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.

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The high medical importance of Crotalus snakes is unquestionable, as this genus is the second in frequency of ophidian accidents in many countries, including Brazil. With a relative less complex composition compared to other genera venoms, as those from the Bothrops genus, the Crotalus genus venom from South America is composed basically by the neurotoxin crotoxin (a phospholipase A2), the thrombin-like gyroxin (a serinoprotease), a very potent aggregating protein convulxin, and a myotoxic polypeptide named crotamine. Interestingly not all Crotalus snakes express crotamine, which was first described in early 50s due to its ability to immobilize animal hind limbs, contributing therefore to the physical immobilization of preys and representing an important advantage for the envenoming efficacy, and consequently, for the feeding and survival of these snakes in nature. Representing about 10–25% of the dry weight of the crude venom of crotamine-positive rattlesnakes, the polypeptide crotamine is also suggested to be of importance for antivenom therapy, although the contribution of this toxin to the main symptoms of envenoming process remains far unknown until now. Herein, we concomitantly performed in vitro and in vivo assays to show for the first time the dose-dependent response of crotamine-triggered hind limbs paralysis syndrome, up to now believed to be observable only at high (sub-lethal) concentrations of crotamine. In addition, ex vivo assay performed with isolated skeletal muscles allowed us to suggest here that compounds active on voltage-sensitive sodium and/or potassium ion channels could both affect the positive inotropic effect elicited by crotamine in isolated diaphragm, besides also affecting the hind limbs paralysis syndrome imposed by crotamine in vivo. By identifying the potential molecular targets of this toxin, our data may contribute to open new roads for translational studies aiming to improve the snakebite envenoming treatment in human. Interestingly, we also demonstrate that the intraplantal or intraperitoneal (ip) injections of crotamine in mice do not promote pain. Therefore, this work may also suggest the profitable utility of non-toxic analogs of crotamine as a potential tool for targeting voltage-gated ion channels in skeletal muscles, aiming its potential use in the therapy of neuromuscular dysfunctions and envenoming therapy.

Background: Experimental autoimmune encephalomyelitis (EAE) is the most commonly used and clinically relevant murine model for human multiple sclerosis (MS), a demyelinating autoimmune disease characterized by mononuclear cell infiltration into the central nervous system (CNS). The aim of the present study was to appraise the alterations, poorly documented in the literature, which may occur at the peripheral nervous system (PNS) level. Methods: To this purpose, a multiple evaluation of peripheral nerve excitability was undertaken, by means of a minimally invasive electrophysiological method, in EAE mice immunized with the myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide, an experimental model for MS that reproduces, in animals, the anatomical and behavioral alterations observed in humans with MS, including CNS inflammation, demyelination of neurons, and motor abnormalities. Additionally, the myelin sheath thickness of mouse sciatic nerves was evaluated using transmission electronic microscopy.