title: Camphor modulates TRPV3 cation channels activity by interacting with critical pore-region cysteine residues.
abstract: 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.
authors.affiliation: Department of Pharmacy, Havelian Campus, Hazara University, Abbottabad, Pakistan. azhar.sherkheli@daad-alumni.de
authors.forename: Muhammad Azhar
authors.initials: MA
authors.lastname: Sherkheli
authors.forename: Angela K
authors.initials: AK
authors.lastname: Vogt-Eisele
authors.forename: Kirsten
authors.initials: K
authors.lastname: Weber
authors.forename: Hanns
authors.initials: H
authors.lastname: Hatt
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001896:Boron Compounds / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002164:Camphor / Q000494:pharmacology*; D003545:Cysteine / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D039821:Monoterpenes / Q000494:pharmacology; D050916:TRPV Cation Channels / Q000819:agonists / Q000378:metabolism*; D014981:Xenopus
chemical_list: D001896:Boron Compounds; D018076:DNA, Complementary; D039821:Monoterpenes; D050916:TRPV Cation Channels; C486760:Trpv3 protein, mouse; D002164:Camphor; C109986:2-aminoethoxydiphenyl borate; D003545:Cysteine; D002118:Calcium; C534305:2-methyl-5-(1-methylethenyl)cyclohexanol