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Deciphering Subtype-Selective Modulations in TRPA1 Biosensor Channels.

Kozai D, Sakaguchi R, Ohwada T, Mori Y - Curr Neuropharmacol (2015)

Bottom Line: More recently, we found that a novel N-nitrosamine compound activates TRPA1 by S-nitrosylation (the addition of a nitric oxide (NO) group to cysteine thiol), and does so with significant selectivity over other NO-sensitive TRP channels.It is proposed that this subtype selectivity is conferred through synergistic effects of electrophilic cysteine transnitrosylation and molecular recognition of the non-electrophilic moiety on the N-nitrosamine.In this review, we describe the molecular pharmacology of these TRPA1 modulators and discuss their modulatory mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura Campus, Nishikyoku, Kyoto 615-8510, Japan. mori@sbchem.kyoto-u.ac.jp.

ABSTRACT
The transient receptor potential (TRP) proteins are a family of ion channels that act as cellular sensors. Several members of the TRP family are sensitive to oxidative stress mediators. Among them, TRPA1 is remarkably susceptible to various oxidants, and is known to mediate neuropathic pain and respiratory, vascular and gastrointestinal functions, making TRPA1 an attractive therapeutic target. Recent studies have revealed a number of modulators (both activators and inhibitors) that act on TRPA1. Endogenous mediators of oxidative stress and exogenous electrophiles activate TRPA1 through oxidative modification of cysteine residues. Non-electrophilic compounds also activate TRPA1. Certain non-electrophilic modulators may act on critical non-cysteine sites in TRPA1. However, a method to achieve selective modulation of TRPA1 by small molecules has not yet been established. More recently, we found that a novel N-nitrosamine compound activates TRPA1 by S-nitrosylation (the addition of a nitric oxide (NO) group to cysteine thiol), and does so with significant selectivity over other NO-sensitive TRP channels. It is proposed that this subtype selectivity is conferred through synergistic effects of electrophilic cysteine transnitrosylation and molecular recognition of the non-electrophilic moiety on the N-nitrosamine. In this review, we describe the molecular pharmacology of these TRPA1 modulators and discuss their modulatory mechanisms.

No MeSH data available.


Related in: MedlinePlus

Selective S-nitrosylation of human TRPA1 by a novel N-nitrosamine. Chemical structures of NNO-ABBH1 (a) and nonelectrophilicanalogs (b). (c) The chemical mechanism underlying the transnitrosylating action of NNO-ABBH1 on protein thiol group. (d)Intracellular Ca2+ concentration ([Ca2+]i) measurements using fura-2. Maximum rises in [Ca2+]i (∆ [Ca2+]i) evoked by 300 µM NNO-ABBH1in HEK 293T cells expressing TRPA1, TRPV1, TRPV3, TRPV4, TRPC5, TRPC1/TRPC5, TRPC4/TRPC5, or vector. (n = 47–107). ***P < 0.001 compared to vector. (e) ∆ [Ca2+]i evoked by 300 µM SNAP in HEK 293T cells expressing TRPA1, TRPV1, TRPV3, TRPV4, TRPC5, TRPC1/TRPC5, TRPC4/TRPC5, or vector (n = 22–111). *P < 0.05 and ***P < 0.001 compared to vector. Reproduced from Fig. 1 of [76]with permission.
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Figure 4: Selective S-nitrosylation of human TRPA1 by a novel N-nitrosamine. Chemical structures of NNO-ABBH1 (a) and nonelectrophilicanalogs (b). (c) The chemical mechanism underlying the transnitrosylating action of NNO-ABBH1 on protein thiol group. (d)Intracellular Ca2+ concentration ([Ca2+]i) measurements using fura-2. Maximum rises in [Ca2+]i (∆ [Ca2+]i) evoked by 300 µM NNO-ABBH1in HEK 293T cells expressing TRPA1, TRPV1, TRPV3, TRPV4, TRPC5, TRPC1/TRPC5, TRPC4/TRPC5, or vector. (n = 47–107). ***P < 0.001 compared to vector. (e) ∆ [Ca2+]i evoked by 300 µM SNAP in HEK 293T cells expressing TRPA1, TRPV1, TRPV3, TRPV4, TRPC5, TRPC1/TRPC5, TRPC4/TRPC5, or vector (n = 22–111). *P < 0.05 and ***P < 0.001 compared to vector. Reproduced from Fig. 1 of [76]with permission.

