Binding and Activating of Analgesic Crotalphine with Human TRPA1
Abstract
:1. Introduction
2. Methods
2.1. Protein Expression and Purification
2.2. Cryo-EM Sample Preparation and Data Acquisition
2.3. Cryo-EM Data Processing
2.4. Model Building
2.5. Patch-Clamp Electrophysiology in HEK293 Cells
2.6. Microscale Thermophoresis (MST)
3. Results
3.1. The Function and Structure of TRPA1
3.2. Cryo-EM Structure of TRPA1-Crotalphine Complex
3.3. Interaction Between Crotalphine and TRPA1
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Diver, M.M.; Lin King, J.V.; Julius, D.; Cheng, Y. Sensory TRP channels in three dimensions. Annu. Rev. Biochem. 2022, 91, 629–649. [Google Scholar] [CrossRef] [PubMed]
- Schaefer, M. Homo- and heteromeric assembly of TRP channel subunits. Pflug. Arch.—Eur. J. Physiol. 2005, 451, 35–42. [Google Scholar] [CrossRef]
- Nilius, B.; Owsianik, G. The transient receptor potential family of ion channels. Genome Biol. 2011, 12, 218. [Google Scholar] [CrossRef]
- Gees, M.; Owsianik, G.; Nilius, B.; Voets, T. TRP channels. Compr. Physiol. 2012, 2, 563–608. [Google Scholar] [CrossRef]
- Zhang, M.; Ma, Y.; Ye, X.; Zhang, N.; Pan, L.; Wang, B. TRP (transient receptor potential) ion channel family: Structures, biological functions and therapeutic interventions for diseases. Signal Transduct. Target. Ther. 2023, 8, 261. [Google Scholar] [CrossRef]
- Raisinghani, M.; Zhong, L.; Jeffry, J.A.; Bishnoi, M.; Pabbidi, R.M.; Pimentel, F.; Cao, D.S.; Evans, M.S.; Premkumar, L.S. Activation characteristics of transient receptor potential ankyrin 1 and its role in nociception. Am. J. Physiol. Cell Physiol. 2011, 301, C587–C600. [Google Scholar] [CrossRef] [PubMed]
- Wei, Y.; Cai, J.; Zhu, R.; Xu, K.; Li, H.; Li, J. Function and therapeutic potential of transient receptor potential ankyrin 1 in fibrosis. Front. Pharmacol. 2022, 13, 1014041. [Google Scholar] [CrossRef] [PubMed]
- Chen, J.; Hackos, D.H. TRPA1 as a drug target--promise and challenges. Naunyn-Schmiedeberg’s Arch. Pharmacol. 2015, 388, 451–463. [Google Scholar] [CrossRef]
- Bessac, B.F.; Jordt, S.E. Breathtaking TRP channels: TRPA1 and TRPV1 in airway chemosensation and reflex control. Physiology 2008, 23, 360–370. [Google Scholar] [CrossRef]
- Moran, M.M. TRP channels as potential drug targets. Annu. Rev. Pharmacol. Toxicol. 2018, 58, 309–330. [Google Scholar] [CrossRef]
- Macpherson, L.J.; Xiao, B.; Kwan, K.Y.; Petrus, M.J.; Dubin, A.E.; Hwang, S.; Cravatt, B.; Corey, D.P.; Patapoutian, A. An ion channel essential for sensing chemical damage. J. Neurosci. Off. J. Soc. Neurosci. 2007, 27, 11412–11415. [Google Scholar] [CrossRef] [PubMed]
- McNamara, C.R.; Mandel-Brehm, J.; Bautista, D.M.; Siemens, J.; Deranian, K.L.; Zhao, M.; Hayward, N.J.; Chong, J.A.; Julius, D.; Moran, M.M.; et al. TRPA1 mediates formalin-induced pain. Proc. Natl. Acad. Sci. USA 2007, 104, 13525–13530. [Google Scholar] [CrossRef]
- Ruparel, N.B.; Patwardhan, A.M.; Akopian, A.N.; Hargreaves, K.M. Desensitization of transient receptor potential ankyrin 1 (TRPA1) by the TRP vanilloid 1-selective cannabinoid arachidonoyl-2 chloroethanolamine. Mol. Pharmacol. 2011, 80, 117–123. [Google Scholar] [CrossRef]
- Muller, C.; Morales, P.; Reggio, P.H. Cannabinoid Ligands Targeting TRP Channels. Front. Mol. Neurosci. 2019, 11, 487. [Google Scholar] [CrossRef] [PubMed]
