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- W4313396935 abstract "During ischemia, the decreased activity of Ca2+ ATPases pumps caused by the drop in available Mg2 + -ATP and the endoplasmic reticulum (ER) calcium leakage resulting from the autonomous activity ion channels both account for ER Ca2+ leakage. This latter induces Ca2+ accumulation in both the cytosol and mitochondria. In non-excitable cells, the main Ca2+ release channel is the inositol 1,4,5-trisphosphate receptor (IP3Rs). Yet it still remains unclear whether IP3Rs participate in ER Ca2+ leakage during ischemia. By mean of the development of a Ca2+ -binding biosensor addressed to the outer mitochondrial membrane, we assessed the endogenous activity of IP3R in mitochondria-associated membranes (MAMs) during ischemia. •N33D3cpv construction by molecular biology: exchange of D1 by D3 Ca2 + −binding domain in N33D1cpv plasmid.•Cell culture of wt and TKO Hela cells. Lipofection of biosensors-encoding plasmids: D1cpv and D3cpv (cytosol), N33D1cpv and N33D3cpv (OMM).•Immunoblotting.•Wield field fluorescence imaging of Ca2 + −binding sensors in cells stimulated either by Na + -ATP, Cyclopiazonic acid or Oxygen/Glucose deprivation (OGD). We assembled a new mitochondrial-surface GFP-based Ca2+ indicator derived from the GFP-based Ca2+ biosensor N33D1cpv, with a higher affinity for Ca2+ allowing sensitive Ca2+ measurements at the cytosolic surface of the OMM. Comparative analysis demonstrated the superiority of this sensor over N33D1cpv to measure low increase in [Ca2+] in MAM, regardless the stimulus used (i.e.: Na + -ATP, Cyclopiazonic acid or OGD). We next showed that either the genetic suppression or a pharmacological inhibition of endogenous IP3R activity reduced the speed of ER Ca2+ leakage during hypoxia with a greater effect in MAMs than in the cytosol. By mean of the new N33D3cpv biosensor, we confirmed that, during hypoxia, the autonomous IP3R activity during hypoxia participates to the ER Ca2+ leakage towards MAMs leading ultimately to mitochondrial Ca2+ overload." @default.
- W4313396935 created "2023-01-06" @default.
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- W4313396935 date "2022-12-01" @default.
- W4313396935 modified "2023-09-26" @default.
- W4313396935 title "High-sensitivity calcium biosensor on the mitochondrial surface reveals that IP3R channels participate to the reticular Ca2+leak towards mitochondria" @default.
- W4313396935 doi "https://doi.org/10.1016/j.yjmcc.2022.08.070" @default.
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