The contribution of Orai(CRACM)1 and Orai(CRACM)2 channels in store-operated Ca2+ entry and mediator release in human lung mast cells.
<h4>Background</h4>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+) i...
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Public Library of Science (PLoS)
2013-01-01
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| Series: | PLoS ONE |
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| author | Ian Ashmole S Mark Duffy Mark L Leyland Peter Bradding |
| author_facet | Ian Ashmole S Mark Duffy Mark L Leyland Peter Bradding |
| author_sort | Ian Ashmole |
| collection | DOAJ |
| description | <h4>Background</h4>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.<h4>Objective</h4>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.<h4>Methods</h4>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.<h4>Results</h4>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.<h4>Conclusion and clinical relevance</h4>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. |
| format | Article |
| id | doaj-art-2aff6f2b2cbd4076bc2300617dfce688 |
| institution | DOAJ |
| issn | 1932-6203 |
| language | English |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS ONE |
| spelling | doaj-art-2aff6f2b2cbd4076bc2300617dfce6882025-08-20T03:11:25ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-0189e7489510.1371/journal.pone.0074895The contribution of Orai(CRACM)1 and Orai(CRACM)2 channels in store-operated Ca2+ entry and mediator release in human lung mast cells.Ian AshmoleS Mark DuffyMark L LeylandPeter Bradding<h4>Background</h4>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.<h4>Objective</h4>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.<h4>Methods</h4>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.<h4>Results</h4>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.<h4>Conclusion and clinical relevance</h4>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.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0074895&type=printable |
| spellingShingle | Ian Ashmole S Mark Duffy Mark L Leyland Peter Bradding The contribution of Orai(CRACM)1 and Orai(CRACM)2 channels in store-operated Ca2+ entry and mediator release in human lung mast cells. PLoS ONE |
| title | The contribution of Orai(CRACM)1 and Orai(CRACM)2 channels in store-operated Ca2+ entry and mediator release in human lung mast cells. |
| title_full | The contribution of Orai(CRACM)1 and Orai(CRACM)2 channels in store-operated Ca2+ entry and mediator release in human lung mast cells. |
| title_fullStr | The contribution of Orai(CRACM)1 and Orai(CRACM)2 channels in store-operated Ca2+ entry and mediator release in human lung mast cells. |
| title_full_unstemmed | The contribution of Orai(CRACM)1 and Orai(CRACM)2 channels in store-operated Ca2+ entry and mediator release in human lung mast cells. |
| title_short | The contribution of Orai(CRACM)1 and Orai(CRACM)2 channels in store-operated Ca2+ entry and mediator release in human lung mast cells. |
| title_sort | contribution of orai cracm 1 and orai cracm 2 channels in store operated ca2 entry and mediator release in human lung mast cells |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0074895&type=printable |
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