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Vol. 20, Issue 1, 389-399, January 1, 2009
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*Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14850; and
Beth Israel-Deaconess Medical Center, Harvard Medical School, Boston, MA 02215
Submitted November 12, 2007;
Revised September 12, 2008;
Accepted October 29, 2008
Monitoring Editor: Jennifer Lippincott-Schwartz
Activation of store operated Ca2+ entry involves stromal interaction molecule 1 (STIM1), localized to the endoplasmic reticulum (ER), and calcium channel subunit (Orai1/CRACM1), localized to the plasma membrane. Confocal microscopy shows that thapsigargin-mediated depletion of ER Ca2+ stores in RBL mast cells causes a redistribution of STIM1, labeled with monomeric red fluorescent protein (mRFP), to micrometer-scale ER-plasma membrane junctions that contain Orai1/CRACM1, labeled with monomeric Aequorea coerulescens green fluorescent protein (AcGFP). Using fluorescence resonance energy transfer (FRET), we determine that this visualized coredistribution is accompanied by nanoscale interaction of STIM1-mRFP and AcGFP-Orai1/CRACM1. We find that antigen stimulation of immunoglobulin E receptors causes much less Orai1/CRACM1 and STIM1 association, but strong interaction is observed under conditions that prevent refilling of ER stores. Stimulated association monitored by FRET is inhibited by sphingosine derivatives in parallel with inhibition of Ca2+ influx. Similar structural and functional effects are caused by mutation of acidic residues in the cytoplasmic segment of Orai1/CRACM1, suggesting a role for electrostatic interactions via these residues in the coupling of Orai1/CRACM1 to STIM1. Our results reveal dynamic molecular interactions between STIM1 and Orai1/CRACM1 that depend quantitatively on electrostatic interactions and on the extent of store depletion.
Address correspondence to: Barbara Baird (bab13{at}cornell.edu).
Abbreviations used: AcGFP, Aequorea coerulescens green fluorescent protein; CRAC, calcium release-activated calcium; CRACM1, CRAC messenger 1 (also called Orai1); DMS, N,N'-dimethylsphingosine; FRET, fluorescence resonance energy transfer; ICRAC, calcium release-activated calcium current; mRFP, monomeric red fluorescent protein; PH, pleckstrin homology (domain); SERCA, sarcoplasmic/endoplasmic reticulum calcium ATPase; SOC, Store operated Ca2+; STIM1, stromal interaction molecule 1; TMS, N,N,N-trimethylsphingosine.
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