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Functional Transient Receptor Potential Melastatin 7 Channels Are Critical for Human Mast Cell Survival

Rob C. E. Wykes^1,*,, Moonhee Lee, S. Mark Duffy^*, Weidong Yang^*, Elizabeth P. Seward^2,3, and Peter Bradding^2,*

^* Department of Infection, Immunity and Inflammation, Institute for Lung Health, University of Leicester, Leicester, United Kingdom; and Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, United Kingdom

Mast cells play a significant role in the pathophysiology of many diverse diseases such as asthma and pulmonary fibrosis. Ca²⁺ influx is essential for mast cell degranulation and release of proinflammatory mediators, while Mg²⁺ plays an important role in cellular homeostasis. The channels supporting divalent cation influx in human mast cells have not been identified, but candidate channels include the transient receptor potential melastatin (TRPM) family. In this study, we have investigated TRPM7 expression and function in primary human lung mast cells (HLMCs) and in the human mast cell lines LAD2 and HMC-1, using RT-PCR, patch clamp electrophysiology, and RNA interference. Whole cell voltage-clamp recordings revealed a nonselective cation current that activated spontaneously following loss of intracellular Mg²⁺. The current had a nonlinear current-voltage relationship with the characteristic steep outward rectification associated with TRPM7 channels. Reducing external divalent concentration from 3 to 0.3 mM dramatically increased the size of the outward current, whereas the current was markedly inhibited by elevated intracellular Mg²⁺ (6 mM). Ion substitution experiments revealed cation selectivity and Ca²⁺ permeability. RT-PCR confirmed the presence of mRNA for TRPM7 in HLMC, LAD2, and HMC-1 cells. Adenoviral-mediated knockdown of TRPM7 in HLMC with short hairpin RNA and in HMC-1 with short interfering RNA markedly reduced TRPM7 currents and induced cell death, an effect that was not rescued by raising extracellular Mg²⁺. In summary, HLMC and human mast cell lines express the nonselective cation channel TRPM7 whose presence is essential for cell survival.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Current address: Department of Physiology M211, Northwestern University, 303 East Chicago Avenue, Chicago, IL 60611.

² E.P.S. and P.B. are joint senior authors for this work.

³ Address correspondence and reprint requests to Dr. Elizabeth P. Seward, Department of Biomedical Science, Alfred Denny Building, University of Sheffield, Western Bank, Sheffield S10 2TN, U.K. E-mail address: e.p.seward{at}sheffield.ac.uk

⁴ Abbreviations used in this paper: TRP, transient receptor potential; TRPM, TRP melastatin; TRPV, TRP vanilloid; HLMC, human lung mast cell; siRNA, short interfering RNA; shRNA, short hairpin RNA; eGFP, enhanced GFP; [Ca²⁺]_i, intracellular Ca²⁺ concentration; Vm, reversal potential; I_CRAC, Ca²⁺ release-activated Ca²⁺ current.

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