Abstract
Background and purposePyrazole derivatives have recently been suggested as selective blockers of transient receptor potential cation (TRPC) channels but their ability to distinguish between the TRPC and Orai pore complexes is ill-defined. This study was designed to characterize a series of pyrazole derivatives in terms of TRPC/Orai selectivity and to delineate consequences of selective suppression of these pathways for mast cell activation.Experimental approachPyrazoles were generated by microwave-assisted synthesis and tested for effects on Ca2+ entry by Fura-2 imaging and membrane currents by patch-clamp recording. Experiments were performed in HEK293 cells overexpressing TRPC3 and in RBL-2H3 mast cells, which express classical store-operated Ca2+ entry mediated by Orai channels. The consequences of inhibitory effects on Ca2+ signalling in RBL-2H3 cells were investigated at the level of both degranulation and nuclear factor of activated T-cells activation.Key ResultsPyr3, a previously suggested selective inhibitor of TRPC3, inhibited Orai1- and TRPC3-mediated Ca2+ entry and currents as well as mast cell activation with similar potency. By contrast, Pyr6 exhibited a 37-fold higher potency to inhibit Orai1-mediated Ca2+ entry as compared with TRPC3-mediated Ca2+ entry and potently suppressed mast cell activation. The novel pyrazole Pyr10 displayed substantial selectivity for TRPC3-mediated responses (18-fold) and the selective block of TRPC3 channels by Pyr10 barely affected mast cell activation.Conclusions and ImplicationsThe pyrazole derivatives Pyr6 and Pyr10 are able to distinguish between TRPC and Orai-mediated Ca2+ entry and may serve as useful tools for the analysis of cellular functions of the underlying Ca2+ channels.
Highlights
Changes in cytosolic Ca2+ concentration control a broad range of cell- and tissue-specific processes reaching from B-cell activation, mast cell degranulation and cardiac pathologies, to cell proliferation and gene expression
HEK293 cells seeded out in adequate density were transiently transfected by lipofection using FuGENE® (Roche, Vienna, Austria) according to manufacturer’s protocol with an n-terminally YFP-tagged TRPC3 cDNA clone to be used as receptor operated Ca2+ entry’ (ROCE) model or with a CFP-tagged stromal interaction molecule 1 (STIM1) and YFP-tagged Orai1 clone to reconstitute the Ca2+ release-activated current (CRAC) pore in HEK293 cells (Muik et al, 2008)
Potency and selectivity of pyrazole compounds in ROCE and store operated Ca2+ entry’ (SOCE) model systems The group of Mori reported a high selectivity of Pyr3 for TRPC3 channels as compared to channels formed by other transient receptor potential channels (TRPCs) species (Kiyonaka et al, 2009)
Summary
Changes in cytosolic Ca2+ concentration control a broad range of cell- and tissue-specific processes reaching from B-cell activation, mast cell degranulation and cardiac pathologies, to cell proliferation and gene expression. Characterization of the mechanisms that govern Ca2+ channel function has resulted in a commonly accepted distinction between ‘receptor operated Ca2+ entry’ (ROCE) pathways that take place in response to receptor agonist/ligandinduced phospholipase C-mediated phosphoinositol-4,5bisphosphate hydrolysis formation (Hofmann et al, 1999; Lemonnier et al, 2008) and ‘store operated Ca2+ entry’ (SOCE), which is activated as a consequence of depletion of endoplasmic reticulum (ER) Ca2+ stores. Until the discovery of stromal interaction molecule 1 (STIM1) and Orai as key components of the latter process (Zhang et al, 2005; Prakriya et al, 2006), the family of canonical transient receptor potential channels (TRPCs) (Pedersen et al, 2005; Nilius et al, 2007; Abramowitz and Birnbaumer, 2009) has been considered the prime candidates for both Ca2+ entry pathways. Experiments were performed in HEK293 cells overexpressing TRPC3 and in RBL-2H3 mast cells, which express classical store-operated Ca2+ entry mediated by Orai channels. The consequences of inhibitory effects on Ca2+ signalling in RBL-2H3 cells were investigated at the level of both degranulation and nuclear factor of activated T-cells activation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
