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.