Ca2+-store-dependent and -independent reversal of Stim1 localization and function.
Stim1 responds to depletion of ER Ca2+ stores by rearranging from tubular structures throughout the ER into punctate structures near the plasma membrane, where it activates Orai store-operated Ca2+ entry (SOCE) channels. However, the mechanism and structural determinants of the localization and reversal of Stim1 puncta formation are poorly understood. Using HEK293 cells expressing Stim1 tagged with enhanced yellow fluorescent protein (EYFP-Stim1), we show that the basis for SOCE termination is the reversal of the punctate Stim1 localization, which absolutely depends on SOCE-dependent store refilling. We also describe rapid, store-independent reversal of EYFP-Stim1 punctae by the ML-9 inhibitor of myosin-light-chain kinase (MLCK). ML-9 similarly inhibited SOCE and the Ca2+-release-activated Ca2+ (CRAC) current. Reversal by ML-9 resulted in full re-establishment of the tubular EYFP-Stim1 localization. A constitutively active EF-hand mutant of EYFP-Stim1 was also reversed by ML-9, regardless of the Ca2+ store content. Inhibition by ML-9 was not due to MLCK inhibition as other inhibitors of MLCK had no effect. Finally, we provide evidence that EYFP-Stim1 punctae form in specific predetermined cellular loci. We conclude that SOCE is tightly coupled to formation of Stim1 puncta, and both SOCE and puncta formation involve a dynamic, reversible signaling complex that probably consists of components in addition to Stim1 and Orai channels.
Smyth
Jeremy T
JT
Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, PO Box 12233, Research Triangle Park, NC 27709, USA.
Dehaven
Wayne I
WI
Bird
Gary S
GS
Putney
James W
JW
D001381:Azepines / Q000494:pharmacology; D001426:Bacterial Proteins / Q000032:analysis; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002460:Cell Line; D004553:Electric Conductivity; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009154:Mutation; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors; D009363:Neoplasm Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D000071737:Stromal Interaction Molecule 1
D001381:Azepines; D001426:Bacterial Proteins; D015220:Calcium Channels; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; C054562:yellow fluorescent protein, Bacteria; C056218:ML 9; D009219:Myosin-Light-Chain Kinase; D002118:Calcium