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Orai1 and CRAC channel dependence of VEGF-activated Ca2+ entry and endothelial tube formation.

ABSTRACT: RATIONALE:Orai1 and the associated calcium release-activated calcium (CRAC) channel were discovered in the immune system. Existence also in endothelial cells has been suggested, but the relevance to endothelial biology is mostly unknown. OBJECTIVE:The aim of this study was to investigate the relevance of Orai1 and CRAC channels to vascular endothelial growth factor (VEGF) and endothelial tube formation. METHODS AND RESULTS:In human umbilical vein endothelial cells, Orai1 disruption by short-interfering RNA or dominant-negative mutant Orai1 inhibited calcium release-activated (store-operated) calcium entry, VEGF-evoked calcium entry, cell migration, and in vitro tube formation. Expression of exogenous wild-type Orai1 rescued the tube formation. VEGF receptor-2 and Orai1 partially colocalized. Orai1 disruption also inhibited calcium entry and tube formation in endothelial progenitor cells from human blood. A known blocker of the immune cell CRAC channel (3-fluoropyridine-4-carboxylic acid (2',5'-dimethoxybiphenyl-4-yl)amide) was a strong blocker of store-operated calcium entry in endothelial cells and inhibited calcium entry evoked by VEGF in 3 types of human endothelial cell. The compound lacked effect on VEGF-evoked calcium-release, STIM1 clustering, and 2 types of transient receptor potential channels, TRPC6 and TRPV4. Without effect on cell viability, the compound inhibited human endothelial cell migration and tube formation in vitro and suppressed angiogenesis in vivo in the chick chorioallantoic membrane. The compound showed 100-fold greater potency for endothelial compared with immune cell calcium entry. CONCLUSIONS:The data suggest positive roles for Orai1 and CRAC channels in VEGF-evoked calcium entry and new opportunity for chemical modulation of angiogenesis.

SUBMITTER: Li J

PROVIDER: S-EPMC3512576 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Orai1 and CRAC channel dependence of VEGF-activated Ca2+ entry and endothelial tube formation.

Li Jing J Cubbon Richard M RM Wilson Lesley A LA Amer Mohamed S MS McKeown Lynn L Hou Bing B Majeed Yasser Y Tumova Sarka S Seymour Victoria A L VA Taylor Hilary H Stacey Martin M O'Regan David D Foster Richard R Porter Karen E KE Kearney Mark T MT Beech David J DJ

Circulation research 20110324 10

<h4>Rationale</h4>Orai1 and the associated calcium release-activated calcium (CRAC) channel were discovered in the immune system. Existence also in endothelial cells has been suggested, but the relevance to endothelial biology is mostly unknown.<h4>Objective</h4>The aim of this study was to investigate the relevance of Orai1 and CRAC channels to vascular endothelial growth factor (VEGF) and endothelial tube formation.<h4>Methods and results</h4>In human umbilical vein endothelial cells, Orai1 di ...[more]

PMID: 21441136

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Project description:Orai1, the pore subunit of Ca(2+) release-activated Ca(2+) channels, has four transmembrane segments (TMs). The first segment, TMI, lines the pore and plays an important role in channel activation and ion permeation. TMIII, on the other hand, does not line the pore but still regulates channel gating and permeation properties. To understand the role of TMIII, we have mutated and characterized several residues in this domain. Mutation of Trp-176 to Cys (W176C) and Gly-183 to Ala (G183A) had dramatic effects. Unlike wild-type channels, which exhibit little outward current and are activated by STIM1, W176C mutant channels exhibited a large outward current at positive potentials and were constitutively active in the absence of STIM1. G183A mutant channels also exhibited substantial outward currents but were active only in the presence of 2-aminoethoxydiphenyl borate (2-APB), irrespective of STIM1. With W176C mutant channels inward, monovalent currents were blocked by Ca(2+) with a high affinity similar to the wild type, but the Ca(2+)-dependent blocking of outward currents differed in the two cases. Although a 50% block of the WT outward current required 250 ?m Ca(2+), more than 6 mm was necessary to have the same effect on W176C mutant channels. In the presence of extracellular Ca(2+), W176C and G183A outward currents developed slowly in a voltage-dependent manner, whereas they developed almost instantaneously in the absence of Ca(2+). These changes in permeation and gating properties mimic the changes induced by mutations of Glu-190 in TMIII and Asp-110/Asp-112 in the TMI/TMII loop. On the basis of these data, we propose that TMIII maintains negatively charged residues at or near the selectivity filter in a conformation that facilitates Ca(2+) inward currents and prevents outward currents of monovalent cations. In addition, to controlling selectivity, TMIII may also stabilize channel gating in a closed state in the absence of STIM1 in a Trp-176-dependent manner.

Project description:Store-operated Ca2+ entry occurs through the binding of the endoplasmic reticulum (ER) Ca2+ sensor STIM1 to Orai1, the pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel. Although the essential steps leading to channel opening have been described, fundamental questions remain, including the functional stoichiometry of the CRAC channel. The crystal structure of Drosophila Orai indicates a hexameric stoichiometry, while studies of linked Orai1 concatemers and single-molecule photobleaching suggest that channels assemble as tetramers. We assessed CRAC channel stoichiometry by expressing hexameric concatemers of human Orai1 and comparing in detail their ionic currents to those of native CRAC channels and channels generated from monomeric Orai1 constructs. Cell surface biotinylation results indicated that Orai1 channels in the plasma membrane were assembled from intact hexameric polypeptides and not from truncated protein products. In addition, the L273D mutation depressed channel activity equally regardless of which Orai1 subunit in the concatemer carried the mutation. Thus, functional channels were generated from intact Orai1 hexamers in which all subunits contributed equally. These hexameric Orai1 channels displayed the biophysical fingerprint of native CRAC channels, including the distinguishing characteristics of gating (store-dependent activation, Ca2+-dependent inactivation, open probability), permeation (ion selectivity, affinity for Ca2+ block, La3+ sensitivity, unitary current magnitude), and pharmacology (enhancement and inhibition by 2-aminoethoxydiphenyl borate). Because permeation characteristics depend strongly on pore geometry, it is unlikely that hexameric and tetrameric pores would display identical Ca2+ affinity, ion selectivity, and unitary current magnitude. Thus, based on the highly similar pore properties of the hexameric Orai1 concatemer and native CRAC channels, we conclude that the CRAC channel functions as a hexamer of Orai1 subunits.

Dataset Information

Orai1 and CRAC channel dependence of VEGF-activated Ca2+ entry and endothelial tube formation.

Publications

Orai1 and CRAC channel dependence of VEGF-activated Ca2+ entry and endothelial tube formation.

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