Mitochondrial Ca2+ uptake and not mitochondrial motility is required for STIM1-Orai1-dependent store-operated Ca2+ entry.
Store-operated Ca(2+) entry (SOCE) is established by formation of subplasmalemmal clusters of the endoplasmic reticulum (ER) protein, stromal interacting molecule 1 (STIM1) upon ER Ca(2+) depletion. Thereby, STIM1 couples to plasma membrane channels such as Orai1. Thus, a close proximity of ER domains to the plasma membrane is a prerequisite for SOCE activation, challenging the concept of local Ca(2+) buffering by mitochondria as being essential for SOCE. This study assesses the impact of mitochondrial Ca(2+) handling and motility on STIM1-Orai1-dependent SOCE. High-resolution microscopy showed only 10% of subplasmalemmal STIM1 clusters to be colocalized with mitochondria. Impairments of mitochondrial Ca(2+) handling by inhibition of mitochondrial Na(+)-Ca(2+) exchanger (NCX(mito)) or depolarization only partially suppressed Ca(2+) entry in cells overexpressing STIM1-Orai1. However, SOCE was completely abolished when both NCX(mito) was inhibited and the inner mitochondrial membrane was depolarized, in STIM1- and Orai1-overexpressing cells. Immobilization of mitochondria by expression of mAKAP-RFP-CAAX, a construct that physically links mitochondria to the plasma membrane, affected the Ca(2+) handling of the organelles but not the activity of SOCE. Our observations indicate that mitochondrial Ca(2+) uptake, including reversal of NCX(mito), is fundamental for STIM1-Orai1-dependent SOCE, whereas the proximity of mitochondria to STIM1-Orai1 SOCE units and their motility is not required.
Naghdi
Shamim
S
Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, 8010 Graz, Austria.
Waldeck-Weiermair
Markus
M
Fertschai
Ismene
I
Poteser
Michael
M
Graier
Wolfgang F
WF
Malli
Roland
R
D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D000071740:ORAI1 Protein; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000071737:Stromal Interaction Molecule 1
D015220:Calcium Channels; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D002118:Calcium