The insecticidal neurotoxin Aps III is an atypical knottin peptide that potently blocks insect voltage-gated sodium channels

Abstract

One of the most potent insecticidal venom peptides described to date is Aps III from the venom of the trapdoor spider Apomastus schlingeri. Aps III is highly neurotoxic to lepidopteran crop pests, making it a promising candidate for bioinsecticide development. However, its disulfide-connectivity, three-dimensional structure, and mode of action have not been determined. Here we show that recombinant Aps III (rAps III) is an atypical knottin peptide; three of the disulfide bridges form a classical inhibitor cystine knot motif while the fourth disulfide acts as a molecular staple that restricts the flexibility of an unusually large β hairpin loop that often houses the pharmacophore in this class of toxins. We demonstrate that the irreversible paralysis induced in insects by rAps III results from a potent block of insect voltage-gated sodium channels. Channel block by rAps III is voltage-independent insofar as it occurs without significant alteration in the voltage-dependence of channel activation or steady-state inactivation. Thus, rAps III appears to be a pore blocker that plugs the outer vestibule of insect voltage-gated sodium channels. This mechanism of action contrasts strikingly with virtually all other sodium channel modulators isolated from spider venoms that act as gating modifiers by interacting with one or more of the four voltage-sensing domains of the channel.

Fig. 1. Production and functional analysis of recombinant Aps III

(A) Schematic representation of the pLicC-NSB1 vector used for periplasmic expression of Aps III. The coding region includes a MalE signal sequence (MalE_SS) for periplasmic export, a His₆ affinity tag, an MBP fusion tag, and a codon-optimised gene encoding Aps III, with a TEV protease recognition site inserted between the MBP and toxin coding regions. The locations of key elements of the vector are shown, including the ribosome binding site (RBS). (B) Primary structure of rAps III. The non-native N-terminal Ser residue is highlighted in grey and the triglycine sequence is underlined. The disulfide framework of rAps III as determined in the current study is shown above the amino acid sequence. (C) SDS-PAGE gels illustrating different steps in the purification of rAps III. Lanes are as follows: M, molecular weight markers; lane 1, E. coli cell extract prior to IPTG induction; lane 2, E. coli cell extract after IPTG induction; lane 3, lysate resulting from cell disruption; lane 4, soluble periplasmic extract; lane 5, Ni-NTA beads after loading the cell lysate (the His₆-MBP-Aps III fusion protein is evident at ~49 kDa); lane 6, eluate from washing Ni-NTA resin with loading buffer; lane 7, eluate from washing Ni-NTA resin with 10 mM imidazole; lane 8, eluate from washing Ni-NTA resin with 500 mM imidazole; lane 9, purified fusion protein before TEV cleavage; lane 10, fusion protein sample after TEV protease cleavage, showing complete cleavage of fusion protein to His₆-MBP. (D) RP-HPLC chromatogram showing the final step in the purification of rAps III. The asterisk denotes the peak corresponding to correctly folded rAps III. Inset is a MALDI-TOF MS spectrum showing the [M+H]⁺ ion for the purified recombinant toxin (obs. = 3846.58 Da; calc. = 3846.49Da). (E) Dose-response curve for the paralytic effects of rAps III determined 24 h after injection into sheep blowflies (L. cuprina).

Fig. 2. Effects of rAps III on Na_V channels in cockroach DUM neurons

(Aa–c) Typical effects of increasing concentrations of rAps III on I_Na. Traces show superimposed control (black) and toxin (grey and shaded) current traces elicited by a 50-ms depolarising test pulse (V_test) shown in panel D. The reduction in I_Na is highlighted in the inset of panels Aa–c. (B) Complete block of I_Na by 300 nM TTX, confirming that ionic currents are solely mediated through Na_V channels. Dotted lines represent zero current. (C) Concentration-response relationship of rAps III to inhibit peak I_Na. The percentage block at increasing concentrations of rAps III were fitted with a logistic function (Eq. 1; see Materials and Methods). The median inhibitory concentration (IC₅₀) was determined to be 540 nM. Data points are the mean ± SEM of 5–9 cells.

