Crotalphine is a structural analogue to a novel analgesic peptide that was first identified in the crude venom from the South American rattlesnake Crotalus durissus terrificus. Although crotalphine's analgesic effect is well established, its direct mechanism of action remains unresolved. The aim of the present study was to investigate the effect of crotalphine on ion channels in peripheral pain pathways. We found that picomolar concentrations of crotalphine selectively activate heterologously expressed and native TRPA1 ion channels. TRPA1 activation by crotalphine required intact N-terminal cysteine residues and was followed by strong and long-lasting desensitization of the channel. Homologous desensitization of recombinant TRPA1 and heterologous desensitization in cultured dorsal root ganglia neurons was observed. Likewise, crotalphine acted on peptidergic TRPA1-expressing nerve endings ex vivo as demonstrated by suppression of calcitonin gene-related peptide release from the trachea and in vivo by inhibition of chemically induced and inflammatory hypersensitivity in mice. The crotalphine-mediated desensitizing effect was abolished by the TRPA1 blocker HC030031 and absent in TRPA1-deficient mice. Taken together, these results suggest that crotalphine is the first peptide to mediate antinociception selectively and at subnanomolar concentrations by targeting TRPA1 ion channels.

Department of Anesthesiology University Hospital Erlangen Friedrich Alexander University Erlangen Nurnberg Erlangen Germany

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

Department of Physiology University Hospital RWTH Aachen Aachen Germany

Institute for Molecular Bioscience The University of Queensland St Lucia Australia

Institute of Physiology and Pathophysiology Friedrich Alexander University Erlangen Nurnberg Erlangen Germany

Laboratory of Pain and Signaling Butantan Institute São Paulo Brazil

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Akopian AN, Ruparel NB, Jeske NA, Hargreaves KM. Transient receptor potential TRPA1 channel desensitization in sensory neurons is agonist dependent and regulated by TRPV1-directed internalization. J Physiol 2007;15:175–93.

Averbeck B, Reeh PW, Michaelis M. Modulation of CGRP and PGE2 release from isolated rat skin by alpha-adrenoceptors and kappa-opioid-receptors. Neuroreport 2001;12:2097–100.

Bandell M, Story GM, Hwang SW, Viswanath V, Eid SR, Petrus MJ, Earley TJ, Patapoutian A. Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron 2004;41:849–57.

Brazil V. Do emprego da peçonha em terapêutica. São Paulo: Biologia Médica 1934.

Brierley SM, Castro J, Harrington AM, Hughes PA, Page AJ, Rychkov GY, Blackshaw LA. TRPA1 contributes to specific mechanically activated currents and sensory neuron mechanical hypersensitivity. J Physiol 2011;589:3575–93.

Brigatte P, Hoffmann FA, Bernardi MM, Giorgi R, Fernandes I, Takehara HA, Barros SB, Almeida MG, Cury Y. Tolerance to the antinociceptive effect of crotalus durissus terrificus snake venom in mice is mediated by pharmacodynamic mechanisms. Toxicon 2001;39:1399–410.

Cunha TM, Verri WA Jr, Vivancos GG, Moreira IF, Reis S, Parada CA, Cunha FQ, Ferreira SH. An electronic pressure-meter nociception paw test for mice. Braz J Med Biol Res 2004;37:401–7.

Diochot S, Baron A, Salinas M, Douguet D, Scarzello S, Dabert-Gay AS, Debayle D, Friend V, Alloui A, Lazdunski M, Lingueglia E. Black mamba venom peptides target acid-sensing ion channels to abolish pain. Nature 2012;490:552–55.

Dittert I, Benedikt J, Vyklický L, Zimmermann K, Reeh PW, Vlachová V. Improved superfusion technique for rapid cooling or heating of cultured cells under patch-clamp conditions. J Neurosci Methods 2006;151:178–85.

Eberhardt MJ, Filipovic MR, Leffler A, de la Roche J, Kistner K, Fischer MJ, Fleming T, Zimmermann K, Ivanovic-Burmazovic I, Nawroth PP, Bierhaus A, Reeh PW, Sauer SK. Methylglyoxal activates nociceptors through transient receptor potential channel A1 (TRPA1): a possible mechanism of metabolic neuropathies. J Biol Chem 2012;287:28291–306.

Fischer MJ, Leffler A, Niedermirtl F, Kistner K, Eberhardt M, Reeh PW, Nau C. The general anesthetic propofol excites nociceptors by activating TRPV1 and TRPA1 rather than GABAA receptors. J Biol Chem 2010;285:34781–92.

Giorgi R, Bernardi MM, Cury Y. Analgesic effect evoked by low molecular weight substances extracted from crotalus durissus terrificus venom. Toxicon 1993;31:1257–65.

Gutierrez VP, Konno K, Chacur M, Sampaio SC, Picolo G, Brigatte P, Zambelli VO, Cury Y. Crotalphine induces potent antinociception in neuropathic pain by acting at peripheral opioid receptors. Eur J Pharmacol 2008;594:84–92.

Gutierrez VP, Zambelli VO, Picolo G, Chacur M, Sampaio SC, Brigatte P, Konno K, Cury Y. The peripheral L-arginine-nitric oxide-cyclic GMP pathway and ATP-sensitive K+ channels are involved in the antinociceptive effect of crotalphine on neuropathic pain in rats. Behav Pharmacol 2012;23:14–24.

Hinman A, Chuang HH, Bautista DM, Julius D. TRP channel activation by reversible covalent modification. Proc Natl Acad Sci U S A 2006;103:19564–68.

