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

Autor: Kevin Morgan, Laura R. Sadofsky, Alyn H. Morice, Christopher Crow
Jazyk: angličtina
Rok vydání: 2014
Předmět:
tyrosine kinases
lcsh:Life
lcsh:QR1-502
Biochemistry
lcsh:Microbiology
Transient receptor potential channel
Neuroblastoma
Transient Receptor Potential Channels
HC 030031
2-(1
3-dimethyl-2
6-dioxo-1
2
3
6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide
PP3
4-amino-7-phenylpyrazol[3
4-d]pyrimidine
TRPA1
transient receptor potential
A1TRPM8
transient receptor potential M8
TRPA1 Cation Channel
Transfection
SNP
single nucleotide polymorphism
EC50
Cold Temperature
WS 12
(1R
2S)-N-(4-methoxyphenyl)-5-methyl-2-(1-methylethyl)cyclohexanecarboxamide
src-Family Kinases
Phosphorylation
Proto-oncogene tyrosine-protein kinase Src
TRPM8
AMTB
N-(3-Aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide
PP2
4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3
4-d]pyrimidine
DNA
Complementary
Biophysics
SNP
TRPM Cation Channels
Nerve Tissue Proteins
Biology
S2
TRPA1
Polymorphism
Single Nucleotide
Cell Line
Cell Line
Tumor
Humans
AITC
allyl-isothiocyanate
Molecular Biology
ICL-1
intracellular loop-1
RFU
relative fluorescence unit
Protein Kinase Inhibitors
Original Paper
HEK 293 cells
Cell Biology
Molecular biology
Fusion protein
HEK
human embryonic kidney cells
lcsh:QH501-531
HEK293 Cells
Cell culture
Calcium
Calcium Channels
Protein Processing
Post-Translational
Zdroj: Bioscience Reports
Bioscience Reports, Vol 34, Iss 4, p e00131 (2014)
ISSN: 1573-4935
0144-8463
Popis: 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.
Transient receptor potential channels M8 and A1 sense the cellular environment and activate entry of Ca2+ into the cytoplasm. We identified a TRPA1 (transient receptor potential A1) variant with increased sensitivity and differential modulation of channels by a tyrosine kinase inhibitor in neuroblastoma cells.
Databáze: OpenAIRE
Externí odkaz: