Role of phosphoinositides in STIM1 dynamics and store-operated calcium entry.
Ca2+ entry through store-operated Ca2+ channels involves the interaction at ER-PM (endoplasmic reticulum-plasma membrane) junctions of STIM (stromal interaction molecule) and Orai. STIM proteins are sensors of the luminal ER Ca2+ concentration and, following depletion of ER Ca2+, they oligomerize and translocate to ER-PM junctions where they form STIM puncta. Direct binding to Orai proteins activates their Ca2+ channel function. It has been suggested that an additional interaction of the C-terminal polybasic domain of STIM1 with PM phosphoinositides could contribute to STIM1 puncta formation prior to binding to Orai. In the present study, we investigated the role of phosphoinositides in the formation of STIM1 puncta and SOCE (store-operated Ca2+ entry) in response to store depletion. Treatment of HeLa cells with inhibitors of PI3K (phosphatidylinositol 3-kinase) and PI4K (phosphatidylinositol 4-kinase) (wortmannin and LY294002) partially inhibited formation of STIM1 puncta. Additional rapid depletion of PtdIns(4,5)P2 resulted in more substantial inhibition of the translocation of STIM1-EYFP (enhanced yellow fluorescent protein) into puncta. The inhibition was extensive at a concentration of LY294002 (50 microM) that should primarily inhibit PI3K, consistent with a major role for PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in puncta formation. Depletion of phosphoinositides also inhibited SOCE based on measurement of the rise in intracellular Ca2+ concentration after store depletion. Overexpression of Orai1 resulted in a recovery of translocation of STMI1 into puncta following phosphoinositide depletion and, under these conditions, SOCE was increased to above control levels. These observations support the idea that phosphoinositides are not essential but contribute to STIM1 accumulation at ER-PM junctions with a second translocation mechanism involving direct STIM1-Orai interactions.
The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Crown Street, Liverpool L69 3BX, UK.
Ciara M
CM
Walsh
Michael
M
Chvanov
Lee P
LP
Haynes
Ole H
OH
Petersen
Alexei V
AV
Tepikin
Robert D
RD
Burgoyne
D019870:1-Phosphatidylinositol 4-Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D009025:Morpholines / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D000071740:ORAI1 Protein; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011485:Protein Binding; D021381:Protein Transport / Q000187:drug effects; D000071737:Stromal Interaction Molecule 1; D014162:Transfection; D000077191:Wortmannin
D019870:1-Phosphatidylinositol 4-Kinase; D000255:Adenosine Triphosphate; D000730:Androstadienes; D002118:Calcium; D015220:Calcium Channels; D002867:Chromones; D015544:Inositol 1,4,5-Trisphosphate; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009025:Morpholines; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; D019869:Phosphatidylinositol 3-Kinases; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D000081082:Phosphoinositide-3 Kinase Inhibitors; D000071737:Stromal Interaction Molecule 1; D000077191:Wortmannin; C510746:ORAI1 protein, human; C103530:STIM1 protein, human; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one