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

Abstract

Background: The influx of extracellular Ca(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(2+) influx occurs. However the individual role of each of the three members of the Orai channel family in Ca(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(2+) influx and in the release of β-hexosaminidase (a marker of degranulation) and newly synthesized LTC4 in activated HLMCs. In contrast shRNA knockdown of Orai2 resulted in only marginal reductions of Ca(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(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(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.

Figure 1. Knockdown of Orai channels using recombinant adenoviruses expressing shRNA.

(A) 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) 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.

Figure 2. Knockdown of Orai1 but not Orai2 significantly reduces Ca²⁺ influx in activated HLMCs.

Whole cell patch clamp current-voltage (I–V) curves from HLMCs transduced with recombinant adenoviruses expressing (A) shRNA against luciferase (n=19 cells), or (B) against Orai1 (n=13); and (C) shRNA against luciferase (n=11) or against Orai2 n=14 (D). (●) baseline current, (■) current elicited following dialysis of HLMCs with 30 µM IP₃ for 4 min, (○) current following the addition of 1 µM GSK-7975A. Currents were recorded in 2 mM external Ca²⁺ and are shown as mean ± SEM. *p=0.0292 compared with corresponding curve in (B).

Figure 3. The effect of Orai channel knockdown on the release of HLMC (A) β-hexosaminidase (degranulation) and (B) LTC₄.

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.

Figure 4. The effect of dominant negative mutations of Orai1 (E106Q), Orai2 (E80Q) and Orai3 (E81Q) on HLMC degranulation.

(A) 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) 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.

Figure 5. Expression of dominant negative mutations of Orai1-3 greatly reduce Ca²⁺ influx into activated HLMCs.

Subtracted whole cell patch clamp current-voltage (I–V) curves from HLMCs transduced with recombinant adenoviruses expressing (A) GFP n=14 cells (●) or Orai1 E106Q n=15 (○), (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.

Figure 6. The effect of FcεRI-dependent HLMC activation on levels of Orai transcripts.

Cells were activated with αFcεR1 antibody and the levels of Orai1-3 transcripts determined by quantitative RT-PCR (A) after activation for 1 hour and (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.