Orai/CRACM1 and KCa3.1 ion channels interact

open access articleBACKGROUND: Orai/CRACM1 ion channels provide the major Ca(2+) influx pathway for FcεRI-dependent human lung mast cell (HLMC) mediator release. The Ca(2+)-activated K(+) channel KCa3.1 modulates Ca(2+) influx and the secretory response through hyperpolarisation of the plasma membrane. We hypothesised that there is a close functional and spatiotemporal interaction between these Ca(2+)- and K(+)-selective channels. RESULTS: Activation of FcεRI-dependent HLMC KCa3.1 currents was dependent on the presence of extracellular Ca(2+), and attenuated in the presence of the selective Orai blocker GSK-7975A. Currents elicited by the KCa3.1 opener 1-EBIO were also attenuated by GSK-7975A. The Orai1 E106Q dominant-negative mutant ablated 1-EBIO and FcεRI-dependent KCa3.1 currents in HLMCs. Orai1 but not Orai2 was shown to co-immunoprecipitate with KCa3.1 when overexpressed in HEK293 cells, and Orai1 and KCa3.1 were seen to co-localise in the HEK293 plasma membrane using confocal microscopy. CONCLUSION: KCa3.1 activation in HLMCs is highly dependent on Ca(2+) influx through Orai1 channels, mediated via a close spatiotemporal interaction between the two channels

Identification of Conserved Hydrophobic C-Terminal Residues of the Human Papillomavirus Type 1 E1∧E4 Protein Necessary for E4 Oligomerisationin Vivo

AbstractPrevious studies have shown that human papillomavirus (HPV) E4 proteins undergo oligomerisation, although the precise sequences involved have not been identified. Using the yeast two-hybrid system we have identified HPV 1 E4 sequences that are critical to multimerisation. Fusion proteins were created by linking wild-type and mutant E4 proteins to a LexA DNA-binding domain or a B42 transactivation domain. HPV 1 E4:E4 interactions were examined by expression of these fusion proteins inSaccharomyces cerevisiae.This assay showed that (1) amino acid residues 95 to 115 at the carboxy-terminus were critical for oligomerisation and (2) hydrophobic residues (isoleucine 107, phenylalanine 114) in this domain are major determinants in the formation of oligomers. Interestingly, the carboxy-terminal domain shares homology with other E4 proteins of cutaneous HPV types and, furthermore, positions 107 and 114 are conserved residues. Substitution of the conserved aspartate amino acids (residues 110 and 112) did not abrogate E4 oligomerisation. Chemical cross-linking of wart and recombinant (baculovirus-expressed) HPV 1 E4 protein indicated that in solution this viral protein forms complexes consistent in size with either trimers or tetramers. These complexes were resistant to urea denaturation and are not dependent on the formation of disulphide linkages. A mutant protein containing a deletion of residues 110 to 115 was unable to form oligomers following cross-linking supporting a role for this region in mediating E4:E4 interactions. We conclude that oligomerisation of the HPV 1 E4 protein is likely to be mediated by carboxy-terminal residues and that conserved hydrophobic residues of this domain play a major role in E4 oligomerisation

Expression of dominant negative mutations of Orai1-3 greatly reduce Ca²⁺ influx into activated HLMCs.

<p>Subtracted whole cell patch clamp current-voltage (I–V) curves from HLMCs transduced with recombinant adenoviruses expressing (<b>A</b>) GFP n=14 cells (●) or Orai1 E106Q n=15 (○), (<b>B</b>) GFP n=11 (●) or Orai2 E80Q n=14 (○), and (C) GFP n=11 (●) or Orai3 E81Q n=13 (○). Currents (mean ± SEM) were recorded in 2 mM external Ca²⁺ following dialysis with 3 µM IP₃ for 4 min.</p

The effect of dominant negative mutations of Orai1 (E106Q), Orai2 (E80Q) and Orai3 (E81Q) on HLMC degranulation.

<p>(<b>A</b>) Transduction of HLMCs with Orai dominant-negative mutants had no effect on cell viability after 4 days compared to GFP control virus (n=5). (<b>B</b>) β-hexosaminidase degranulation was essentially abolished following transduction of HLMC with Orai dominant negative mutants. Mean ± SEM of net percentage release observed in cells transduced with adenoviruses expressing the indicated protein. n=4-7, * p<0.05 compared to GFP control.</p

The contribution of Orai(CRACM)1 and Orai(CRACM)2 channels in store-operated Ca2+ entry and mediator release in human lung mast cells

Background: The influx of extracellular Ca[superscript 2+] into mast cells is critical for the FcεR1-dependent release of preformed granule-derived mediators and newly synthesised autacoids and cytokines. The Orai(CRACM) ion channel family provide the major pathway through which this Ca[superscript 2+] influx occurs. However the individual role of each of the three members of the Orai channel family in Ca[superscript 2+] influx and mediator release has not been defined in human mast cells. Objective: To assess whether there might be value in targeting individual Orai family members for the inhibition of FcεRI-dependent human lung mast cells (HLMC) mediator release. Methods: We used an adenoviral delivery system to transduce HLMCs with shRNAs targeted against Orai1 and Orai2 or with cDNAs directing the expression of dominant-negative mutations of the three known Orai channels. Results: shRNA-mediated knockdown of Orai1 resulted in a significant reduction of approximately 50% in Ca[superscript 2+] influx and in the release of β-hexosaminidase (a marker of degranulation) and newly synthesized LTC[subscript 4] in activated HLMCs. In contrast shRNA knockdown of Orai2 resulted in only marginal reductions of Ca[superscript 2+] influx, degranulation and LTC4 release. Transduced dominant-negative mutants of Orai1, -2 and -3 markedly reduced Orai currents and completely inhibited HLMC degranulation suggesting that Orai channels form heteromultimers in HLMCs, and that Orai channels comprise the dominant Ca[superscript 2+] influx pathway following FceRI-dependent HLMC activation. Inhibition of Orai currents did not alter HLMC survival. In addition we observed a significant down-regulation of the level of CRACM3 mRNA transcripts together with a small increase in the level of CRACM1 and CRACM2 transcripts following a period of sustained HLMC activation. Conclusion and Clinical Relevance: Orai1 plays an important role in Ca[superscript 2+] influx and mediator release from HLMCs. Strategies which target Orai1 will effectively inhibit FcεRI-dependent HLMC activation, but spare off-target inhibition of Orai2 in other cells and body systems

