Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 283, Issue 38, Pages 26252-26262Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M802239200
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Funding
- National Institutes of Health [HL55426, AI058173]
- American Heart Association
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Store-operated channels (SOCs) mediate Ca2+ entry signals in response to endoplasmic reticulum (ER) Ca2+ depletion in most cells. STIM1 senses decreased ER luminal Ca2+ through its EF-hand Ca2+-binding motif and aggregates in near-plasma membrane (PM) ER junctions to activate PM Orai1, the functional SOC. STIM1 is also present in the PM, although its role there is unknown. STIM1-mediated coupling was examined using the stable EF20 HEK293 cell line expressing the STIM1-D76A/E87A EF-hand mutant (STIM1(EF)) deficient in Ca2+ binding. EF20 cells were viable despite constitutive Ca2+ entry, allowing study of SOC activation without depleting ER Ca2+. STIM1(EF) was exclusively in stable near-PM junctions, 3.5-fold larger than formed with STIM1(WT). STIMEF-expressing cells had normal ER Ca2+ levels but substantially reduced ER Ca2+ leak. Expression of antiapoptotic Bcl-2 proteins (BCl-2, MCL-1, BCL-XL) were increased 2-fold in EF20 cells, probably reflecting survival of EF20 cells but not accounting for decreased ER Ca2+ leak. Surface biotinylation and streptavidin pull-down of cells expressing STIM1(WT) or STIM1(EF) revealed strong PM interactions of both proteins. Although surface expression of STIM1(WT) was clearly detectable, STIM1(EF) was undetectable at the cell surface. Thus, the Ca2+ binding-defective STIM1(EF) mutant exists exclusively in aggregates within near-PM junctions but, unlike STIM1WT, is not trafficked to the PM. Although not inserted in the PM, external application of a monoclonal anti-N-terminal STIM1 antibody blocked constitutive STIMEF-mediated Ca2+ entry, but only in cells expressing endogenous STIM1(WT) and not in DT40 STIM1 knock-out cells devoid of STIMWT. This suggests that PM-STIM1 may play a regulatory role in SOC activation.
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