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First published online 19 February 2008
doi: 10.1242/jcs.023903

This Article Figures Only Full Text Full Text (PDF) Supplementary Material All Versions of this Article: jcs.023903v1 121/6/762    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Google Scholar Articles by Smyth, J. T. Articles by Putney, J. W. PubMed PubMed Citation Articles by Smyth, J. T. Articles by Putney, J. W., Jr

Ca²⁺-store-dependent and -independent reversal of Stim1 localization and function

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

^* Author for correspondence (e-mail: putney{at}niehs.nih.gov)

Accepted 13 December 2007

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.

Key words: Stim1, ML-9, Inhibitors, Store-operated channels

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