Genome-wide RNAi screen of Ca ²⁺ influx identifies genes that regulate Ca ²⁺ release-activated Ca ²⁺ channel activity

TLDR

Stim is essential for store‑operated Ca²⁺ influx and CRAC channel activity, and olf186‑F is a conserved four‑transmembrane protein family spanning species from C. elegans to humans. The study aimed to identify genes that regulate CRAC channel activity by performing an unbiased genome‑wide RNAi screen in Drosophila S2 cells. Using a genome‑wide RNAi screen in Drosophila S2 cells, the authors identified 75 genes whose knockdown strongly inhibited thapsigargin‑induced Ca²⁺ influx.

Abstract

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.