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MmTX1 and MmTX2 from coral snake venom potently modulate GABAA receptor activity.

ABSTRACT: GABAA receptors shape synaptic transmission by modulating Cl(-) conductance across the cell membrane. Remarkably, animal toxins that specifically target GABAA receptors have not been identified. Here, we report the discovery of micrurotoxin1 (MmTX1) and MmTX2, two toxins present in Costa Rican coral snake venom that tightly bind to GABAA receptors at subnanomolar concentrations. Studies with recombinant and synthetic toxin variants on hippocampal neurons and cells expressing common receptor compositions suggest that MmTX1 and MmTX2 allosterically increase GABAA receptor susceptibility to agonist, thereby potentiating receptor opening as well as desensitization, possibly by interacting with the ?(+)/?(-) interface. Moreover, hippocampal neuron excitability measurements reveal toxin-induced transitory network inhibition, followed by an increase in spontaneous activity. In concert, toxin injections into mouse brain result in reduced basal activity between intense seizures. Altogether, we characterized two animal toxins that enhance GABAA receptor sensitivity to agonist, thereby establishing a previously unidentified class of tools to study this receptor family.

SUBMITTER: Rosso JP

PROVIDER: S-EPMC4345566 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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MmTX1 and MmTX2 from coral snake venom potently modulate GABAA receptor activity.

Rosso Jean-Pierre JP Schwarz Jürgen R JR Diaz-Bustamante Marcelo M Céard Brigitte B Gutiérrez José M JM Kneussel Matthias M Pongs Olaf O Bosmans Frank F Bosmans Frank F Bougis Pierre E PE

Proceedings of the National Academy of Sciences of the United States of America 20150209 8

GABAA receptors shape synaptic transmission by modulating Cl(-) conductance across the cell membrane. Remarkably, animal toxins that specifically target GABAA receptors have not been identified. Here, we report the discovery of micrurotoxin1 (MmTX1) and MmTX2, two toxins present in Costa Rican coral snake venom that tightly bind to GABAA receptors at subnanomolar concentrations. Studies with recombinant and synthetic toxin variants on hippocampal neurons and cells expressing common receptor comp ...[more]

PMID: 25675485

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Project description:BACKGROUND: Micrurus corallinus (coral snake) is a tropical forest snake belonging to the family Elapidae. Its venom shows a high neurotoxicity associated with pre- and post-synaptic toxins, causing diaphragm paralysis, which may result in death. In spite of a relatively small incidence of accidents, serum therapy is crucial for those bitten. However, the adequate production of antiserum is hampered by the difficulty in obtaining sufficient amounts of venom from a small snake with demanding breeding conditions. In order to elucidate the molecular basis of this venom and to uncover possible immunogens for an antiserum, we generated expressed sequences tags (ESTs) from its venom glands and analyzed the transcriptomic profile. In addition, their immunogenicity was tested using DNA immunization. RESULTS: A total of 1438 ESTs were generated and grouped into 611 clusters. Toxin transcripts represented 46% of the total ESTs. The two main toxin classes consisted of three-finger toxins (3FTx) (24%) and phospholipases A(2) (PLA(2)s) (15%). However, 8 other classes of toxins were present, including C-type lectins, natriuretic peptide precursors and even high-molecular mass components such as metalloproteases and L-amino acid oxidases. Each class included an assortment of isoforms, some showing evidence of alternative splicing and domain deletions. Five antigenic candidates were selected (four 3FTx and one PLA(2)) and used for a preliminary study of DNA immunization. The immunological response showed that the sera from the immunized animals were able to recognize the recombinant antigens. CONCLUSION: Besides an improvement in our knowledge of the composition of coral snake venoms, which are very poorly known when compared to Old World elapids, the expression profile suggests abundant and diversified components that may be used in future antiserum formulation. As recombinant production of venom antigens frequently fails due to complex disulfide arrangements, DNA immunization may be a viable alternative. In fact, the selected candidates provided an initial evidence of the feasibility of this approach, which is less costly and not dependent on the availability of the venom.