- Chen, J.; Joshi, S.K.; DiDomenico, S.; Perner, R.J.; Mikusa, J.P.; Gauvin, D.M.; Segreti, J.A.; Han, P.; Zhang, X.-F.; Niforatos, W.; et al. Selective blockade of TRPA1 channel attenuates pathological pain without altering noxious cold sensation or body temperature regulation. Pain 2011, 152, 1165–1172. [Google Scholar] [CrossRef]
- Eid, S.R.; Crown, E.D.; Moore, E.L.; Liang, H.A.; Choong, K.C.; Dima, S.; Henze, D.A.; Kane, S.A.; Urban, M.O. HC-030031, a TRPA1 selective antagonist, attenuates inflammatory- and neuropathy-induced mechanical hypersensitivity. Mol. Pain 2008, 4, 48. [Google Scholar] [CrossRef]
- Bandell, M.; Story, G.M.; Hwang, S.W.; Viswanath, V.; Eid, S.R.; Petrus, M.J.; Earley, T.J.; Patapoutian, A. Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron 2004, 41, 849–857. [Google Scholar] [CrossRef] [PubMed]
- Jordt, S.E.; Bautista, D.M.; Chuang, H.H.; McKemy, D.D.; Zygmunt, P.M.; Högestätt, E.D.; Meng, I.D.; Julius, D. Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1. Nature 2004, 427, 260–265. [Google Scholar] [CrossRef]
- Hinman, A.; Chuang, H.H.; Bautista, D.M.; Julius, D. TRP channel activation by reversible covalent modification. Proc. Natl. Acad. Sci. USA 2006, 103, 19564–19568. [Google Scholar] [CrossRef]
- Macpherson, L.J.; Dubin, A.E.; Evans, M.J.; Marr, F.; Schultz, P.G.; Cravatt, B.F.; Patapoutian, A. Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines. Nature 2007, 445, 541–545. [Google Scholar] [CrossRef]
- Bahia, P.K.; Parks, T.A.; Stanford, K.R.; Mitchell, D.A.; Varma, S.; Stevens, S.M., Jr.; Taylor-Clark, T.E. The exceptionally high reactivity of Cys 621 is critical for electrophilic activation of the sensory nerve ion channel TRPA1. J. Gen. Physiol. 2016, 147, 451–465. [Google Scholar] [CrossRef] [PubMed]
- Suo, Y.; Wang, Z.; Zubcevic, L.; Hsu, A.L.; He, Q.; Borgnia, M.J.; Ji, R.R.; Lee, S.Y. Structural Insights into Electrophile Irritant Sensing by the Human TRPA1 Channel. Neuron 2020, 105, 882–894.e5. [Google Scholar] [CrossRef]
- Zhao, J.; Lin King, J.V.; Paulsen, C.E.; Cheng, Y.; Julius, D. Irritant-evoked activation and calcium modulation of the TRPA1 receptor. Nature 2020, 585, 141–145. [Google Scholar] [CrossRef]
- Chi, H.; Zhang, X.; Chen, X.; Fang, S.; Ding, Q.; Gao, Z. Sanguinarine is an agonist of TRPA1 channel. Biochem. Biophys. Res. Commun. 2021, 534, 226–232. [Google Scholar] [CrossRef]
- Liu, C.; Reese, R.; Vu, S.; Rougé, L.; Shields, S.D.; Kakiuchi-Kiyota, S.; Chen, H.; Johnson, K.; Shi, Y.P.; Chernov-Rogan, T.; et al. A Non-covalent Ligand Reveals Biased Agonism of the TRPA1 Ion Channel. Neuron 2021, 109, 273–284.e4. [Google Scholar] [CrossRef]
- Chernov-Rogan, T.; Gianti, E.; Liu, C.; Villemure, E.; Cridland, A.P.; Hu, X.; Ballini, E.; Lange, W.; Deisemann, H.; Li, T.; et al. TRPA1 modulation by piperidine carboxamides suggests an evolutionarily conserved binding site and gating mechanism. Proc. Natl. Acad. Sci. USA 2019, 116, 26008–26019. [Google Scholar] [CrossRef]
- Konno, K.; Picolo, G.; Gutierrez, V.P.; Brigatte, P.; Zambelli, V.O.; Camargo, A.C.; Cury, Y. Crotalphine, a novel potent analgesic peptide from the venom of the South American rattlesnake Crotalus durissus terrificus. Peptides 2008, 29, 1293–1304. [Google Scholar] [CrossRef] [PubMed]
- Gutierrez, V.P.; Konno, K.; Chacur, M.; Sampaio, S.C.; Picolo, G.; Brigatte, P.; Zambelli, V.O.; Cury, Y. Crotalphine induces potent antinociception in neuropathic pain by acting at peripheral opioid receptors. Eur. J. Pharmacol. 2008, 594, 84–92. [Google Scholar] [CrossRef] [PubMed]
