On how to eat hyper-toxic frogs - energetic variations of the voltage-gated sodium channel of potential prey

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Several studies have proposed that predator-prey interactions promote an evolutionary arms race. The prey must avoid being predated and the predator must consequently overcome those recently acquired characteristics. One example is the interaction between Phyllobates terribilis, a hyper-toxic frog, and its predator the snake Erythrolamprus epinephelus. Two consecutive point mutations were found on the voltage gated sodium channel (NaV) of the snake, the target protein of the batrachotoxin (BTX), the frog’s lethal defense. Protein fragments containing the mutations were inserted in complete NaV sequences from three different snakes, Thamnophis sirtalis, Python bovaris, and Ophiophagus hannah. The proteins were then modeled to compare their 3D structure and electrostatic potential energy with the original proteins. Even though no structural changes were found, significant electrostatic variations did occur. This was particularly the case in T. sirtalis, the more closely related species. Electrostatic energy is important for protein-ligand interaction because it determines how much energy is available for bonding. Electrostatic variation in the proteins containing the mutations could explain the resistance to BTX mechanism.


Ha sido propuesto en varios estudios que las interacciones depredador-presa se comportan de acuerdo con un modelo evolutivo de carrera armamentista, en donde una presa debe generar barreras para evitar ser depredada y su contraparte debe sobreponerse a estas barreras. Un ejemplo poco estudiado es el caso de la serpiente Erythrolamprus epinephelus que se alimenta de la rana hipertóxica Phyllobates terribilis. Se reportaron en la serpiente dos mutaciones puntuales consecutivas en el canal de sodio voltaje dependiente (NaV), proteína blanco de la batracotoxina (BTX). Fragmentos de ésta proteína fueron insertados en secuencias de NaV completas de tres especies de serpientes diferentes, Thamnophis sirtalis,

Python bovaris, y Ophiophagus hannah. Las proteínas fueron modeladas para

comparar su estructura tridimensional y potencial electrostático con las proteínas originales. A pesar de no encontrar cambios estructurales, se encontraron variaciones electrostáticas significativas, especialmente en el caso de T. sirtalis, la especie más cercanamente emparentada. La energía electrostática es importante para la interacción entre las proteínas y los ligandos, dado que determina cuanta energía libre hay para generar enlaces. Es por esto que las variaciones electrostáticas en las proteínas que contienen las mutaciones pueden estar explicando el mecanismo de resistencia a la BTX.


Voltage gated sodium channel, protein, electrostatic energy, resistance, batrachotoxin, point mutations.



It has been proposed by several authors that the predator-prey interaction is one of the most strong selective pressures (Oksanen et al., 1981; Brodie & Brodie, 1999 & Abrams, 2000). Prey benefit from acquiring or developing different techniques to decrease the probability of being predated while the predators need to overcome them. Predator-prey interactions have therefore been proposed to follow the dynamics proposed by the Red Queen Hypothesis, suggesting an arms race-like relationship between the interactive species (Van Valen, 1974; Brodie & Brodie, 1999). When prey contain very noxious or lethal toxins, predators are expected to modify their behaviour or physiology in order to be able to consume it without risks, for example throughout molecular adaptation (Williams et al., 2003). It has been demonstrated that different point mutations in the target protein have allowed predators to ingest toxic prey without any negative effects (Feldman, 2012).

A poorly known predator-prey system includes the hypertoxic frog Phyllobates

terribilis (Myers et al., 1978), from the family Dendrobatidae, who secretes

batrachotoxin (BTX) through the skin (Daly & Witkop, 1965), and the snake Erythrolamprus (Liophis) epinephelus (Cope, 1862). BTX is a lethal alkaloid compound that targets voltage-dependent sodium channels (Nav), interrupting the correct ion transport through cellular membranes, causing death through muscular paralysis. As seen in other systems, just like with bufanotoxin and tetrodotoxin (TTX) in toads and salamanders (Geffeney et al., 2005), two point mutations were found in

E. epinephelus´s Nav P-Loop of the IV domain (Ramirez, 2014 unpublished). These


to BTX, due to the congruence with TTX and pyrhenoid resistance mutations reported by several authors (Rinkevich et al., 2013 & Wang & Wang, 1999).

The functional role of these mutations in BTX resistance can not be ascertained until the mutated voltage-gated channel is cloned and assessed in vitro. A first approach is to compare the differences in 3D structure of proteins and energetic variations in the protein-ligand interaction. Resistance to BTX could be hypothesised in two ways: firstly, a change in structure could prevent the ligand from interacting with the binding site. Secondly, a change in energetic properties could be related to free energy available for viable interactions; point mutations could alter proteic energetic fields, decreasing free energy for bonding and the isoelectric constants, both relevant in the protein-ligand interaction (Ujvari et al., 2015).


DNA sequences of S6 and p-loop from the four domains of the voltage gated sodium channel of E. epinephelus were obtained from Ramirez (2014). The DNA was then translated into amino acids using Geneious 7.0. To obtain complete E. epinephelus protein sequence chimeras, these fragments were aligned with the complete proteins

of Thamnophis sirtalis (AY851744.1), Python bovaris (XP_007432184) and

Ophiophagus hannah (ETE69867), using TCoffee Expresso tool and then inserted in

the complete protein sequences in their corresponding locus, replacing the original S6s and P-Loops (Fig 1.) (Table 1).

The protein sequences, originals and chimeras, were modeled using Swiss-Model by ExPASy web server, using the homology method (Biasini et al. 2014; Arnold et al.


