Stimuli that trigger release of calcium from the endoplasmic reticulum (ER) through inositol trisphosphate (IP3) receptors can deplete this intracellular calcium store and trigger store-operated calcium (SOC) influx from the plasma membrane. Liou et al. designed a siRNA screen against 2304 proteins selected on the basis of the presence of signaling domains in their primary sequence. These were tested to identify proteins that, when depleted, reduced the plateau phase of calcium influx after exposure of cells to histamine (which promotes calcium release through IP3 receptors) in the presence of thapsigargin (to inhibit calcium reloading of the ER). siRNA for stromal interaction molecules 1 and 2 (STIM1 and STIM2), which have a single transmembrane domain and a single EF-hand calcium-binding motif, suppressed the sustained calcium signal without decreasing the initial transient calcium signal. siRNA for STIM1 or STIM2 reduced the calcium influx observed after extracellular calcium was added after stores were depleted in the absence of extracellular calcium (calcium add-back assay). Overexpression of a yellow fluorescent protein (YFP)-STIM1 resulted in increased opening of the SOC influx channel, based on a Mn2+ quench assay, in which Mn2+ entering through the SOC influx channel quenches the Fura-2 signal. YFP-STIM1 was localized to the ER, and store depletion caused redistribution of this signal to punctate structures near the cell periphery even in the absence of extracellular calcium, which suggests that store depletion (rather than subsequent calcium influx through the plasma membrane) may be the cause of the redistribution. Mutation of the EF-hand domain caused redistribution of YFP-STIM1 to the punctate structures in the absence of store depletion and promoted calcium influx in the absence of store depletion, and store depletion did not cause any additional influx. In cells transfected to express a wild-type cyan fluorescent protein (CFP)-STIM1 and the EF-hand YFP-STIM1, store depletion triggered redistribution of the CFP-STIM1 to the YFP-STIM1-positive punctate structures. Thus, it appears that SOC influx may involve the rapid redistribution of ER proteins to a punctate intracellular structure in close proximity to the membrane, which may mediate a direct coupling between proteins in these intracellular structures with the channels in the plasma membrane.
J. Liou, M. L. Kim, W. D. Heo, J. T. Jones, J. W. Myers, J. E. Ferrell Jr., T. Meyer, STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx. Current Biol. 15, 1235-1241 (2005). [PubMed]