title: Orai and TRPC channel characterization in FcεRI-mediated calcium signaling and mediator secretion in human mast cells.
abstract: Inappropriate activation of mast cells via the FcεRI receptor leads to the release of inflammatory mediators and symptoms of allergic disease. Calcium influx is a critical regulator of mast cell signaling and is required for exocytosis of preformed mediators and for synthesis of eicosanoids, cytokines and chemokines. Studies in rodent and human mast cells have identified Orai calcium channels as key contributors to FcεRI-initiated mediator release. However, until now the role of TRPC calcium channels in FcεRI-mediated human mast cell signaling has not been published. Here, we show evidence for the expression of Orai 1,2, and 3 and TRPC1 and 6 in primary human lung mast cells and the LAD2 human mast cell line but, we only find evidence of functional contribution of Orai and not TRPC channels to FcεRI-mediated calcium entry. Calcium imaging experiments, utilizing an Orai selective antagonist (Synta66) showed the contribution of Orai to FcεRI-mediated signaling in human mast cells. Although, the use of a TRPC3/6 selective antagonist and agonist (GSK-3503A and GSK-2934A, respectively) did not reveal evidence for TRPC6 contribution to FcεRI-mediated calcium signaling in human mast cells. Similarly, inactivation of STIM1-regulated TRPC1 in human mast cells (as tested by transfecting cells with STIM1-KK684-685EE - TRPC1 gating mutant) failed to alter FcεRI-mediated calcium signaling in LAD2 human mast cells. Mediator release assays confirm that FcεRI-mediated calcium influx through Orai is necessary for histamine and TNFαrelease but is differentially involved in the generation of cytokines and eicosanoids.
authors.affiliation: Department of Biomedical Science, University of Sheffield Western Bank, Sheffield, UK.
authors.forename: Hannah E
authors.initials: HE
authors.lastname: Wajdner
authors.affiliation: Department of Biomedical Science, University of Sheffield Western Bank, Sheffield, UK.
authors.forename: Jasmine
authors.initials: J
authors.lastname: Farrington
authors.affiliation: Department of Biomedical Science, University of Sheffield Western Bank, Sheffield, UK.
authors.forename: Claire
authors.initials: C
authors.lastname: Barnard
authors.affiliation: Academic Unit of Respiratory Medicine, University of Sheffield The Royal Hallamshire Hospital, Sheffield, UK.
authors.forename: Peter T
authors.initials: PT
authors.lastname: Peachell
authors.affiliation: Metabolic Pathways and Cardiovascular Unit, GlaxoSmithKline, King of Prussia, Pennsylvania.
authors.forename: Christine G
authors.initials: CG
authors.lastname: Schnackenberg
authors.affiliation: Metabolic Pathways and Cardiovascular Unit, GlaxoSmithKline, King of Prussia, Pennsylvania.
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Marino
authors.affiliation: Metabolic Pathways and Cardiovascular Unit, GlaxoSmithKline, King of Prussia, Pennsylvania.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Xu
authors.affiliation: Respiratory Therapy Area Unit, GlaxoSmithKline, Stevenage, UK.
authors.forename: Karen
authors.initials: K
authors.lastname: Affleck
authors.affiliation: Respiratory Therapy Area Unit, GlaxoSmithKline, Stevenage, UK.
authors.forename: Malcolm
authors.initials: M
authors.lastname: Begg
authors.affiliation: Department of Biomedical Science, University of Sheffield Western Bank, Sheffield, UK e.p.seward@sheffield.ac.uk.
authors.forename: Elizabeth P
authors.identifier: http://orcid.org/0000-0001-5311-1343
authors.initials: EP
authors.lastname: Seward
mesh_terms: D002118:Calcium / Q000378:metabolism*; D000071739:Calcium Release Activated Calcium Channels / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002460:Cell Line; D006801:Humans; D008168:Lung / Q000166:cytology / Q000378:metabolism; D008407:Mast Cells / Q000166:cytology / Q000378:metabolism*; D017455:Receptors, IgE / Q000378:metabolism*; D050052:TRPC Cation Channels / Q000378:metabolism*
chemical_list: D000071739:Calcium Release Activated Calcium Channels; D017455:Receptors, IgE; D050052:TRPC Cation Channels; D002118:Calcium
keywords: 
  STIM
  ; 
  TRPC
  ; Calcium; FcεRI; Orai; cytokine; human; mast cell