Figures and data
Pore domain mutations found in extremely TTX-resistant snakes.
a, The pore-forming domain of NaV1.4 is highly conserved across disparate phyla, although some populations of Thamnophis spp. exhibit mutations in pore domains III and IV very closely opposed to the selectivity filter (SF) (GenBank: Rattus norvegicus NM_013178.1 and Homo sapiens NP_000325.4)23. b,e, Snakes in both species carrying pore mutations are extremely resistant to TTX relative to their conserved counterparts. Resistance is measured here as the median dose required to reduce snake locomotor activity to 50%, scaled relative to the dose at which a mouse of equal mass would succumb (50% Mass-Adjusted Mouse Unit, MAMU50). Whereas a dose of 1 MAMU subdues mammals, mutant populations of Th. atratus (c) and Th. sirtalis (f) are 250X more resistant to TTX. We designate snakes carrying conserved pore sequences as TTX-sensitive despite being somewhat more resistant than mammals (Th. atratus: 1.35 MAMU50 and Th. sirtalis: 4.16 MAMU50). Medians are presented as neither resistant population shows variation in this metric and the variation in sensitive populations is imperceptible at the scale presented. Pore domain mutations deform the TTX binding site (d, g), thereby conferring resistance. However, the high degree of conservation at these substitution sites implies a high degree of functional significance that likely underlies a phenotypic trade-off in this system.
Sodium channel unitary conductance is reduced in two independently evolved pore domain mutants that confer TTX resistance.
a, Tertiary structures of skeletal muscle voltage-gated sodium channels from the extracellular face (Homo sapiens structure, PDB: 6agf) showing the TTX-sensitive NaV1.4+ (black) as well as the TTX-resistant Th. atratus NaV1.4EPN (blue) and Th. sirtalis NaV1.4LVNV (red) mutations. Movement of the voltage-sensing domains (VSDs) dilates the sodium-permeant central pore by tugging on the pore-forming domains (PFDs), the surface of which forms the binding site for TTX. b, Representative families of macroscopic sodium currents recorded from Rattus clones carrying identical mutations. c, Mutagenized rat channels are hardly affected by relatively large doses of TTX. d, Current magnitude among TTX-resistant sodium channels is reduced without any differences in (e) voltage-dependence of activation, inactivation or peak window current (e inset). f, Current-variance relationships demonstrate deficits in unitary current (cord conductance, g) among TTX-resistant sodium channels without any concomitant differences in sodium channel number (N, h) or peak-open probability (Po,max, i). All p-values presented are calculated by Dunn’s post-hoc pairwise comparison test after Kruskal-Wallis nonparametric ANOVA. Significantly different pairwise comparisons denoted by solid bars and their associated p-value; nonsignificant results (n.s.) denoted by dashed lines.
Muscle performance is reduced in two snake species carrying independently evolved TTX-resistance mutations in NaV1.4.
a,e, Mean effect of tetrodotoxin concentration [TTX] on snake skeletal muscle transient force (±sem). Data are grouped by species and genotype: Th. atratus (a) carrying TTX-sensitive NaV1.4+ (black, N=16, n=64) and TTX-resistant NaV1.4EPN (blue, N=10, n=40) and Th. sirtalis (e) carrying TTX-sensitive NaV1.4+ (black, N=23, n=92) and TTX-resistant NaV1.4LVNV (red, N=11, n=44). b,f, Transient muscular contractions (mean±sem) with electric field stimulus onset and duration indicated by a vertical black line (grouped as above). c,g, Tetanic muscular contractions (mean±sem) with a 2 second stimulus indicated by the black line (c, Th.atratus NaV1.4+N=15, NaV1.4EPN N=10; g, Th. sirtalis NaV1.4+N=17, NaV1.4LVNV N=11). d,h, In both snake species, skeletal muscles carrying ancestral sodium channel pore sequences exhibit greater force while mutant muscles display orders of magnitude greater TTX resistance but weaker force. This is the clearest evidence of a trade-off in populations that have coevolved with tetrodotoxic newts. X-error caps exaggerated for visibility. i,j, The temporal progression of transient contractions reveals variable timing between genotypes within species. Relevant metrics of contraction chronology include time to 10% contraction (F0.1max), time to peak first derivative (
Network of interaction energies (ΔG, kj/mol) between key residues in sodium channels.
Toxin-sensitive channels (left column) display different interaction energies compared to toxin-resistant channels (NaV1.4LVNV: rows A, B, & C; NaV1.4EPN row D). The left two columns show key scenes in the protein while the right two columns show simplified schematics demonstrating the interaction energies between residues of interest.
Scenes in the voltage-gated sodium channel depicting interfacing areas between the pore loops and the S5/S6 helices.
The Domain III (A) pore loops (dark blue) interact with the S5/S6 helices (pale blue) via hydrophobic interactions from the interior aspect of the reentrant loops and the superior aspect of the S5/S6 helices. The insert shows how these same DIII S5/S6 helices interact with the inactivation particle (yellow). Similarly, the Domain IV (B) pore loops (red) interact with the DIV S5/S6 helices (pink), also through hydrophobic interactions. The DIV S5/S6 helices also directly interface with the inactivation particle. Therefore, the pore loops are indirectly linked to the inactivation gate via hydrophobic interactions.
