CRACM/Orai ion channel expression and function in human lung mast cells

Ian Ashmole,S Mark Duffy,Mark L Leyland,Valerie S Morrison,Malcolm Begg,Peter Bradding

doi:10.1016/j.jaci.2012.01.070

Abstract

BackgroundInflux of extracellular Ca2+ into human lung mast cells (HLMCs) is essential for the FcεRI-dependent release of preformed granule-derived mediators and newly synthesized autacoids and cytokines. However, the identity of the ion channels underlying this Ca2+ influx is unknown. The recently discovered members of the CRACM/Orai ion channel family that carries the Ca2+ release–activated Ca2+ current are candidates.ObjectivesTo investigate the expression and function of CRACM channels in HLMCs.MethodsCRACM mRNA, protein, and functional expression were examined in purified HLMCs and isolated human bronchus.ResultsCRACM1, -2, and -3 mRNA transcripts and CRACM1 and -2 proteins were detectable in HLMCs. A CRACM-like current was detected following FcεRI-dependent HLMC activation and also in HLMCs dialyzed with 30 μM inositol triphosphate. The Ca2+-selective current obtained under both conditions was blocked by 10 μM La3+ and Gd3+, known blockers of CRACM channels, and 2 distinct and specific CRACM-channel blockers—GSK-7975A and Synta-66. Both blockers reduced FcεRI-dependent Ca2+ influx, and 3 μM GSK-7975A and Synta-66 reduced the release of histamine, leukotriene C4, and cytokines (IL-5/-8/-13 and TNFα) by up to 50%. Synta-66 also inhibited allergen-dependent bronchial smooth muscle contraction in ex vivo tissue.ConclusionsThe presence of CRACM channels, a CRACM-like current, and functional inhibition of HLMC Ca2+ influx, mediator release, and allergen-induced bronchial smooth muscle contraction by CRACM-channel blockers supports a role for CRACM channels in FcεRI-dependent HLMC secretion. CRACM channels are therefore a potential therapeutic target in the treatment of asthma and related allergic diseases.

Highlights

Influx of extracellular Ca21 into human lung mast cells (HLMCs) is essential for the FcεRI-dependent release of preformed granule-derived mediators and newly synthesized autacoids and cytokines
HLMCs express CRACM currents following store depletion with IP3 To investigate whether HLMCs expressed a Ca21 current induced by store depletion, cells were dialyzed with 30 mM IP3
Studies of knockout mice lacking CRACM1 function have shown that CRACM channels are essential for the influx of extracellular Ca21 into rodent mast cells following their activation.[22]

Summary

Introduction

Influx of extracellular Ca21 into human lung mast cells (HLMCs) is essential for the FcεRI-dependent release of preformed granule-derived mediators and newly synthesized autacoids and cytokines. The Ca21-selective current obtained under both conditions was blocked by 10 mM La31 and Gd31, known blockers of CRACM channels, and 2 distinct and specific CRACM-channel blockers—GSK-7975A and Synta-66. Both blockers reduced FcεRI-dependent Ca21 influx, and 3 mM GSK-7975A and Synta-66 reduced the release of histamine, leukotriene C4, and cytokines (IL-5/-8/-13 and TNFa) by up to 50%. Synta-66 inhibited allergen-dependent bronchial smooth muscle contraction in ex vivo tissue. Conclusions: The presence of CRACM channels, a CRACM-like current, and functional inhibition of HLMC Ca21 influx, mediator release, and allergen-induced bronchial smooth muscle contraction by CRACM-channel blockers supports a role for CRACM channels in FcεRI-dependent HLMC secretion. CRACM channels are a potential therapeutic target in the treatment of asthma and related allergic diseases. (J Allergy Clin Immunol 2012;129:1628-35.)

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