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

• Published in: Journal of allergy and clinical immunology 2012-06, Vol.129 (6), p.1628-1635.e2
• Main Authors: Ashmole, Ian, PhD, Duffy, S. Mark, PhD, Leyland, Mark L., PhD, Morrison, Valerie S., BSc, Begg, Malcolm, PhD, Bradding, Peter, DM
• Format: Article
• Language: English
• Subjects: Allergens - immunology; Allergic diseases; Allergy and Immunology; Asthma; Biological and medical sciences; Bronchi - drug effects; Bronchi - metabolism; Bronchus; Ca2; Calcium; Calcium (extracellular); Calcium - metabolism; Calcium Channel Blockers - metabolism; Calcium Channel Blockers - pharmacology; Calcium channels; Calcium Channels - genetics; Calcium Channels - metabolism; Calcium currents; Calcium influx; Cell culture; Cell Line; Chronic obstructive pulmonary disease, asthma; CRACM; cytokine; Cytokines; Endoplasmic reticulum; Experiments; Fundamental and applied biological sciences. Psychology; Fundamental immunology; GSK-7975A; Histamine; Humans; Immunoglobulin E - metabolism; Immunopathology; Inositol trisphosphate; Interleukin 5; Ion channels; leukotriene C4; Leukotrienes; Lung; Lung - cytology; Lung - metabolism; mast cell; Mast cells; Mast Cells - metabolism; Mechanisms of Allergy and Clinical Immunology; Medical sciences; Muscle contraction; Muscle Contraction - drug effects; Muscle, Smooth - drug effects; Muscle, Smooth - metabolism; Orai; Pneumology; Protein expression; Proteins; RNA, Messenger - metabolism; Rodents; Sarcoidosis. Granulomatous diseases of unproved etiology. Connective tissue diseases. Elastic tissue diseases. Vasculitis; Smooth muscle; Synta-66
• ISSN: 0091-6749; 1097-6825; 1097-6825
• DOI: 10.1016/j.jaci.2012.01.070

Background Influx 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. Objectives To investigate the expression and function of CRACM channels in HLMCs. Methods CRACM mRNA, protein, and functional expression were examined in purified HLMCs and isolated human bronchus. Results CRACM1, -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. Conclusions The 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.