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.
The endoplasmic reticulum (ER) plays a pivotal role in the regulation of cytosolic Ca(2+) concentrations ([Ca(2+)](cyt)) and hence in insulin secretion from pancreatic beta-cells. However, the molecular mechanisms involved in both the uptake and release of Ca(2+) 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 Ca(2+) fluxes across the ER membrane in single live MIN6 beta-cells, we have imaged changes in the ER intralumenal free Ca(2+) concentration ([Ca(2+)](ER)) using ER-targeted cameleons. Resting [Ca(2+)](ER) (approximately 250 micromol/l) was markedly reduced after suppression (by approximately 40%) of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)-2b but not the SERCA3 isoform by microinjection of antisense oligonucleotides, implicating SERCA2b as the principle ER Ca(2+)-ATPase in this cell type. Nutrient secretagogues that elevated [Ca(2+)](cyt) also increased [Ca(2+)](ER), an effect most marked at the cell periphery, whereas inositol 1,4,5-trisphosphate-generating agents caused a marked and homogenous lowering of [Ca(2+)](ER). Demonstrating the likely presence of ryanodine receptors (RyRs), caffeine and 4-chloro-3-ethylphenol both caused an almost complete emptying of ER Ca(2+) and marked increases in [Ca(2+)](cyt). Furthermore, photolysis of caged cyclic ADP ribose increased [Ca(2+)](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 beta-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 Ca(2+) changes in the beta-cell and thus in the regulation of insulin secretion.
Varadi
Aniko
A
Department of Biochemistry, University of Bristol, Bristol, U.K.
Rutter
Guy A
GA
D001896:Boron Compounds; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002733:Chlorophenols / Q000494:pharmacology; D018679:Cholinergic Agonists / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005456:Fluorescent Dyes; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007340:Insulinoma; D007515:Islets of Langerhans / Q000378:metabolism*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010777:Photochemistry; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013194:Staining and Labeling; D014162:Transfection; D014407:Tumor Cells, Cultured
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D015220:Calcium Channels; D002733:Chlorophenols; D018679:Cholinergic Agonists; D005456:Fluorescent Dyes; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007328:Insulin; D010726:Phosphodiesterase Inhibitors; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C091710:4-chloro-3-ethylphenol; D002110:Caffeine; D002217:Carbachol; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506949:ATP2A3 protein, human; C506950:Atp2a3 protein, mouse; D000252:Calcium-Transporting ATPases; D002118:Calcium