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

Abstract

Background: Influx of extracellular Ca(2+) 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 Ca(2+) influx is unknown. The recently discovered members of the CRACM/Orai ion channel family that carries the Ca(2+) release-activated Ca(2+) 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 Ca(2+)-selective current obtained under both conditions was blocked by 10 μM La(3+) and Gd(3+), known blockers of CRACM channels, and 2 distinct and specific CRACM-channel blockers-GSK-7975A and Synta-66. Both blockers reduced FcεRI-dependent Ca(2+) influx, and 3 μM GSK-7975A and Synta-66 reduced the release of histamine, leukotriene C(4), 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 Ca(2+) 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.

Fig 1

HLMCs express CRACM1, CRACM2, and CRACM3 mRNAs. RT-PCR of CRACM1, -2, and -3 and rig/S15 mRNA transcripts using RNA purified from HLMCs (A) and from HMC-1 cells (B) in the presence (+) or absence (−) of reverse transcriptase. C, Quantitative RT-PCR of CRACM mRNA transcripts in HLMCs relative to β-actin transcripts (mean ± SEM; n = 6-8). CRACM1 versus CRACM2, P = .0068.

Fig 2

HLMCs express CRACM1 and CRACM2 proteins. A, B, and C, Representative Western blots using the indicated antibodies and lysates of HMC-1 and HLMCs (Fig 2, A and B) or A-20 cells and HLMCs (Fig 2, C). Three independent mast cell lysates were analyzed. D, Western blots using the indicated antibodies and lysates of HEK293 cells transiently transfected with vectors directing the expression of CRACM1-Myc (lane 1), CRACM2-Myc (lane 2), or CRACM3-Myc (lane 3).

Fig 3

HLMCs express CRACM-channel currents when dialyzed with IP₃. Subtracted whole-cell patch-clamp current-voltage (I-V) curves from HLMCs 4 minutes after dialysis with IP₃ in 2 mM external Ca²⁺ (mean ± SEM; n = 42 cells) (A) and in 2 mM and then 10 mM external Ca²⁺ (mean ± SEM; n = 6) (B). C, Representative raw current traces from a single cell in the presence of IP₃ (voltage protocol shown inset). Inhibition of IP₃-dependent whole-cell current by (D) GSK-7975A and (E) Synta-66 (n = 4-6) (a negative value implies a contribution from CRACM channels to the baseline whole-cell current) and (F) 10 μM La³⁺ (n = 12) and (G) 10 μM Gd³⁺.

Fig 4

HLMCs express CRACM-channel currents following FcεRI-dependent activation. Whole-cell patch-clamp current-voltage (I-V) curves of HLMCs within 4 minutes of FcεRI-dependent activation in (A) 2 mM external Ca²⁺ (mean ± SEM; n = 37 cells) and (B) 2 mM and then 10 mM external Ca²⁺ (mean ± SEM; n = 7). C, Representative raw current following FcεRI-dependent activation. Inhibition of IgE-dependent CRACM currents by (D) 1 μM GSK-7975A, (E) 1 μM Synta-66, and (F) 10 μM La³⁺ (n = 11 cells). G, Attenuation of the FcεRI-dependent increase in [Ca²⁺]_i in HLMCs by 1 μM Synta-66.

Fig 5

The CRACM-channel blockers GSK-7975A and Synta-66 inhibit HLMC histamine (A), LTC₄(B), and cytokine release (C) dose-dependently following FcεRI-dependent activation. Mean ± SEM for percentage inhibition (n = 5 for histamine and LTC₄, n = 3 for cytokines). P < .05 for all conditions except Synta-66 TNFα analyzed by repeated-measures ANOVA on raw data. ∗P < .05 compared with control using Bonferroni post hoc test.

Fig 6

The CRACM-channel blocker Synta-66 attenuates allergen-dependent human bronchial smooth muscle contraction (n = 4). P = .02 for rightward shift in allergen EC₅₀.