Isolation of human TRPA1 channel from transfected HEK293 cells and identification of alkylation sites after sulfur mustard exposure.
Agonists of hTRPA1
Amino acid modifications
HETE
Hydroxyethylthioethyl-moiety
Immunomagnetic separation
μLC-ESI MS/HR MS
Journal
Archives of toxicology
ISSN: 1432-0738
Titre abrégé: Arch Toxicol
Pays: Germany
ID NLM: 0417615
Informations de publication
Date de publication:
02 2023
02 2023
Historique:
received:
05
08
2022
accepted:
03
11
2022
pubmed:
14
11
2022
medline:
25
1
2023
entrez:
13
11
2022
Statut:
ppublish
Résumé
Transient receptor potential (TRP) channels are important in the sensing of pain and other stimuli. They may be triggered by electrophilic agonists after covalent modification of certain cysteine residues. Sulfur mustard (SM) is a banned chemical warfare agent and its reactivity is also based on an electrophilic intermediate. The activation of human TRP ankyrin 1 (hTRPA1) channels by SM has already been documented, however, the mechanism of action is not known in detail. The aim of this work was to purify hTRPA1 channel from overexpressing HEK293 cells for identification of SM-induced alkylation sites. To confirm hTRPA1 isolation, Western blot analysis was performed showing a characteristic double band at 125 kDa. Immunomagnetic separation was carried out using either an anti-His-tag or an anti-hTRPA1 antibody to isolate hTRPA1 from lysates of transfected HEK293 cells. The identity of the channel was confirmed by micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry. Following SM exposure, hTRPA1 channel modifications were found at Cys
Identifiants
pubmed: 36371551
doi: 10.1007/s00204-022-03411-1
pii: 10.1007/s00204-022-03411-1
pmc: PMC9859856
doi:
Substances chimiques
Mustard Gas
T8KEC9FH9P
TRPA1 Cation Channel
0
Cysteine
K848JZ4886
Chemical Warfare Agents
0
TRPA1 protein, human
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
429-439Subventions
Organisme : Deutsche Forschungsgemeinschaft
ID : GRK2338
Informations de copyright
© 2022. The Author(s).
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