Recent studies by our group and others demonstrated a required and conserved role of Stim in store-operated Ca<SUP>2+</SUP> (SOC) influx and Ca<SUP>2+</SUP> release-activated Ca<SUP>2+</SUP> (CRAC) channel activity. Using an unbiased genome-wide RNAi screen in Drosophila S2 cells, 75 hits were identified that strongly inhibited Ca<SUP>2+</SUP> influx upon store emptying by thapsigargin (TG). Among these hits are 11 predicted trans membrane proteins, including Stim and one, olf186-F, that upon RNAi-mediated knockdown exhibited a profound reduction of TG-evoked Ca<SUP>2+</SUP> entry and CRAC current, and upon over expression a three- fold augmentation of CRAC current. CRAC currents were further increased to eight-fold higher than control and developed more rapidly when olf186-F was co-transfected with Stim. olf186-F is a member of a highly conserved family of four-trans membrane spanning proteins with homo logs from C.elegans to human. The ER Ca<SUP>2+</SUP> pump SERCA and the SNARE protein Syntaxin5 were also required for CRAC channel activity, consistent with a signaling pathway in which Stim senses Ca<SUP>2+</SUP> depletion within the ER, trans locates to the plasma membrane, and interacts with olf186-F to trigger CRAC channel activity.
CRAC CHANNEL AND MODULATOR SCREENING METHODS