2006 & Sali, 1995). The predicted model PDB files were then analyzed using DeepView Swiss PDB-Viewer. The three pairs of proteins, the original and chimeras

E. epinephelus, were treated as replicates. Proteins were then pairwise overlapped

to observe structural changes and to measure the distance between alpha carbon atoms with a root square mean (RMS) measurement (Clore et al., 1987).

To estimate energetic variations, proteins were studied individually. Electrostatic potential fields were calculated for each protein using a Poisson-Boltzman distribution with a dielectric constant of 4.00 for the protein and assuming a solvent ionic strenght of 0.00mol/L, as in aqueous conditions. Values for electrostatic force and total forces were obtained from the whole protein and for the mutation loci in order to observe both local and general effects of the point mutations. Additionally, isoelectric constant was calculated using ProtParam by ExPASy for every protein. Pairwise comparisons were carried out between the original sequence and the E.

epinephelus transformed sequences. As the three template models are not

equivalently related to E. epinephelus, genetic distance was measured between all the sequences in order to calibrate the differences, using Python as the most ancient representative.


To properly validate the structural comparisons, we created 3D models based on ion transporter protein template, with an average homology of 30% (± 3), assuring the presence of all functional structures, like the pore and the extracellular loops. Overlapped proteins showed a highly conserved transmembrane region, while extracellular loops did have large variations (Fig 2). When comparing alpha carbon


RMS (Fig. 3), the relatedness of the focal species to E. epinephelus was correlated with the structural similarity of the protein; indeed the structures were not significantly different when compared to T. sirtalis.

Energetic variations do follow a different pattern. When observing electrostatic potential fields, all chimeras showed higher potential fields than the original protein, except for the positive field in Python (Fig. 4). In terms of electrostatic energy, the pattern is opposite to the one found in structural comparisons; the phylogenetically closest species, T. sirtalis, is the one with largest effects on its energetic characteristics (Fig 5). Finally, isoelectric constant comparisons show no statistical differences among the species studied here.


Regarding protein structural comparisons, among species differences can be explained by phylogenetic distance rather than an eventual effect of mutations on the protein folding patterns. The larger the distance, the more different the structures were. Even if the NaV sequence and structure were biologically constrained due to its important physiological role, p-loops do vary between species and there is a phylogenetic inertia that has larger effects than two consecutive point mutations.

On the other hand, electrostatic energy differences were larger among more closely related species, showing a greater importance and effects on T. sirtalis. This could represent a possible mechanism for resistance, where two consecutive point mutations have a greater effect on two really similar structures. Electrostatic energy is relevant for protein-ligand interaction because it refers to how much energy


available the protein has for bonding (Sharp & Honing, 1990). The point mutations D4i17S and G4i18D are located in the p-loop; a single mutation can alter the entire protein´s force field, taking into consideration all biological and evolutionary constraints (Feldman, 2012). Broad changes in protein function due to two consecutive point mutations has also been reported in Nav1.4 β1. The mutations, also found on a p-loop, could alter the entire subunits functionality (Scior, 2015). Rinkevich et al. (2013) also reported that two mutations could increase resistance to pyrethroid compounds compared to just one mutation’s effect.

Even though both point mutations increase the electrostatic energy of the entire protein and of each loci, they both present different natures. The first mutation D4i17S changes from a charged amino acid to a polar one, but then the consecutive locus, G4i18D, reverses again from a polar amino acid to the same charged one. Even though the overall charge of the protein seems to have non significant changes, its behavior does change due to changes in the interaction of internal charged groups and minor shifts in its conformational energy. This behavior shows that the relevant electrostatic energy is not only the sum of individual electrostatic energies (Sharp & Honig, 1990).



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género Phyllobates: Caracterización de secuencias del gen codificante para el canal de sodio voltaje dependiente. Undergraduate thesis. Universidad Nacional de Colombia

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Table 1. Genetic distance between the templates, Thamnophis, Cobra and Python, and the chimera E. epinephelus sequences.


Thamnophis Mutated Cobra Mutated Python


s Cobra Python

Mutated Thamnophis


Mutated Cobra

0.316 0

Mutated Python

0.295 0.157 0

Thamnophis 0.007 0.325 0.304 0

Cobra 0.376 0.045 0.209 0.372 0


Figure 1. Mutant chimeras procedure, the original E. epinephelus fragments from S6 fragments and P-Loops from the four NaV domains, shown in orange, replace analogous loci on the original sequences from Thamnophis, Ophiophagus and Python.





Figure 2. Overlapping pairs of the structural models obtained from Swiss Model

Prot. A. Python in yellow and the mutated E. epinephelus in orange. The point mutations are highlighted in blue. B. Ophiophagus hannah protein in light purple and the composed E. epinephelus in dark purple. Mutations are coloured green. C. Thamnophis sirtalis protein model in light green and E. epinephelus in dark green, mutations are shown in orange.


Fig 3. Graph showing differences in structural comparison between original proteins

and mutated ones. The Y axis shows genetic distance between all sequences using

Python as the most ancient state. X axis represents root square means measured in

Amstrongs. Green dots represent original proteins while purple dots represent the mutated and assumed resistant proteins. Dotted grey lines join pairs of proteins from the same original species.


Fig 4. Graphs showing A. positive electrostatic potential field and B. Negative electrostatic potential field (kJ/mol) of the three pairwise comparisons. In green, original protein sequences, in purple, chimera protein sequences. Grey lines join pairwise sequences, the original and the transformed E. epinephelus.


Fig 5. Electrostatic energy variations (kJ/mol) in A. the whole protein B. the first point mutation D4i17S and C. in the second point mutation G4i18D. The y axis shows the genetic distance between the protein and Python sequence.





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