Dataset Information

Distinct gating mechanism of SOC channel involving STIM-Orai coupling and an intramolecular interaction of Orai in Caenorhabditis elegans.

ABSTRACT: Store-operated calcium entry (SOCE), an important mechanism of Ca2+ signaling in a wide range of cell types, is mediated by stromal interaction molecule (STIM), which senses the depletion of endoplasmic reticulum Ca2+ stores and binds and activates Orai channels in the plasma membrane. This inside-out mechanism of Ca2+ signaling raises an interesting question about the evolution of SOCE: How did these two proteins existing in different cellular compartments evolve to interact with each other? We investigated the gating mechanism of Caenorhabditis elegans Orai channels. Our analysis revealed a mechanism of Orai gating by STIM binding to the intracellular 2-3 loop of Orai in C. elegans that is radically different from Orai gating by STIM binding to the N and C termini of Orai in mammals. In addition, we found that the conserved hydrophobic amino acids in the 2-3 loop of Orai1 are important for the oligomerization and gating of channels and are regulated via an intramolecular interaction mechanism mediated by the N and C termini of Orai1. This study identifies a previously unknown SOCE mechanism in C. elegans and suggests that, while the STIM-Orai interaction is conserved between invertebrates and mammals, the gating mechanism for Orai channels differs considerably.

SUBMITTER: Kim KM

PROVIDER: S-EPMC5960282 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Distinct gating mechanism of SOC channel involving STIM-Orai coupling and an intramolecular interaction of Orai in Caenorhabditis elegans.

Kim Kyu Min KM Wijerathne Tharaka T Hur Jin-Hoe JH Kang Uk Jung UJ Kim Ihn Hyeong IH Kweon Yeong Cheon YC Lee Ah Reum AR Jeong Su Ji SJ Lee Sang Kwon SK Lee Yoon Young YY Sim Bo-Woong BW Lee Jong-Hee JH Baig Chunggi C Kim Sun-Uk SU Chang Kyu-Tae KT Lee Kyu Pil KP Park Chan Young CY

Proceedings of the National Academy of Sciences of the United States of America 20180430 20

Store-operated calcium entry (SOCE), an important mechanism of Ca2+ signaling in a wide range of cell types, is mediated by stromal interaction molecule (STIM), which senses the depletion of endoplasmic reticulum Ca2+ stores and binds and activates Orai channels in the plasma membrane. This inside-out mechanism of Ca2+ signaling raises an interesting question about the evolution of SOCE: How did these two proteins existing in different cellular compartments evolv ...[more]

PMID: 29712850

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Project description:Ca(2+)-release-activated Ca(2+) (CRAC) channels underlie sustained Ca(2+) signalling in lymphocytes and numerous other cells after Ca(2+) liberation from the endoplasmic reticulum (ER). RNA interference screening approaches identified two proteins, Stim and Orai, that together form the molecular basis for CRAC channel activity. Stim senses depletion of the ER Ca(2+) store and physically relays this information by translocating from the ER to junctions adjacent to the plasma membrane, and Orai embodies the pore of the plasma membrane calcium channel. A close interaction between Stim and Orai, identified by co-immunoprecipitation and by Förster resonance energy transfer, is involved in the opening of the Ca(2+) channel formed by Orai subunits. Most ion channels are multimers of pore-forming subunits surrounding a central channel, which are preassembled in the ER and transported in their final stoichiometry to the plasma membrane. Here we show, by biochemical analysis after cross-linking in cell lysates and intact cells and by using non-denaturing gel electrophoresis without cross-linking, that Orai is predominantly a dimer in the plasma membrane under resting conditions. Moreover, single-molecule imaging of green fluorescent protein (GFP)-tagged Orai expressed in Xenopus oocytes showed predominantly two-step photobleaching, again consistent with a dimeric basal state. In contrast, co-expression of GFP-tagged Orai with the carboxy terminus of Stim as a cytosolic protein to activate the Orai channel without inducing Ca(2+) store depletion or clustering of Orai into punctae yielded mostly four-step photobleaching, consistent with a tetrameric stoichiometry of the active Orai channel. Interaction with the C terminus of Stim thus induces Orai dimers to dimerize, forming tetramers that constitute the Ca(2+)-selective pore. This represents a new mechanism in which assembly and activation of the functional ion channel are mediated by the same triggering molecule.

Dataset Information

Distinct gating mechanism of SOC channel involving STIM-Orai coupling and an intramolecular interaction of Orai in Caenorhabditis elegans.

Publications

Distinct gating mechanism of SOC channel involving STIM-Orai coupling and an intramolecular interaction of Orai in <i>Caenorhabditis elegans</i>.

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