Genome-wide RNAi screen of Ca(2+) influx identifies genes that regulate Ca(2+) release-activated Ca(2+) channel activity.
Recent studies by our group and others demonstrated a required and conserved role of Stim in store-operated Ca(2+) influx and Ca(2+) release-activated Ca(2+) (CRAC) channel activity. By using an unbiased genome-wide RNA interference screen in Drosophila S2 cells, we now identify 75 hits that strongly inhibited Ca(2+) influx upon store emptying by thapsigargin. Among these hits are 11 predicted transmembrane proteins, including Stim, and one, olf186-F, that upon RNA interference-mediated knockdown exhibited a profound reduction of thapsigargin-evoked Ca(2+) entry and CRAC current, and upon overexpression a 3-fold augmentation of CRAC current. CRAC currents were further increased to 8-fold higher than control and developed more rapidly when olf186-F was cotransfected with Stim. olf186-F is a member of a highly conserved family of four-transmembrane spanning proteins with homologs from Caenorhabditis elegans to human. The endoplasmic reticulum (ER) Ca(2+) pump sarco-/ER calcium ATPase (SERCA) and the single transmembrane-soluble N-ethylmaleimide-sensitive (NSF) attachment receptor (SNARE) protein Syntaxin5 also were required for CRAC channel activity, consistent with a signaling pathway in which Stim senses Ca(2+) depletion within the ER, translocates to the plasma membrane, and interacts with olf186-F to trigger CRAC channel activity.
Zhang
Shenyuan L
SL
Department of Physiology and Biophysics, University of California, Irvine, CA 92697, USA.
Yeromin
Andriy V
AV
Zhang
Xiang H-F
XH
Yu
Ying
Y
Safrina
Olga
O
Penna
Aubin
A
Roos
Jack
J
Stauderman
Kenneth A
KA
Cahalan
Michael D
MD
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D049750:Genome, Insect; D006801:Humans; D018408:Patch-Clamp Techniques; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D019284:Thapsigargin / Q000378:metabolism
D015220:Calcium Channels; D029721:Drosophila Proteins; D004791:Enzyme Inhibitors; D012330:RNA, Double-Stranded; D011993:Recombinant Fusion Proteins; D019284:Thapsigargin; D002118:Calcium