Project description:BACKGROUND: Snake venom is shaped by the ecology and evolution of venomous species, and signals of positive selection in toxins have been consistently documented, reflecting the role of venoms as an ecologically critical phenotype. New World coral snakes (Elapidae) are represented by three genera and over 120 species and subspecies that are capable of causing significant human morbidity and mortality, yet coral-snake venom composition is poorly understood in comparison to that of Old World elapids. High-throughput sequencing is capable of identifying thousands of loci, while providing characterizations of expression patterns and the molecular evolutionary forces acting within the venom gland. RESULTS: We describe the de novo assembly and analysis of the venom-gland transcriptome of the eastern coral snake (Micrurus fulvius). We identified 1,950 nontoxin transcripts and 116 toxin transcripts. These transcripts accounted for 57.1% of the total reads, with toxins accounting for 45.8% of the total reads. Phospholipases A(2) and three-finger toxins dominated expression, accounting for 86.0% of the toxin reads. A total of 15 toxin families were identified, revealing venom complexity previously unknown from New World coral snakes. Toxins exhibited high levels of heterozygosity relative to nontoxins, and overdominance may favor gene duplication leading to the fixation of advantageous alleles. Phospholipase A(2) expression was uniformly distributed throughout the class while three-finger toxin expression was dominated by a handful of transcripts, and phylogenetic analyses indicate that toxin divergence may have occurred following speciation. Positive selection was detected in three of the four most diverse toxin classes, suggesting that venom diversification is driven by recurrent directional selection. CONCLUSIONS: We describe the most complete characterization of an elapid venom gland to date. Toxin gene duplication may be driven by heterozygote advantage, as the frequency of polymorphic toxin loci was significantly higher than that of nontoxins. Diversification among toxins appeared to follow speciation reflecting species-specific adaptation, and this divergence may be directly related to dietary shifts and is suggestive of a coevolutionary arms race.

Project description:Millions of years of evolution have fine-tuned the ability of venom peptides to rapidly incapacitate both prey and potential predators. Toxicofera reptiles are characterized by serous-secreting mandibular or maxillary glands with heightened levels of protein expression. These glands are the core anatomical components of the toxicoferan venom system, which exists in myriad points along an evolutionary continuum. Neofunctionalisation of toxins is facilitated by positive selection at functional hotspots on the ancestral protein and venom proteins have undergone dynamic diversification in helodermatid and varanid lizards as well as advanced snakes. A spectacular point on the venom system continuum is the long-glanded blue coral snake (Calliophis bivirgatus), a specialist feeder that preys on fast moving, venomous snakes which have both a high likelihood of prey escape but also represent significant danger to the predator itself. The maxillary venom glands of C. bivirgatus extend one quarter of the snake's body length and nestle within the rib cavity. Despite the snake's notoriety its venom has remained largely unstudied. Here we show that the venom uniquely produces spastic paralysis, in contrast to the flaccid paralysis typically produced by neurotoxic snake venoms. The toxin responsible, which we have called calliotoxin (δ-elapitoxin-Cb1a), is a three-finger toxin (3FTx). Calliotoxin shifts the voltage-dependence of NaV1.4 activation to more hyperpolarised potentials, inhibits inactivation, and produces large ramp currents, consistent with its profound effects on contractile force in an isolated skeletal muscle preparation. Voltage-gated sodium channels (NaV) are a particularly attractive pharmacological target as they are involved in almost all physiological processes including action potential generation and conduction. Accordingly, venom peptides that interfere with NaV function provide a key defensive and predatory advantage to a range of invertebrate venomous species including cone snails, scorpions, spiders, and anemones. Enhanced activation or delayed inactivation of sodium channels by toxins is associated with the extremely rapid onset of tetanic/excitatory paralysis in envenomed prey animals. A strong selection pressure exists for the evolution of such toxins where there is a high chance of prey escape. However, despite their prevalence in other venomous species, toxins causing delay of sodium channel inhibition have never previously been described in vertebrate venoms. Here we show that NaV modulators, convergent with those of invertebrates, have evolved in the venom of the long-glanded coral snake. Calliotoxin represents a functionally novel class of 3FTx and a structurally novel class of NaV toxins that will provide significant insights into the pharmacology and physiology of NaV. The toxin represents a remarkable case of functional convergence between invertebrate and vertebrate venom systems in response to similar selection pressures. These results underscore the dynamic evolution of the Toxicofera reptile system and reinforces the value of using evolution as a roadmap for biodiscovery.

Dataset Information

MmTX1 and MmTX2 from coral snake venom potently modulate GABAA receptor activity.

Publications

MmTX1 and MmTX2 from coral snake venom potently modulate GABAA receptor activity.

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