Abstract
BackgroundThe influx of extracellular Ca2+ 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 Ca2+ influx occurs. However the individual role of each of the three members of the Orai channel family in Ca2+ influx and mediator release has not been defined in human mast cells.ObjectiveTo 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.MethodsWe 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.ResultsshRNA-mediated knockdown of Orai1 resulted in a significant reduction of approximately 50% in Ca2+ 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 Ca2+ 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 Ca2+ 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 RelevanceOrai1 plays an important role in Ca2+ 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.
Highlights
Mast cells play a critical role in the development of asthma and related allergic diseases [1]
Ca2+ influx into human mast cells is essential for the release of mediators such as histamine, tryptase, leukotrienes and cytokines following their activation through the high affinity IgEreceptor (FcεRI)
We have recently shown that members of the Orai ion channel family play a major role in FcεRI-dependent Ca2+ influx in human lung mast cells [15]
Summary
Mast cells play a critical role in the development of asthma and related allergic diseases [1]. Mast cell activation leads to the release of a battery of mediators including preformed granule-derived mediators such as histamine and proteases and newly synthesized prostaglandins, leukotrienes and cytokines. Excess release of these mediators as a result of aberrant activation contributes to allergic disease states. The individual role of each of the three members of the Orai channel family in Ca2+ 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. Strategies which target Orai will effectively inhibit FcεRI-dependent HLMC activation, but spare off-target inhibition of Orai in other cells and body systems
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