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Kozai et al.
molecular recognition. However, it is unclear to what extent the selective transnitrosylating modulators of proteins. Our results
transnitrosylation site and the nonelectrophilic group-activating also suggest that an important concept in designing such
site overlap in TRPA1. NNO-ABBH1 may be considered as a pharmacological agents is that they should contain both
hybrid activator, apparently combining covalent binding to a transnitrosylating moiety and a skeleton that imbues target
reactive thiols with noncovalent molecular recognition in the protein selectivity via molecular recognition.
same binding pocket of TRPA1.
Our [Ca21]i measurements revealed that TRPV1, TRPV3,
Acknowledgments
The authors thank F. Karaki for providing NNO-ABBH5 and
performance of the Griess assay. The authors thank M. X. Mori,
N. Ogawa, and T. Kuwamura for experimental advice and helpful
discussions.
TRPV4, TRPC5, TRPC1/TRPC5, and TRPC4/TRPC5 failed to
respond to 300 mM NNO-ABBH1. In addition, S-nitrosylation
of TRPV1 proteins was not induced by NNO-ABBH1. This
may be due to the absence in these TRP channels of structural
features required for the binding of NNO-ABBH1. Consider-
ing the nature of transnitrosylation, the compounds must be
able to reach the target site cysteine residues for trans-
nitrosylation. Our findings suggest that molecular recognition
is critical for this. Indeed, TRPC5 and TRPV1 failed to respond
to the nonelectrophilic derivative NMe-ABBH, suggesting that
these proteins may lack the molecular recognition site for the
ABBH skeleton. However, we cannot exclude the possibility
that the ABBH compound binds to, but does not activate, these
channels. Importantly, the site for S-nitrosylation by SNAP
varies among TRP channels, being on the N-terminal side of
the pore region in TRPC5 and TRPV1, but in the cytoplasmic
N-terminal region of TRPA1 (Yoshida et al., 2006; Takahashi
et al., 2008; Miyamoto et al., 2009).
In addition to transnitrosylation via direct protein–protein
interaction, selectivity of S-nitrosylation is provided by a
proximity effect of the subcellular colocalization of NOS and
the targets of the generated NO, exemplified by the N-methyl-
D-aspartate receptor/NO signaling module (Hess et al., 2005).
We have demonstrated that TRPC5 is activated by nitro-
sylation via colocalized endothelial NOS (eNOS) following
ATP receptor stimulation in endothelial cells (Yoshida et al.,
2006). It is known that eNOS localizes in caveolae, a special-
ized lipid raft domain, and interacts with caveolin-1, a major
coat protein of caveolae (García-Cardeña et al., 1996a,b; Shaul
et al., 1996). In addition, caveolin-1 is known to interact with
TRPC1, which can form heteromers with TRPC5 (Lockwich
et al., 2000; Strübing et al., 2001). Therefore, it is possible that
eNOS and TRPC5 are in close proximity due to these in-
teractions in caveolae. It has been reported that the activation of
TRPV1 by ligands in endothelial cells may trigger Ca21-depen-
dent signaling, leading to enhanced TRPV1 phosphorylation,
the formation of a TRPV1-eNOS complex, activation of eNOS,
and NO production (Ching et al., 2011). Production of NO is
known to activate TRPA1 in dorsal root ganglion neurons
(Miyamoto et al., 2009), although it is unknown whether NOS
binds to TRPA1. Thus, TRPC5 and TRPV1 can achieve NO
signaling specificity through subcellular colocalization with
NOS, instead of through transnitrosylation.
Authorship Contributions
Participated in research design: Kozai, Takahashi, Mori, Ohwada.
Conducted experiments: Kozai, Kabasawa, Ebert, Firman, Otani,
Ohwada.
Contributed new reagents or analytic tools: Kabasawa, Firman,
Otani, Ohwada.
Performed data analysis: Kozai, Kabasawa, Ohwada.
Wrote or contributed to the writing of the manuscript: Kozai, Ebert,
Kiyonaka, Otani, Numata, Takahashi, Mori, Ohwada.
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Our studies using the WI-38 human fibroblast cell line
revealed that endogenous TRPA1 is activated through trans-
nitrosylation by ABBH N-nitrosamines. Recently, it has been
shown that TRPA1 is natively expressed in endothelial cells
as well, and allyl isothiocyanate–induced TRPA1 activation
in endothelial cells elicits vasodilation of cerebral arteries
(Earley et al., 2009). Thus, transnitrosylating agents ABBH
N-nitrosamines will be useful for investigating the effect of
transnitrosylation-induced TRPA1 activation on vasodilation.
In conclusion, our finding that N-nitroso derivatives of
ABBH are selective activators of TRPA1 channels and act via
transnitrosylation presents the opportunity for developing