Mentions: To develop transnitrosylation-based subtype-selective activators of TRP channels, it is necessary to first identify a synthetic NO donor that has only the transnitrosylative reactivity. However, SNAP(S-nitroso-N-acetyl-DL-penicillamine) and NOR3 ((±)-(E)-4-ethyl-2-(E)-hydroxyimino-5-nitro-3-hexenamide) are NO-releasing donors. In addition, it is suggested that nitroglycerin also releases NO by its metabolism [147]. S-Nitrosoglutathione is known to be a biological transnitrosylating agent, but also releases NO [148, 149]. In contrast, the ABBH N-nitrosamines constitute a new class of NO donors that, at physiological pH and temperature, transnitrosylate thiols to generate S-nitrosothiols without releasing NO [150-152]. Surprisingly, our intracellular Ca2+ imaging measurements have demonstrated that N-nitroso-2-exo,3-exo-ditrifluoromethyl-7-azabenzobicyclo [2.2.1] heptane (NNO-ABBH1) induces robust Ca2+ influx via recombinant human TRPA1 channels, but not via other SNAP-activated TRP channels, suggesting that NNO-ABBH1 selectively S-nitrosylates TRPA1 [76] (Fig. 4). A modified labeling assay to biochemically identify protein S-nitrosothiol [153] showed that SNAP S-nitrosylates both TRPA1 and TRPV1, but NNO-ABBH1 S-nitrosylates only TRPA1. TRPA1 activation by NNO-ABBH1 is suppressed by specific cysteine mutations but not by NO scavenging, indicating that transnitrosylation underlies the activation of TRPA1 by NNO-ABBH1. This is supported by a positive correlation of N–NO bond reactivity and TRPA1-activating potency in a congeneric series of ABBH N-nitrosamines. Cys540, Cys641, and Cys665 of human TRPA1 are involved in its modification by NNO-ABBH1. Cys641 and Cys665 are also required for responsiveness to SNAP [32, 53], indicating that Cys540 may be a unique target for NNO-ABBH1.


Deciphering Subtype-Selective Modulations in TRPA1 Biosensor Channels.

Kozai D, Sakaguchi R, Ohwada T, Mori Y - Curr Neuropharmacol (2015)

Selective S-nitrosylation of human TRPA1 by a novel N-nitrosamine. Chemical structures of NNO-ABBH1 (a) and nonelectrophilicanalogs (b). (c) The chemical mechanism underlying the transnitrosylating action of NNO-ABBH1 on protein thiol group. (d)Intracellular Ca2+ concentration ([Ca2+]i) measurements using fura-2. Maximum rises in [Ca2+]i (∆ [Ca2+]i) evoked by 300 µM NNO-ABBH1in HEK 293T cells expressing TRPA1, TRPV1, TRPV3, TRPV4, TRPC5, TRPC1/TRPC5, TRPC4/TRPC5, or vector. (n = 47–107). ***P < 0.001 compared to vector. (e) ∆ [Ca2+]i evoked by 300 µM SNAP in HEK 293T cells expressing TRPA1, TRPV1, TRPV3, TRPV4, TRPC5, TRPC1/TRPC5, TRPC4/TRPC5, or vector (n = 22–111). *P < 0.05 and ***P < 0.001 compared to vector. Reproduced from Fig. 1 of [76]with permission.
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Related In: Results  -  Collection

