The Contribution of Orai(CRACM)1 and Orai(CRACM)2 Channels in Store-Operated Ca(2+) Entry and Mediator Release in Human Lung Mast Cells

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 LTC(4) in activated HLMCs. In contrast shRNA knockdown of Orai2 resulted in only marginal reductions of Ca(2+) influx, degranulation and LTC(4) 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.