- Machado, F.C.; Zambelli, V.O.; Fernandes, A.C.; Heimann, A.S.; Cury, Y.; Picolo, G. Peripheral interactions between cannabinoid and opioid systems contribute to the antinociceptive effect of crotalphine. Br. J. Pharmacol. 2014, 171, 961–972. [Google Scholar] [CrossRef]
- Ribeiro Lopes, F.S.; Giardini, A.C.; Martins Sant’anna, M.B.; Picolo, G. The analgesic effect of Crotalphine involves the activation of central opioid and cannabinoid receptors and the participation of microglia. Toxicon 2019, 168, S32. [Google Scholar] [CrossRef]
- Bressan, E.; Touska, F.; Vetter, I.; Kistner, K.; Kichko, T.I.; Teixeira, N.B.; Picolo, G.; Cury, Y.; Lewis, R.J.; Fischer, M.J.M.; et al. Crotalphine desensitizes TRPA1 ion channels to alleviate inflammatory hyperalgesia. Pain 2016, 157, 2504–2516. [Google Scholar] [CrossRef] [PubMed]
- Punjani, A.; Rubinstein, J.L.; Fleet, D.J.; Brubaker, M.A. cryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 2017, 14, 290–296. [Google Scholar] [CrossRef] [PubMed]
- Emsley, P.; Lohkamp, B.; Scott, W.G.; Cowtan, K. Features and development of Coot. Acta Crystallogr. Sect. D Biol. Crystallogr. 2010, 66 Pt 4, 486–501. [Google Scholar] [CrossRef] [PubMed]
- Liebschner, D.; Afonine, P.V.; Baker, M.L.; Bunkóczi, G.; Chen, V.B.; Croll, T.I.; Hintze, B.; Hung, L.W.; Jain, S.; McCoy, A.J.; et al. Macromolecular structure determination using X-rays, neutrons and electrons: Recent developments in Phenix. Acta Crystallogr. Sect. D Struct. Biol. 2019, 75 Pt 10, 861–877. [Google Scholar] [CrossRef]
- Paulsen, C.E.; Armache, J.P.; Gao, Y.; Cheng, Y.; Julius, D. Structure of the TRPA1 ion channel suggests regulatory mechanisms. Nature 2015, 520, 511–517. [Google Scholar] [CrossRef]
- Zhang, K.; Julius, D.; Cheng, Y. A step-by-step protocol for capturing conformational snapshots of ligand gated ion channels by single-particle cryo-EM. STAR Protoc. 2022, 3, 101732. [Google Scholar] [CrossRef]
- Wang, L.; Cvetkov, T.L.; Chance, M.R.; Moiseenkova-Bell, V.Y. Identification of in vivo disulfide conformation of TRPA1 ion channel. J. Biol. Chem. 2012, 287, 6169–6176. [Google Scholar] [CrossRef]
- Eberhardt, M.J.; Filipovic, M.R.; Leffler, A.; de la Roche, J.; Kistner, K.; Fischer, M.J.; Fleming, T.; Zimmermann, K.; Ivanovic-Burmazovic, I.; Nawroth, P.P.; et al. Methylglyoxal activates nociceptors through transient receptor potential channel A1 (TRPA1): A possible mechanism of metabolic neuropathies. J. Biol. Chem. 2012, 287, 28291–28306. [Google Scholar] [CrossRef]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Kang, M.; Zhang, Y.; Ding, X.; Xu, J.; Pang, X. Binding and Activating of Analgesic Crotalphine with Human TRPA1. Membranes 2025, 15, 187. https://doi.org/10.3390/membranes15060187
Kang M, Zhang Y, Ding X, Xu J, Pang X. Binding and Activating of Analgesic Crotalphine with Human TRPA1. Membranes. 2025; 15(6):187. https://doi.org/10.3390/membranes15060187
Chicago/Turabian StyleKang, Mingmin, Yanming Zhang, Xiufang Ding, Jianfu Xu, and Xiaoyun Pang. 2025. "Binding and Activating of Analgesic Crotalphine with Human TRPA1" Membranes 15, no. 6: 187. https://doi.org/10.3390/membranes15060187
APA StyleKang, M., Zhang, Y., Ding, X., Xu, J., & Pang, X. (2025). Binding and Activating of Analgesic Crotalphine with Human TRPA1. Membranes, 15(6), 187. https://doi.org/10.3390/membranes15060187