title: Functional and Structural Divergence in Human TRPV1 Channel Subunits by Oxidative Cysteine Modification.
abstract: Transient receptor potential vanilloid 1 (TRPV1) channel is a tetrameric protein that acts as a sensor for noxious stimuli such as heat and for diverse inflammatory mediators such as oxidative stress to mediate nociception in a subset of sensory neurons. In TRPV1 oxidation sensing, cysteine (Cys) oxidation has been considered as the principle mechanism; however, its biochemical basis remains elusive. Here, we characterize the oxidative status of Cys residues in differential redox environments and propose a model of TRPV1 activation by oxidation. Through employing a combination of non-reducing SDS-PAGE, electrophysiology, and mass spectrometry we have identified the formation of subunit dimers carrying a stable intersubunit disulfide bond between Cys-258 and Cys-742 of human TRPV1 (hTRPV1). C258S and C742S hTRPV1 mutants have a decreased protein half-life, reflecting the role of the intersubunit disulfide bond in supporting channel stability. Interestingly, the C258S hTRPV1 mutant shows an abolished response to oxidants. Mass spectrometric analysis of Cys residues of hTRPV1 treated with hydrogen peroxide shows that Cys-258 is highly sensitive to oxidation. Our results suggest that Cys-258 residues are heterogeneously modified in the hTRPV1 tetrameric complex and comprise Cys-258 with free thiol for oxidation sensing and Cys-258, which is involved in the disulfide bond for assisting subunit dimerization. Thus, the hTRPV1 channel has a heterogeneous subunit composition in terms of both redox status and function.
authors.affiliation: From the Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.
authors.forename: Nozomi
authors.initials: N
authors.lastname: Ogawa
authors.affiliation: From the Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.
authors.forename: Tatsuki
authors.initials: T
authors.lastname: Kurokawa
authors.affiliation: From the Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Fujiwara
authors.affiliation: From the Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.
authors.forename: Onur Kerem
authors.initials: OK
authors.lastname: Polat
authors.affiliation: From the Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.
authors.forename: Heba
authors.initials: H
authors.lastname: Badr
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, and.
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Takahashi
authors.affiliation: From the Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan, Department of Technology and Ecology, Hall of Global Environmental Studies, Kyoto University, Kyoto 615-8510, Japan mori@sbchem.kyoto-u.ac.jp.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Mori
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry*; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D051381:Rats; D050916:TRPV Cation Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D050916:TRPV Cation Channels; C494482:TRPV1 protein, human; D006861:Hydrogen Peroxide; D003545:Cysteine
keywords: dimerization; disulfide bond; pain; post-translational modification (PTM); reactive oxygen species (ROS); redox regulation; transient receptor potential channels (TRP channels)