title: 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.
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.
authors: *Respiratory Medicine, Centre for Cardiovascular and Metabolic Research, Daisy Building, University of Hull and Hull York Medical School, Castle Hill Hospital, Cottingham HU16 5JQ, East Yorkshire, U.K.
authors: Kevin
authors: K
authors: Morgan
authors: *Respiratory Medicine, Centre for Cardiovascular and Metabolic Research, Daisy Building, University of Hull and Hull York Medical School, Castle Hill Hospital, Cottingham HU16 5JQ, East Yorkshire, U.K.
authors: Laura R
authors: LR
authors: Sadofsky
authors: *Respiratory Medicine, Centre for Cardiovascular and Metabolic Research, Daisy Building, University of Hull and Hull York Medical School, Castle Hill Hospital, Cottingham HU16 5JQ, East Yorkshire, U.K.
authors: Christopher
authors: C
authors: Crow
authors: *Respiratory Medicine, Centre for Cardiovascular and Metabolic Research, Daisy Building, University of Hull and Hull York Medical School, Castle Hill Hospital, Cottingham HU16 5JQ, East Yorkshire, U.K.
authors: Alyn H
authors: AH
authors: Morice
mesh_terms: D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics*; D002460:Cell Line; D045744:Cell Line, Tumor; D003080:Cold Temperature; D018076:DNA, Complementary / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D009419:Nerve Tissue Proteins / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000235:genetics*; D009447:Neuroblastoma / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000235:genetics; D000074025:TRPA1 Cation Channel; D050053:TRPM Cation Channels / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics*; D050051:Transient Receptor Potential Channels / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000235:genetics*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors*
chemical_list: D015220:Calcium Channels; D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; D047428:Protein Kinase Inhibitors; D000074025:TRPA1 Cation Channel; C473350:TRPA1 protein, human; D050053:TRPM Cation Channels; C425966:TRPM8 protein, human; D050051:Transient Receptor Potential Channels; D019061:src-Family Kinases; D002118:Calcium