Pain-producing animal venoms contain evolutionarily honed toxins that can be exploited to study and manipulate somatosensory and nociceptive signaling pathways. From a functional screen, we have identified a secreted phospholipase A2 (sPLA2)-like protein, BomoTx, from the Brazilian lancehead pit viper (Bothrops moojeni). BomoTx is closely related to a group of Lys49 myotoxins that have been shown to promote ATP release from myotubes through an unknown mechanism. Here we show that BomoTx excites a cohort of sensory neurons via ATP release and consequent activation of P2X₂ and/or P2X₃ purinergic receptors. We provide pharmacological and electrophysiological evidence to support pannexin hemichannels as downstream mediators of toxin-evoked ATP release. At the behavioral level, BomoTx elicits nonneurogenic inflammatory pain, thermal hyperalgesia, and mechanical allodynia, of which the latter is completely dependent on purinergic signaling. Thus, we reveal a role of regulated endogenous nucleotide release in nociception and provide a detailed mechanism of a pain-inducing Lys49 myotoxin from Bothrops species, which are responsible for the majority of snake-related deaths and injuries in Latin America.

Pain-producing animal venoms contain evolutionarily honed toxins that can be exploited to study and manipulate somatosensory and nociceptive signaling pathways. From a functional screen, we have identified a secreted phospholipase A2 (sPLA2)-like protein, BomoTx, from the Brazilian lancehead pit viper (Bothrops moojeni). BomoTx is closely related to a group of Lys49 myotoxins that have been shown to promote ATP release from myotubes through an unknown mechanism. Here we show that BomoTx excites a cohort of sensory neurons via ATP release and consequent activation of P2X₂ and/or P2X₃ purinergic receptors. We provide pharmacological and electrophysiological evidence to support pannexin hemichannels as downstream mediators of toxin-evoked ATP release. At the behavioral level, BomoTx elicits nonneurogenic inflammatory pain, thermal hyperalgesia, and mechanical allodynia, of which the latter is completely dependent on purinergic signaling. Thus, we reveal a role of regulated endogenous nucleotide release in nociception and provide a detailed mechanism of a pain-inducing Lys49 myotoxin from Bothrops species, which are responsible for the majority of snake-related deaths and injuries in Latin America.

Background

Beta-cardiotoxin (β-CTX), the three-finger toxin isolated from king cobra (Ophiophagus hannah) venom, possesses β-blocker activity as indicated by its negative chronotropy and its binding property to both β-1 and β-2 adrenergic receptors and has been proposed as a novel β-blocker candidate. Previously, β-CTX was isolated and purified by FPLC. Here, we present an alternative method to purify this toxin. In addition, we tested its cytotoxicity against different mammalian muscle cell types and determined the impact on cardiac function in isolated cardiac myocyte so as to provide insights into the pharmacological action of this protein.

Methods

β-CTX was isolated from the crude venom of the Thai king cobra using reverse-phased and cation exchange HPLC. In vitro cellular viability MTT assays were performed on mouse myoblast (C2C12), rat smooth muscle (A7r5), and rat cardiac myoblast (H9c2) cells. Cell shortening and calcium transient dynamics were recorded on isolated rat cardiac myocytes over a range of β-CTX concentration.

Results

Purified β-CTX was recovered from crude venom (0.53% w/w). MTT assays revealed 50% cytotoxicity on A7r5 cells at 9.41 ± 1.14 µM (n = 3), but no cytotoxicity on C2C12 and H9c2 cells up to 114.09 µM. β-CTX suppressed the extend of rat cardiac cell shortening in a dose-dependent manner; the half-maximal inhibition concentration was 95.97 ± 50.10 nM (n = 3). In addition, the rates of cell shortening and re-lengthening were decreased in β-CTX treated myocytes concomitant with a prolongation of the intracellular calcium transient decay, indicating depression of cardiac contractility secondary to altered cardiac calcium homeostasis.

Conclusion

We present an alternative purification method for β-CTX from king cobra venom. We reveal cytotoxicity towards smooth muscle and depression of cardiac contractility by this protein. These data are useful to aid future development of pharmacological agents derived from β-CTX.

Natural products that elicit discomfort or pain represent invaluable tools for probing molecular mechanisms underlying pain sensation. Plant-derived irritants have predominated in this regard, but animal venoms have also evolved to avert predators by targeting neurons and receptors whose activation produces noxious sensations. As such, venoms provide a rich and varied source of small molecule and protein pharmacophores that can be exploited to characterize and manipulate key components of the pain-signalling pathway. With this in mind, here we perform an unbiased in vitro screen to identify snake venoms capable of activating somatosensory neurons. Venom from the Texas coral snake (Micrurus tener tener), whose bite produces intense and unremitting pain, excites a large cohort of sensory neurons. The purified active species (MitTx) consists of a heteromeric complex between Kunitz- and phospholipase-A2-like proteins that together function as a potent, persistent and selective agonist for acid-sensing ion channels (ASICs), showing equal or greater efficacy compared with acidic pH. MitTx is highly selective for the ASIC1 subtype at neutral pH; under more acidic conditions (pH < 6.5), MitTx massively potentiates (>100-fold) proton-evoked activation of ASIC2a channels. These observations raise the possibility that ASIC channels function as coincidence detectors for extracellular protons and other, as yet unidentified, endogenous factors. Purified MitTx elicits robust pain-related behaviour in mice by activation of ASIC1 channels on capsaicin-sensitive nerve fibres. These findings reveal a mechanism whereby snake venoms produce pain, and highlight an unexpected contribution of ASIC1 channels to nociception.

Snakes possess a unique sensory system for detecting infrared radiation, enabling them to generate a 'thermal image' of predators or prey. Infrared signals are initially received by the pit organ, a highly specialized facial structure that is innervated by nerve fibres of the somatosensory system. How this organ detects and transduces infrared signals into nerve impulses is not known. Here we use an unbiased transcriptional profiling approach to identify TRPA1 channels as infrared receptors on sensory nerve fibres that innervate the pit organ. TRPA1 orthologues from pit-bearing snakes (vipers, pythons and boas) are the most heat-sensitive vertebrate ion channels thus far identified, consistent with their role as primary transducers of infrared stimuli. Thus, snakes detect infrared signals through a mechanism involving radiant heating of the pit organ, rather than photochemical transduction. These findings illustrate the broad evolutionary tuning of transient receptor potential (TRP) channels as thermosensors in the vertebrate nervous system.