Location and function of STIM1 in the activation of Ca²⁺ entry signals

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 Ca ²⁺-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.STIM^EF-expressing cells had normal ER Ca ²⁺ 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 STIM1^WT, is not trafficked to the PM. Although not inserted in the PM, external application of a monoclonal anti-N-terminal STIM1 antibody blocked constitutive STIM^EF-mediated Ca²⁺ entry, but only in cells expressing endogenous STIM1^WT and not in DT40 STIM1 knock-out cells devoid of STIM^WT. This suggests that PM-STIM1 may play a regulatory role in SOC activation.