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Figure 4: Selective S-nitrosylation of human TRPA1 by a novel N-nitrosamine. Chemical structures of NNO-ABBH1 (a) and nonelectrophilicanalogs (b). (c) The chemical mechanism underlying the transnitrosylating action of NNO-ABBH1 on protein thiol group. (d)Intracellular Ca2+ concentration ([Ca2+]i) measurements using fura-2. Maximum rises in [Ca2+]i (∆ [Ca2+]i) evoked by 300 µM NNO-ABBH1in HEK 293T cells expressing TRPA1, TRPV1, TRPV3, TRPV4, TRPC5, TRPC1/TRPC5, TRPC4/TRPC5, or vector. (n = 47–107). ***P < 0.001 compared to vector. (e) ∆ [Ca2+]i evoked by 300 µM SNAP in HEK 293T cells expressing TRPA1, TRPV1, TRPV3, TRPV4, TRPC5, TRPC1/TRPC5, TRPC4/TRPC5, or vector (n = 22–111). *P < 0.05 and ***P < 0.001 compared to vector. Reproduced from Fig. 1 of [76]with permission.
Mentions: To develop transnitrosylation-based subtype-selective activators of TRP channels, it is necessary to first identify a synthetic NO donor that has only the transnitrosylative reactivity. However, SNAP(S-nitroso-N-acetyl-DL-penicillamine) and NOR3 ((±)-(E)-4-ethyl-2-(E)-hydroxyimino-5-nitro-3-hexenamide) are NO-releasing donors. In addition, it is suggested that nitroglycerin also releases NO by its metabolism [147]. S-Nitrosoglutathione is known to be a biological transnitrosylating agent, but also releases NO [148, 149]. In contrast, the ABBH N-nitrosamines constitute a new class of NO donors that, at physiological pH and temperature, transnitrosylate thiols to generate S-nitrosothiols without releasing NO [150-152]. Surprisingly, our intracellular Ca2+ imaging measurements have demonstrated that N-nitroso-2-exo,3-exo-ditrifluoromethyl-7-azabenzobicyclo [2.2.1] heptane (NNO-ABBH1) induces robust Ca2+ influx via recombinant human TRPA1 channels, but not via other SNAP-activated TRP channels, suggesting that NNO-ABBH1 selectively S-nitrosylates TRPA1 [76] (Fig. 4). A modified labeling assay to biochemically identify protein S-nitrosothiol [153] showed that SNAP S-nitrosylates both TRPA1 and TRPV1, but NNO-ABBH1 S-nitrosylates only TRPA1. TRPA1 activation by NNO-ABBH1 is suppressed by specific cysteine mutations but not by NO scavenging, indicating that transnitrosylation underlies the activation of TRPA1 by NNO-ABBH1. This is supported by a positive correlation of N–NO bond reactivity and TRPA1-activating potency in a congeneric series of ABBH N-nitrosamines. Cys540, Cys641, and Cys665 of human TRPA1 are involved in its modification by NNO-ABBH1. Cys641 and Cys665 are also required for responsiveness to SNAP [32, 53], indicating that Cys540 may be a unique target for NNO-ABBH1.

Bottom Line: More recently, we found that a novel N-nitrosamine compound activates TRPA1 by S-nitrosylation (the addition of a nitric oxide (NO) group to cysteine thiol), and does so with significant selectivity over other NO-sensitive TRP channels.It is proposed that this subtype selectivity is conferred through synergistic effects of electrophilic cysteine transnitrosylation and molecular recognition of the non-electrophilic moiety on the N-nitrosamine.In this review, we describe the molecular pharmacology of these TRPA1 modulators and discuss their modulatory mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura Campus, Nishikyoku, Kyoto 615-8510, Japan. mori@sbchem.kyoto-u.ac.jp.

ABSTRACT
The transient receptor potential (TRP) proteins are a family of ion channels that act as cellular sensors. Several members of the TRP family are sensitive to oxidative stress mediators. Among them, TRPA1 is remarkably susceptible to various oxidants, and is known to mediate neuropathic pain and respiratory, vascular and gastrointestinal functions, making TRPA1 an attractive therapeutic target. Recent studies have revealed a number of modulators (both activators and inhibitors) that act on TRPA1. Endogenous mediators of oxidative stress and exogenous electrophiles activate TRPA1 through oxidative modification of cysteine residues. Non-electrophilic compounds also activate TRPA1. Certain non-electrophilic modulators may act on critical non-cysteine sites in TRPA1. However, a method to achieve selective modulation of TRPA1 by small molecules has not yet been established. More recently, we found that a novel N-nitrosamine compound activates TRPA1 by S-nitrosylation (the addition of a nitric oxide (NO) group to cysteine thiol), and does so with significant selectivity over other NO-sensitive TRP channels. It is proposed that this subtype selectivity is conferred through synergistic effects of electrophilic cysteine transnitrosylation and molecular recognition of the non-electrophilic moiety on the N-nitrosamine. In this review, we describe the molecular pharmacology of these TRPA1 modulators and discuss their modulatory mechanisms.

No MeSH data available.


Related in: MedlinePlus