Effects of STIM1 invalidation on cell calcium homeostasis measured with genetically encoded fluorescent indicators.

Hélène Jousset (1), Maud Frieden(1), Oliver Hartley(2), and Nicolas Demaurex(1).

Changes in the intracellular free Ca2+ concentration regulate many cellular functions such as cardiac contraction, hormone secretion, gene transcription, and cell death. In non-excitable cells, Ca2+ signals are generated by the controlled release of Ca2+ from the endoplasmic reticulum (ER), and the depletion of the ER, in turn, activates store-operated Ca2+ channels (SOC) at the plasma membrane. The mechanism that activates SOC channels in response to ER depletion has long been elusive. Recently, STIM 1, a single transmembrane ER protein that relocates into puncta near the plasma membrane upon Ca2+ store depletion, has been proposed to function as an ER Ca2+ sensor that activates store-operated calcium channels (Roos JCB 169:435, 2005; Liou, Current Biology 15:1235, 2005). To assess the Ca2+ signaling role of STIM1, we used siRNAs and genetically encoded yellow cameleon Ca2+ indicators (YC) to measure Ca2+ changes in the cytosol and beneath the plasma membrane in HeLa cells. Cells were co-transfected with YC together with siRNAs and Ca2+ responses measured both at the single-cell level by Ca2+ imaging or in cell populations with a 96 well plate fluorescence reader. To validate the assay, we used a siRNA directed against the histamine receptor (siHRH1). The cytosolic Ca2+ signals elicited by histamine were inhibited by 95% by siHRH1 while ATP induced-calcium signals were not altered, indicating that the invalidation was efficient and specific. When a siRNA directed against STIM1 was used (siSTIM1), the delayed phase of the histamine-induced Ca2+ response, which reflects the influx of Ca2+ across SOC channels, was inhibited by 45%. Moreover, when the SERCA ATPase inhibitor thapsigargin was used to fully deplete ER Ca2+ stores, store-operated Ca2+ influx was inhibited by 85%. In contrast, the amplitude of the Ca2+ signal measured in Ca2+ free medium was not altered, indicating that Ca2+ release from the ER was not affected by STIM1 invalidation. In conclusion, the use of genetic Ca2+ indicators to measure the activity of siRNAs provides a very efficient functional assay, because almost all fluorescent cells receive at least one copy of the interfering RNA. The lack of effects of STIM1 on Ca2+ release indicates that STIM1 does not interfere with the Ca2+ release process, but is involved in signal transduction between the ER and SOC channels.