Ibrahim MM, Porreca F, Lai J, Albrecht PJ, Rice FL, Khodorova A, Davar G, Makriyannis A, Vanderah TW, Mata HP, Malan TP Jr. CB2 cannabinoid receptor activation produces antinociception by stimulating peripheral release of endogenous opioids. Proc Natl Acad Sci U S A 2005;102:3093–8.

Jiang WJ, Liang YX, Han LP, Qiu PX, Yuan J, Zhao SJ. Purification and characterization of a novel antinociceptive toxin from cobra venom (Naja naja atra). Toxicon 2008;52:638–46.

Kichko TI, Reeh PW. TRPV1 controls acid- and heat-induced calcitonin gene-related peptide release and sensitization by bradykinin in the isolated mouse trachea. Eur J Neurosci 2009;29:1896–904.

Konno K, Picolo G, Gutierrez VP, Brigatte P, Zambelli VO, Camargo AC, Cury Y. Crotalphine, a novel potent analgesic peptide from the venom of the South American rattlesnake crotalus durissus terrificus. Peptides 2008;29:1293–304.

Kwan KY, Allchorne AJ, Vollrath MA, Christensen AP, Zhang DS, Woolf CJ, Corey DP. TRPA1 contributes to cold, mechanical, and chemical nociception but is not essential for hair-cell transduction. Neuron 2006;50:277–89.

Lewis RJ, Sellin M, Poli MA, Norton RS, MacLeod JK, Sheil MM. Purification and characterization of ciguatoxins from moray eel (Lycodontis javanicus, Muraenidae). Toxicon 1991;29:1115–27.

Machado FC, Zambelli VO, Fernandes AC, Heimann AS, Cury Y, Picolo G. Peripheral interactions between cannabinoid and opioid systems contribute to the antinociceptive effect of crotalphine. Br J Pharmacol 2014;171:961–72.

Macpherson LJ, Dubin AE, Evans MJ, Marr F, Schultz PG, Cravatt BF, Patapoutian A. Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines. Nature 2007;445:541–45.

Meller ST, Gebhart GF. Intraplantar zymosan as a reliable, quantifiable model of thermal and mechanical hyperalgesia in the rat. Eur J Pain 1997;1:43–52.

Monaelesser A, Taguet C. Traitement des algies et des tumeurs par le venin de cobra. Bull Acad Med 1933;109:371–76.

Paulsen CE, Armache JP, Gao Y, Cheng Y, Julius D. Structure of the TRPA1 ion channel suggests regulatory mechanisms. Nature 2015;525:552.

Picolo G, Giorgi R, Cury Y. Delta-opioid receptors and nitric oxide mediate the analgesic effect of crotalus durissus terrificus snale venom. Eur J Pharmacol 2000;391:55–62.

Picolo G, Cury Y. Peripheral neuronal nitric oxide synthase activity mediates the antinociceptive effect of Crotalus durissus terrificus snake venom, a delta- and kappa-opioid receptor agonist. Life Sci 2004;75:559–73.

Pu XC, Wong PT, Gopalakrishnakone P. A novel analgesic toxin (hannalgesin) from the venom of king cobra (ophiophagus hannah). Toxicon 1995;33:1425–31.

Reeh PW. Sensory receptors in mammalian skin in an in vitro preparation. Neurosci Lett 1986;66:141–46.

Souza GP. Tratamento de epilepsia pelo veneno da cascavel. Ann Paul Med Cir 1914;1:33.

Sousa SR, Vetter I, Ragnarsson L, Lewis RJ. Expression and pharmacology of endogenous Cav channels in SH-SY5Y human neuroblastoma cells. PLoS One 2013;8:e59293.

Shuster SJ, Riedl M, Li X, Vulchanova L, Elde R. Stimulus-dependent translocation of kappa opioid receptors to the plasma membrane. J Neurosci 1999;19:2658–64.

Vetter I, Lewis RJ. Characterization of endogenous calcium responses in neuronal cell lines. Biochem Pharmacol 2010;79:908–20.

Vetter I, Mozar CA, Durek T, Wingerd JS, Alewood PF, Christie MJ, Lewis RJ. Characterization of Na(v) types endogenously expressed in human SH-SY5Y neuroblastoma cells. Biochem Pharmacol 2012;83:1562–71.

Vetter I, Touska F, Hess A, Hinsbey R, Sattler S, Lampert A, Sergejeva M, Sharov A, Collins LS, Eberhardt M, Engel M, Cabot PJ, Wood JN, Vlachová V, Reeh PW, Lewis RJ, Zimmermann K. Ciguatoxins activate specific cold pain pathways to elicit burning pain from cooling. EMBO J 2012;31:3795–808.

Vilceanu D, Stucky CL. TRPA1 mediates mechanical currents in the plasma membrane of mouse sensory neurons. PLoS One 2010;5:e12177.

Wang H, Ehnert C, Brenner GJ, Woolf CJ. Bradykinin and peripheral sensitization. Biol Chem 2006;387:11–14.

Xiao B, Dubin AE, Bursulaya B, Viswanath V, Jegla TJ, Patapoutian A. Identification of transmembrane domain 5 as a critical molecular determinant of menthol sensitivity in mammalian TRPA1 channels. J Neurosci 2008;28:9640–51.

Xu H, Delling M, Jun J, Clapham D. Oregano, thyme and clove-derived flavors and skin sensitizers activate specific TRP channels. Nat Neurosci 2006;9:628–35.

Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. PAIN 1983;16:109–10.