Article

Genome-wide RNAi screen of Ca2+ influx identifies genes that regulate Ca2+ release-activated Ca2+ channel activity

Department of Physiology and Biophysics, University of California, Irvine, CA 92697, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 07/2006; 103(24):9357-62. DOI: 10.1073/pnas.0603161103
Source: PubMed

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.

Download full-text

Full-text

Available from: Ying Yu, Jul 04, 2015
0 Followers
 · 
125 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: STIM proteins populate and expand cortical ER sheets to mediate store-operated Ca(2+) entry (SOCE) by trapping and gating Orai channels in ER-PM clusters. A longer splice variant, STIM1L, forms permanent ER-PM clusters and mediates rapid influx in muscle. Here, we used electron microscopy, TIRF, and Ca(2+) imaging to establish the trafficking and signaling properties of the two STIM1 isoforms in Stim1(-/-)/Stim2(-/-) fibroblasts. Unlike STIM1, STIM1L was poorly recruited into ER-PM clusters and did not mediate store-dependent expansion of cortical ER cisternae. Removal of the STIM1 lysine-rich tail prevented store-dependent cluster enlargement, while inhibition of cytosolic Ca(2+) elevations or removal of the STIM1L actin binding domain had no impact on cluster expansion. Finally, STIM1L restored robust, but not accelerated SOCE and clustered with Orai1 channels more slowly than STIM1 following store depletion. These results indicate that STIM1L does not mediate rapid SOCE but can trap and gate Orai1 channels efficiently without remodeling cortical ER cisternae. The ability of STIM proteins to induce cortical ER formation is dispensable for SOCE and requires the lysine-rich tail of STIM1 involved in binding to phosphoinositides.
    Journal of Cell Science 03/2015; 128(8). DOI:10.1242/jcs.164228 · 5.33 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Transepithelial bicarbonate secretion plays a key role in the maintenance of fluid and protein secretion from epithelial cells and the protection of the epithelial cell surface from various pathogens. Epithelial bicarbonate secretion is mainly under the control of cAMP and calcium signaling. While the physiological roles and molecular mechanisms of cAMP-induced bicarbonate secretion are relatively well defined, those induced by calcium signaling remain poorly understood in most epithelia. The present review summarizes the current status of knowledge on the role of calcium signaling in epithelial bicarbonate secretion. Specifically, this review introduces how cytosolic calcium signaling can increase bicarbonate secretion by regulating membrane transport proteins and how it synergizes with cAMP-induced mechanisms in epithelial cells. In addition, tissue-specific variations in the pancreas, salivary glands, intestines, bile ducts, and airways are discussed. We hope that the present report will stimulate further research into this important topic. These studies will provide the basis for future medicines for a wide spectrum of epithelial disorders including cystic fibrosis, Sjögren's syndrome, and chronic pancreatitis.
    Cell calcium 06/2014; DOI:10.1016/j.ceca.2014.02.002 · 4.21 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Orai proteins contribute to Ca(2+) entry into cells through both store-dependent, Ca(2+) release-activated Ca(2+) (CRAC) channels (Orai1) and store-independent, arachidonic acid (AA)-regulated Ca(2+) (ARC) and leukotriene C4 (LTC4)-regulated Ca(2+) (LRC) channels (Orai1/3 heteromultimers). Although activated by fundamentally different mechanisms, CRAC channels, like ARC and LRC channels, require stromal interacting molecule 1 (STIM1). The role of endoplasmic reticulum-resident STIM1 (ER-STIM1) in CRAC channel activation is widely accepted. Although ER-STIM1 is necessary and sufficient for LRC channel activation in vascular smooth muscle cells (VSMCs), the minor pool of STIM1 located at the plasma membrane (PM-STIM1) is necessary for ARC channel activation in HEK293 cells. To determine whether ARC and LRC conductances are mediated by the same or different populations of STIM1, Orai1, and Orai3 proteins, we used whole-cell and perforated patch-clamp recording to compare AA- and LTC4-activated currents in VSMCs and HEK293 cells. We found that both cell types show indistinguishable nonadditive LTC4- and AA-activated currents that require both Orai1 and Orai3, suggesting that both conductances are mediated by the same channel. Experiments using a nonmetabolizable form of AA or an inhibitor of 5-lipooxygenase suggested that ARC and LRC currents in both cell types could be activated by either LTC4 or AA, with LTC4 being more potent. Although PM-STIM1 was required for current activation by LTC4 and AA under whole-cell patch-clamp recordings in both cell types, ER-STIM1 was sufficient with perforated patch recordings. These results demonstrate that ARC and LRC currents are mediated by the same cellular populations of STIM1, Orai1, and Orai3, and suggest a complex role for both ER-STIM1 and PM-STIM1 in regulating these store-independent Orai1/3 channels.
    The Journal of General Physiology 03/2014; 143(3):345-59. DOI:10.1085/jgp.201311084 · 4.57 Impact Factor