title: Functional transient receptor potential melastatin 7 channels are critical for human mast cell survival.
abstract: 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.
authors.affiliation: Department of Infection, Immunity and Inflammation, Institute for Lung Health, University of Leicester, Leicester, United Kingdom.
authors.forename: Rob C E
authors.initials: RC
authors.lastname: Wykes
authors.forename: Moonhee
authors.initials: M
authors.lastname: Lee
authors.forename: S Mark
authors.initials: SM
authors.lastname: Duffy
authors.forename: Weidong
authors.initials: W
authors.lastname: Yang
authors.forename: Elizabeth P
authors.initials: EP
authors.lastname: Seward
authors.forename: Peter
authors.initials: P
authors.lastname: Bradding
mesh_terms: D058666:Adrenergic beta-2 Receptor Agonists; D002413:Cations, Divalent / Q000378:metabolism; D016923:Cell Death / Q000235:genetics / Q000276:immunology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics / Q000276:immunology; D006801:Humans; D008168:Lung / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D008407:Mast Cells / Q000166:cytology* / Q000276:immunology / Q000378:metabolism*; D008564:Membrane Potentials / Q000235:genetics / Q000276:immunology*; D018408:Patch-Clamp Techniques; D017346:Protein Serine-Threonine Kinases; D034622:RNA Interference / Q000276:immunology; D012333:RNA, Messenger / Q000096:biosynthesis; D018343:Receptors, Adrenergic, beta-2 / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050053:TRPM Cation Channels / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014161:Transduction, Genetic