δ-Conotoxin SuVIA suggests an evolutionary link between ancestral predator defence and the origin of fish-hunting behaviour in carnivorous cone snails

Abstract

Some venomous cone snails feed on small fishes using an immobilizing combination of synergistic venom peptides that target Kv and Nav channels. As part of this envenomation strategy, δ-conotoxins are potent ichtyotoxins that enhance Nav channel function. δ-Conotoxins belong to an ancient and widely distributed gene superfamily, but any evolutionary link from ancestral worm-eating cone snails to modern piscivorous species has not been elucidated. Here, we report the discovery of SuVIA, a potent vertebrate-active δ-conotoxin characterized from a vermivorous cone snail (Conus suturatus). SuVIA is equipotent at hNaV1.3, hNaV1.4 and hNaV1.6 with EC50s in the low nanomolar range. SuVIA also increased peak hNaV1.7 current by approximately 75% and shifted the voltage-dependence of activation to more hyperpolarized potentials from -15 mV to -25 mV, with little effect on the voltage-dependence of inactivation. Interestingly, the proximal venom gland expression and pain-inducing effect of SuVIA in mammals suggest that δ-conotoxins in vermivorous cone snails play a defensive role against higher order vertebrates. We propose that δ-conotoxins originally evolved in ancestral vermivorous cones to defend against larger predators including fishes have been repurposed to facilitate a shift to piscivorous behaviour, suggesting an unexpected underlying mechanism for this remarkable evolutionary transition.

Keywords: conotoxin; defence; molecular evolution; predation; venom.

Figure 1.

Isolation and sequence characterization of SuVIA. (a) Fractionation of the venom extract from C. suturatus using a Thermo Hypercil-C₁₈ 4.6 × 150 mm column eluted with a linear gradient from 0 to 80% of buffer B, over 80 min at 1 ml min⁻¹. The arrow indicated where SuVIA eluted. (b) The peak indicated by the arrow was further characterized on a Thermo Hypersil-C₁₈ 2.1 × 150 mm column using a linear gradient from 20 to 80% of buffer B, over 60 min at 0.3 ml min⁻¹. (c,d) Single monoisotopic mass of 2742.16 Da (M + H) was detected using 4700 MALDI-TOF MS. (e) The amino acid sequence of the SuVIA was obtained by Edman degradation and tandem mass spectrometry. The calculated monoisotopic mass of 2741.157 Da matched the above-detected mass.

Figure 2.

Native SuVIA activates hNa_V1.3, hNa_V1.4, hNa_V1.6 and hNa_V1.7. The activity of SuVIA at human sodium channel isoforms Na_V1.3, Na_V1.4, Na_V1.6 and Na_V1.7 was assessed using a fluorescent membrane potential assay in stably transfected HEK293 cells. SuVIA was approximately equipotent at hNa_V1.3 (EC50 3.98 ± 0.97 nM), hNa_V1.4 (EC50 4.99 ± 0.92 nM), hNa_V1.6 (EC50 1.27 ± 0.56 nM) and hNa_V1.7 (EC50 2.42 ± 0.12 nM). Data are expressed as mean ± s.e.m. from n = 3 wells and is representative of three independent experiments.

Figure 3.

SuVIA (5 nM) (grey) alters the electrophysiological properties of human Na_V1.7 (black). (a) A representative trace of hNa_V1.7 showing an increase in peak current induced by SuVIA. (b) Current–voltage relationship in the presence and absence of SuVIA. Peak current is increased and the voltage of activation of hNa_V1.7 is shifted by 10 mV to more hyperpolarized potentials in the presence of SuVIA. (c) The conductance—voltage relationship is shifted towards more hyperpolarized potentials by SuVIA. Control V₅₀: −41.38 ± 0.28 mV, SuVIA V₅₀: −50.85 ± 0.44 mV. (d) The voltage of inactivation is slightly altered by SuVIA although peak current at hyperpolarized potentials is increased. Control V₅₀: −65.98 ± 0.4 mV, SuVIA V₅₀: −68.81 ± 0.93 mV. Data are expressed as mean ± s.e.m.; n = 8–9.

Figure 4.

Regional expression of SuVIA in the venom duct and its proposed defensive role. LC-MS trace of the distal venom extract (a) shows a low expression level of SuVIA, whereas it is abundant in the proximal extract (b). (c) Based on its proximal expression profile and its mode of action probably inducing pain, we propose a defensive role for δ-conotoxins to deter potential vertebrate predators such as fishes.

Figure 5.

Nocifensive behaviour elicited by intraplantar administration of SuVIA. Shallow subcutaneous injection of SuVIA (grey; 50 nM, 20 µl) into the hindpaw of C57BL/6 mice elicited nocifensive behaviour evidenced by increased licking of the affected hind paw compared with saline (black).