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Transient receptor potential vanilloid 4 (TRPV4) channel as a target of crotamiton and its bimodal effects

  • Ion channels, receptors and transporters
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Abstract

The sensation of itching can be defined as “an unpleasant cutaneous sensation that provokes a desire to scratch.” The perception of itching is not critical for the maintenance of life, but persistent itching can be extremely irritating and decreases the quality of life. Crotamiton (N-ethyl-o-crotonotoluidide) has been used as an anti-itch agent for humans for around 70 years. In spite of the long use of crotamiton, its mechanism of action remains unknown. We hypothesized that crotamiton might have effects on transient receptor potential (TRP) channels expressed in the peripheral nervous system and the skin. We first examined the effects of crotamiton on TRP channels by whole-cell patch-clamp recordings. We found that crotamiton strongly inhibited TRPV (vanilloid) 4 channels followed by large currents after crotamiton washout. In mice, crotamiton inhibited itch-related behaviors induced by a TRPV4-selective agonist (GSK1016790A). We biophysically investigated the large TRPV4 currents after crotamiton washout. Comparing single-channel open probabilities and current amplitudes of TRPV4, increases in both parameters were found to contribute to the large washout currents of TRPV4. Because the change in current amplitudes suggested pore dilation of TRPV4, we examined this possibility with cation replacement experiments and by measuring changes in reversal potentials. Greater cation influxes and changes in reversal potentials upon crotamiton washout were observed, suggesting that the TRPV4 pore dilated in its uninhibited state. From these results, we identified the molecular target of crotamiton as TRPV4 and demonstrated pore dilation of TRPV4 upon crotamiton washout.

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Acknowledgements

This work was supported by the Japanese Ministry of Education, Culture, Sports, Science and Technology Grants 16K21691 (to H. K.), 15H02501 (to M. T.), and 15H05928 (to M. T.).

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Author notes

  1. Hiroki Kittaka and Yu Yamanoi contributed equally to this work.

Authors and Affiliations

  1. Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi, 444-8787, Japan

    Hiroki Kittaka, Yu Yamanoi & Makoto Tominaga

  2. Department of Physiological Sciences, The Graduate University for Advanced Studies (SOKENDAI), 5-1 Higashiyama, Myodaiji, Okazaki, Aichi, 444-8787, Japan

    Yu Yamanoi & Makoto Tominaga

  3. Research Laboratory, Ikedamohando Co., Ltd., 16 Jinden, Kamiichi, Nakaniikawa, Toyama, 930-0394, Japan

    Yu Yamanoi

  4. Institute for Environmental and Gender-Specific Medicine, Juntendo University, Chiba, 279-0021, Japan

    Makoto Tominaga

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  1. Hiroki Kittaka
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  2. Yu Yamanoi
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Contributions

H. K. and Y. Y. designed and performed the experiments. H. K., Y. Y., and M. T wrote the manuscript.

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Correspondence to Makoto Tominaga.

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This work was supported by the Japanese Ministry of Education, Culture, Sports, Science and Technology Grants 16K21691 (to H. K.), 15H02501 (to M. T.), and 15H05928 (to M. T.).

Electronic supplementary material

Supplementary figure 1

Effects of crotamiton on mTRPA1, mTRPM8, mTRPV1, mTRPV2 and mTRPV3 were investigated by whole cell patch-clamp recordings in the absence of extracellular Ca2+ in HEK293 cells. Holding potential was −60 mV. (JPEG 267 kb).

High resolution image (EPS 2294 kb)

Supplementary figure 2

Representative traces of mTRPV4 currents inhibited by crotamiton both in 2 mM extracellular calcium and extracellular calcium-free conditions by whole cell patch-clamp recordings in HEK293 cells. Ramp pulses from −100 mV to +100 mV for 500 ms were added every 5 s. Holding potential was −60 mV. (JPEG 173 kb).

High resolution image (EPS 1498 kb)

Supplementary figure 3

A, A representative trace of the membrane current in a mock-transfected HEK293T cell upon applications of crotamiton and TRP ligands. B, A representative trace of the membrane current in a HEK293T cell expressing mTRPV4 upon crotamiton application. Holding potential was −60 mV. (JPEG 188 kb).

High resolution image (EPS 1579 kb)

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Kittaka, H., Yamanoi, Y. & Tominaga, M. Transient receptor potential vanilloid 4 (TRPV4) channel as a target of crotamiton and its bimodal effects. Pflugers Arch - Eur J Physiol 469, 1313–1323 (2017). https://doi.org/10.1007/s00424-017-1998-7

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