+]ER (∼250 μmol/l) was markedly reduced after suppression (by ∼40%) of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)-2b but not the SERCA3 isoform by microinjection of antisense oligonucleotides, implicating SERCA2b as the principle ER Ca2+-ATPase in this cell type. Nutrient secretagogues that elevated [Ca2+]cyt also increased [Ca2+]ER, an effect most marked at the cell periphery, whereas inositol 1,4,5-trisphosphate-generating agents caused a marked and homogenous lowering of [Ca2+]ER. Demonstrating the likely presence of ryanodine receptors (RyRs), caffeine and 4-chloro-3-ethylphenol both caused an almost complete emptying of ER Ca2+ and marked increases in [Ca2+]cyt. Furthermore, photolysis of caged cyclic ADP ribose increased [Ca2+]cyt, and this effect was largely abolished by emptying ER/Golgi stores with thapsigargin. Expression of RyR protein in living MIN6, INS-1, and primary mouse β-cells was also confirmed by the specific binding of cell-permeate BODIPY TR-X ryanodine. RyR channels are likely to play an important part in the regulation of intracellular free Ca2+ changes in the β-cell and thus in the regulation of insulin secretion.

• Journal Article Type: Article
• Publication Date: Feb 1, 2002
• Journal: Diabetes
• Print ISSN: 0012-1797
• Electronic ISSN: 1939-327X
• Publisher: American Diabetes Association
• Peer Reviewed: Peer Reviewed
• Volume: 51
• Issue: S1
• Pages: 190-201
• DOI: https://doi.org/10.2337/diabetes.51.2007.S190
• Keywords: dynamic imaging, endoplasmic reticulum Ca2+, insulin-secreting MIN6 cells, recombinant targeted cameleons, sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)-2, ryanodine receptors
• Public URL: https://uwe-repository.worktribe.com/output/1079451
• Publisher URL: http://dx.doi.org/10.2337/diabetes.51.2007.S190