Merck

Camphor modulates TRPV3 cation channels activity by interacting with critical pore-region cysteine residues.

Pakistan journal of pharmaceutical sciences (2013-04-30)
Muhammad Azhar Sherkheli, Angela K Vogt-Eisele, Kirsten Weber, Hanns Hatt
RESUMEN

TRPV3 ion channels mediate thermo-transduction, nociception, inflammation and dermatitis in mammals. TRPV1-4 proteins have been shown to have conserved cysteine-residues in the pore-forming regions. These residues participate in channel activation via S-nitrosylation of channel proteins. Camphor is a commonly used ligand for TRPV3 channels. Thus the knowledge about the potential binding/interacting site(s) for camphor will help to design effective and potent analgesic compounds. In an overlap-extension PCR method, following primer-pairs were used to mutate conserved cysteine-residues in the pore-region of TRPV3 channels; GATTGAGAATcCTCCAAGGACAAAAAGGAC, TRPV3-C612S-Fw and GTCCTTGGAGgACTTCTCAATCAGTCAGTGAGG, TRPV3-C612S-Rv primers pair. And for TRPV3-C619S: GGACTCcAGTTCCTATGGCCAGC, TRPV3-C619S-Fw and GCTGGCCATAgGAACTGGAGTCC, TRPV3-C619S-Rv respectively. All cDNA constructs were confirmed by DNA-sequencing and used to make cRNAs. Oocytes expressing mTRPV3-C619S and mTRPV3-C612S mutant channels were challenged with 2-APB (1 mM), camphor (10 mM) and dihydrocarveol (10 mM) either at -40 mV or +40 mV holding potentials in voltage-clamp experiments. Responses of both mutants to 2-APB were similar to wild-type mTRPV3. Interestingly, responses to camphor were totally lost in mTRPV3-C619S mutant, while responses to dihydrocarveol remained intact. In contrast mTRPV3-C612S displayed slightly altered (16±2 % reduction) phenotype with respect to camphor sensitivity. It is concluded that pore-region cysteines play critical role in camphor sensitivity of TRPV3 ion channels.

MATERIALES
Referencia del producto
Marca
Descripción del producto

Sigma-Aldrich
(±)-Camphor, ≥95.5%
Sigma-Aldrich
(±)-Camphor, meets analytical specification of Ph. Eur., BP, ≥95% (GC), racemic
Sigma-Aldrich
(±)-Camphor, purum, synthetic, ≥95.0% (GC)
Sigma-Aldrich
(1R)-(+)-Camphor, 98%
Sigma-Aldrich
Camphor, 96%
Supelco
(−)-Camphor, analytical standard
Camphor (racemic), European Pharmacopoeia (EP) Reference Standard
Camphor (dl), primary reference standard
Sigma-Aldrich
L-Cysteine, BioUltra, ≥98.5% (RT)
SAFC
L-Cysteine
Sigma-Aldrich
L-Cysteine, from non-animal source, BioReagent, suitable for cell culture, ≥98%
Sigma-Aldrich
D-Camphor, ≥97%, FG
Sigma-Aldrich
(1S)-(−)-Camphor, 95%
Sigma-Aldrich
L-Cysteine, 97%
Sigma-Aldrich
L-Cysteine, ≥97%, FG
Supelco
D-Camphor, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
L-Cysteine, produced by Wacker Chemie AG, Burghausen, Germany, ≥98.0%
Supelco
L-Cysteine, certified reference material, TraceCERT®