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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

From the Department of Biochemistry, University of Bristol, Bristol, U.K.

The endoplasmic reticulum (ER) plays a pivotal role in the regulation of cytosolic Ca²⁺ concentrations ([Ca²⁺]_cyt) and hence in insulin secretion from pancreatic ß-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 ß-cells, we have imaged changes in the ER intralumenal 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 ß-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 ß-cell and thus in the regulation of insulin secretion.

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