The role of the S1-S4 sensor domain in the activation and modulation of the TRPA1 ion channel

L. Zímová1, V. Synytsya1,2, A. Hynková1,2,V. Zíma3, I. Barvík3, V. Vlachová1

1Institute of Physiology of the Czech Academy of Sciences, Department of Cellular Neurophysiology, Prague, Czech Republic

2Faculty of Science, Charles University in Prague, Prague, Czech Republic

3Faculty of Mathematics and Physics, Charles University in Prague, Prague, Czech Republic

Transient receptor potential ankyrin 1 (TRPA1) is a polymodal ion channel sensitive to noxious chemical agents. It is mainly expressed in peripheral nociceptor neurons. TRPA1 agonists include a broad class of electrophiles that activate the channel through covalent modification of reactive cysteines. It has been generally believed that the N-terminus is a key target for electrophiles [1, 2]. However, recent results of Moparthi et al. [3] showed that human TRPA1 is activated by electrophilic compounds even in the absence of the N-terminal domain. When activated, TRPA1 is partially calcium selective and permeating Ca2+ ions critically regulate its gating by promoting both potentiation and inactivation [4]. The molecular details behind these processes remain unknown as well as the nature of weak TRPA1 voltage sensitivity.

Here, we investigate the involvement of TRPA1 sensor domain, the bundle of S1-S4 helices, in electrophile sensitivity. Transmembrane location of this domain requires careful consideration of the contribution of voltage- and Ca2+- dependent modulation of the channel gating. There are 6 cysteine residues within the transmembrane part of each TRPA1 subunit. All of them represent possible targets for covalent modifications during stimulation by membrane permeable electrophilic agonists. Using the site directed mutagenesis and whole-cell electrophysiology we identified C856A mutant to have detrimental effect on TRPA1 gating with remarkable dependence on membrane voltage polarity and agonist character. Moreover, we were able to characterise two more alanine mutations that share very similar unique phenotype. Both are located at the intracellular side of S1-S4 bundle and both are approximately equally embedded in membrane yet probably spatially distant of each other. Thanks to the recently published 3D structure of the TRPA1 channel [5] we were able to build a reliable homology model, which showed that both residues are oriented into the splayed open lower vestibule of the S1-S4 sensor domain. The results allow us to suggest that the shape of these solvated vestibules of TRPA1 channel tetramer is a critical determinant of its voltage sensitivity and, by extension, an essential component of TRPA1 gating machinery.

1. Hinman, Andrew, et al. "TRP channel activation by reversible covalent modification." Proceedings of the National Academy of Sciences 103.51 (2006): 19564-19568.

2. Macpherson, Lindsey J., et al. "Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines." Nature 445.7127 (2007): 541-545.

3. Moparthi, Lavanya, et al. "Human TRPA1 is intrinsically cold-and chemosensitive with and without its N-terminal ankyrin repeat domain." Proceedings of the National Academy of Sciences 111.47 (2014): 16901-16906.

4. Wang, Yuanyuan Y., et al. "The nociceptor ion channel TRPA1 is potentiated and inactivated by permeating calcium ions." Journal of Biological Chemistry 283.47 (2008): 32691-32703.

5. Paulsen, Candice E., et al. "Structure of the TRPA1 ion channel suggests regulatory mechanisms." Nature (2015).