A Truncated Splice-Variant of the FcεRIβ Receptor Subunit Is Critical for Microtubule Formation and Degranulation in Mast Cells

SummaryHuman linkage analyses have implicated the MS4A2-containing gene locus (encoding FcεRIβ) as a candidate for allergy susceptibility. We have identified a truncation of FcεRIβ (t-FcεRIβ) in humans that contains a putative calmodulin-binding domain and thus, we sought to identify the role of this variant in mast cell function. We determined that t-FcεRIβ is critical for microtubule formation and degranulation and that it may perform this function by trafficking adaptor molecules and kinases to the pericentrosomal and Golgi region in response to Ca2+ signals. Mutagenesis studies suggest that calmodulin binding to t-FcεRIβ in the presence of Ca2+ could be critical for t-FcεRIβ function. In addition, gene targeting of t-FcεRIβ attenuated microtubule formation, degranulation, and IL-8 production downstream of Ca2+ signals. Therefore, t-FcεRIβ mediates Ca2+-dependent microtubule formation, which promotes degranulation and cytokine release. Because t-FcεRIβ has this critical function, it represents a therapeutic target for the downregulation of allergic inflammation

CRACM/Orai ion channel expression and function in human lung mast cells

BackgroundInflux of extracellular Ca2+ into human lung mast cells (HLMCs) is essential for the FcεRI-dependent release of preformed granule-derived mediators and newly synthesized autacoids and cytokines. However, the identity of the ion channels underlying this Ca2+ influx is unknown. The recently discovered members of the CRACM/Orai ion channel family that carries the Ca2+ release–activated Ca2+ current are candidates.ObjectivesTo investigate the expression and function of CRACM channels in HLMCs.MethodsCRACM mRNA, protein, and functional expression were examined in purified HLMCs and isolated human bronchus.ResultsCRACM1, -2, and -3 mRNA transcripts and CRACM1 and -2 proteins were detectable in HLMCs. A CRACM-like current was detected following FcεRI-dependent HLMC activation and also in HLMCs dialyzed with 30 μM inositol triphosphate. The Ca2+-selective current obtained under both conditions was blocked by 10 μM La3+ and Gd3+, known blockers of CRACM channels, and 2 distinct and specific CRACM-channel blockers—GSK-7975A and Synta-66. Both blockers reduced FcεRI-dependent Ca2+ influx, and 3 μM GSK-7975A and Synta-66 reduced the release of histamine, leukotriene C4, and cytokines (IL-5/-8/-13 and TNFα) by up to 50%. Synta-66 also inhibited allergen-dependent bronchial smooth muscle contraction in ex vivo tissue.ConclusionsThe presence of CRACM channels, a CRACM-like current, and functional inhibition of HLMC Ca2+ influx, mediator release, and allergen-induced bronchial smooth muscle contraction by CRACM-channel blockers supports a role for CRACM channels in FcεRI-dependent HLMC secretion. CRACM channels are therefore a potential therapeutic target in the treatment of asthma and related allergic diseases

The effect of FcεRI-dependent HLMC activation on levels of Orai transcripts.

<p>Cells were activated with αFcεR1 antibody and the levels of Orai1-3 transcripts determined by quantitative RT-PCR (<b>A</b>) after activation for 1 hour and (<b>B</b>) for 4 hours. Mean ± SEM fold change compared to untreated cells. n=5, * p<0.05, ns not significant. Activation of HLMC for 4 hours results in the down-regulation of Orai3 and up-regulation of Orai1 and -2 transcripts.</p

The effect of Orai channel knockdown on the release of HLMC (A) β-hexosaminidase (degranulation) and (B) LTC₄.

<p>The graphs demonstrate the mean ± SEM release presented as a percentage of that observed in control cells transduced with adenovirus expressing shRNA directed against luciferase. n=6-10 independent donors for β-hexosaminidase, n=5 for LTC₄. * p<0.05, *** p<0.001.</p

Knockdown of Orai channels using recombinant adenoviruses expressing shRNA.

<p>(<b>A</b>) Knockdown efficiency of shRNAs against Orai1 (Orai1 v1) and Orai2 (Orai2 v1) was determined in HLMCs 4 days following transduction with virus by quantitative RT-PCR (n=3 independent experiments using HLMCs from 3 donors), * p<0.05 compared to expression in cells transduced with luciferase control. All other conditions were not significantly different to control. (<b>B</b>) Transduction of HLMCs with adenoviruses expressing shRNAs against Orai1 (Orai1 v1) and Orai2 (Orai2 v1) (n=3 donors), had no significant effect on cell viability after 4 days compared to luciferase control virus.</p