Skip to main content

Research Repository

Advanced Search

Human TRPM8 and TRPA1 pain channels, including a gene variant with increased sensitivity to agonists (TRPA1 R797T), exhibit differential regulation by SRC-tyrosine kinase inhibitor

Morgan, Kevin; Sadofsky, Laura R.; Crow, Christopher; Morice, Alyn H.

Authors

Kevin Morgan

Christopher Crow



Abstract

TRPM8 (transient receptor potential M8) and TRPA1 (transient receptor potential A1) are cold-temperature-sensitive nociceptors expressed in sensory neurons but their behaviour in neuronal cells is poorly understood. Therefore DNA expression constructs containing human TRPM8 or TRPA1 cDNAs were transfected into HEK (human embryonic kidney cells)-293 or SH-SY5Y neuroblastoma cells and G418 resistant clones analysed for effects of agonists and antagonists on intracellular Ca2+ levels. Approximately 51% of HEK-293 and 12% of SH-SY5Y cell clones expressed the transfected TRP channel. TRPM8 and TRPA1 assays were inhibited by probenecid, indicating the need to avoid this agent in TRP channel studies. A double-residue mutation in ICL-1 (intracellular loop-1) of TRPM8 (SV762,763EL, mimicking serine phosphorylation) or one in the C-terminal tail region (FK1045,1046AG, a lysine knockout) retained sensitivity to agonists (WS 12, menthol) and antagonist {AMTB [N-(3-Aminopropyl)-2-[(3- methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide]}. SNP (single nucleotide polymorphism) variants in TRPA1 ICL-1 (R797T, S804N) and TRPA1 fusion protein containing C-terminal (His)10 retained sensitivity to agonists (cinnamaldehyde, allyl-isothiocyanate, carvacrol, eugenol) and antagonists (HC-030031, A967079). One SNP variant, 797T, possessed increased sensitivity to agonists. TRPA1 became repressed in SH-SY5Y clones but was rapidly rescued by Src-family inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine]. Conversely, TRPM8 in SH-SY5Y cells was inhibited by PP2. Further studies utilizing SH-SY5Y may identify structural features of TRPA1 and TRPM8 involved in conferring differential post-translational regulation. © 2014 The Author(s).

Citation

Morgan, K., Sadofsky, L. R., Crow, C., & Morice, A. H. (2014). Human TRPM8 and TRPA1 pain channels, including a gene variant with increased sensitivity to agonists (TRPA1 R797T), exhibit differential regulation by SRC-tyrosine kinase inhibitor. Bioscience Reports, 34(4), 469-478. https://doi.org/10.1042/BSR20140061

Journal Article Type Article
Acceptance Date May 16, 2014
Online Publication Date Jun 30, 2014
Publication Date 2014-08
Deposit Date May 16, 2022
Publicly Available Date May 27, 2022
Journal Bioscience Reports
Print ISSN 0144-8463
Electronic ISSN 1573-4935
Publisher Portland Press
Peer Reviewed Peer Reviewed
Volume 34
Issue 4
Pages 469-478
DOI https://doi.org/10.1042/BSR20140061
Keywords EC50; SNP; TRPA1; TRPM8; Tyrosine kinases
Public URL https://hull-repository.worktribe.com/output/3610095

Files






You might also like



Downloadable Citations