title: Transnitrosylation directs TRPA1 selectivity in N-nitrosamine activators.
abstract: S-Nitrosylation, the addition of a nitrosyl group to cysteine thiols, regulates various protein functions to mediate nitric oxide (NO) bioactivity. Recent studies have demonstrated that selectivity in protein S-nitrosylation signaling pathways is conferred through transnitrosylation, a transfer of the NO group, between proteins via interaction. We previously demonstrated that sensitivity to activation by synthetic NO-releasing agents via S-nitrosylation is a common feature of members of the transient receptor potential (TRP) family of Ca(2+)-permeable cation channels. However, strategies to confer subtype selectivity to nitrosylating agents targeted to TRP channels are yet to be developed. Here, we show selective activation of TRPA1 channels by novel NO donors derived from the ABBH (7-azabenzobicyclo[2.2.1]heptane) N-nitrosamines, which exhibit transnitrosylation reactivity to thiols without releasing NO. The NNO-ABBH1 (N-nitroso-2-exo,3-exo-ditrifluoromethyl-7-azabenzobicyclo[2.2.1]heptane) elicits S-nitrosylation of TRPA1 proteins, and dose-dependently induces robust Ca(2+) influx via both recombinant and native TRPA1 channels, but not via other NO-activated TRP channels. TRPA1 activation by NNO-ABBH1 is suppressed by specific cysteine mutations but not by NO scavenging, suggesting that cysteine transnitrosylation underlies the activation of TRPA1 by NNO-ABBH1. This is supported by the correlation of N-NO bond reactivity and TRPA1-activating potency in a congeneric series of ABBH N-nitrosamines. Interestingly, nonelectrophilic derivatives of ABBH also activate TRPA1 selectively, but less potently, compared with NNO-ABBH1. Thus, ABBH N-nitrosamines confer subtype selectivity on S-nitrosylation in TRP channels through synergetic effects of two chemical processes: cysteine transnitrosylation and molecular recognition of the nonelectrophilic moiety.
authors.affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering (D.K., M.E., S.K., T.N., N.T., Y.M.), Department of Technology and Ecology, Hall of Global Environmental Studies (S.K., T.N., Y.M.), and Advanced Biomedical Engineering Research Unit (N.T.), Kyoto University, Kyoto, Japan; Laboratory of Organic and Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan (Y.K., F., Y.O., T.O.); and Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Tokyo, Japan (S.K., Y.M.).
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kozai
authors.forename: Yoji
authors.initials: Y
authors.lastname: Kabasawa
authors.forename: Maximilian
authors.initials: M
authors.lastname: Ebert
authors.forename: Shigeki
authors.initials: S
authors.lastname: Kiyonaka
authors.forename: Yuko
authors.initials: Y
authors.lastname: Otani
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Numata
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Takahashi
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Mori
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Ohwada
mesh_terms: D001372:Aza Compounds / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D015220:Calcium Channels / Q000378:metabolism*; D057809:HEK293 Cells; D006536:Heptanes / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D009602:Nitrosamines / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D000074025:TRPA1 Cation Channel; D050051:Transient Receptor Potential Channels / Q000378:metabolism*
chemical_list: D001372:Aza Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D015220:Calcium Channels; D006536:Heptanes; D009419:Nerve Tissue Proteins; D020030:Nitric Oxide Donors; D009602:Nitrosamines; D000074025:TRPA1 Cation Channel; C473350:TRPA1 protein, human; D050051:Transient Receptor Potential Channels