Fig. 3. Effects of rAps III on the voltage-dependence of Na_V channel activation in cockroach DUM neurons

Typical families of I_Na recorded prior to (A), and following (B), application of 1 µM rAps III. Nav channel currents were elicited by the test pulse protocol shown in panel F. (C–D) Normalised peak I_Na-V relationships. Currents recorded in the presence of 1 µM rAps III were normalised to the maximum inward I_Na in controls (C), or maximum inward I_Na (D). Data shows I_Na before (closed symbols), and after (open symbols and shaded), application of 1 µM rAps III. Data were fitted with Eq. 2 (see Materials and Methods). As can be seen in panel (D), no significant shifts in the voltage dependence of Na_V channel activation were observed. (E) Fractional block of I_Na by 1 µM rAps III showing lack of voltage dependence. Normalised peak I_Na in the presence of toxin were calculated as a fraction of peak control I_Na and plotted against the test potential. Data were taken from panel C and fitted using linear regression. All data are expressed as the mean ± SEM of 4 cells.

Fig. 4. Effects of rAps III on steady-state Na_V channel inactivation (h_∞)

Steady-state inactivation was determined using a two-pulse protocol (see inset). (A–B) Typical peak I_Na recorded during the test pulse (V_test) are shown following 1-s prepulse potentials (V_prepulse) to −120 mV, −60 mV and −40 mV recorded before (left-hand traces), and following (right-hand shaded traces), perfusion with 30 nM rAps III. Dotted lines represent zero current. (C–D) Peak I_Na, recorded during V_test were expressed as a fraction of maximum control I_Na (C), or normalised to peak I_Na amplitude (D), and plotted against prepulse potential. Panels show the proportion of I_Na that is available for activation under control conditions (closed circles), and during perfusion with 30 nM rAps III (open circles and shaded). The h_∞/V curves were fitted with Eq. 3 (see Materials and Methods). All data are expressed as the mean ± SEM of 3 cells.

Fig. 5. Effect of rAps III on K_V and Ca_V channel currents in cockroach DUM neurons

(A) Whole-cell M-LVA and HVA I_Ba in the absence (black traces), and presence (dark grey and shaded traces), of 1 µM rAps III. M-LVA and HVA I_Ba were activated by a 100 ms V_test to −30 mV and +20 mV, respectively, as shown in the inset in panel C. Perfusion with 1 µM rAps III partially blocked M-LVA (Aa) and HVA (Ab) Ca_V channel currents. Dotted lines represent zero current. (B) Effects of 1 µM rAps III on the voltage-dependence of Ca_V channel activation. Families of Ca_V channel currents were generated by the V_test protocol shown in the inset of panel C. Currents recorded in the presence of 1 µM rAps III were normalised to the maximum inward I_Ba in control (Ba) or maximum inward I_Ba in toxin (Bb). I_Ba-V relationships show current recorded before (closed circles), and after (open circles and shaded), perfusion with 1 µM rAps III. Normalised I–V relationships were fitted using Eq. 2. (C) Typical superimposed global I_K recorded prior to (black traces), and following (dark grey and shaded traces), application of 1 µM rAps III. Currents were generated by 100-ms depolarising test pulses (V_test) as shown in the as shown in the inset in panel C. (D) Effects of rAps III on the voltage-dependence of global K_V channel activation. Families of outward I_K were recorded before (closed circles), and after (open circles and shaded), application of 1 µM rAps III. Families of I_K were generated by the V_test protocol shown in the inset of panel C. Global I_K-V relationships show effects of the toxin on peak (Da) and late (Db) global I_K. Late currents were measured at 100 ms. All data are expressed as the mean ± SEM of 4 cells.

Fig. 6. rAps III inhibits BgNa_V1-mediated sodium currents

(A) Inhibition of BgNa_V1-mediated sodium currents by 1 µM rAps III at a depolarization to −20 mV from a holding voltage of −90 mV. (B) Representative I_Na-V relationship for BgNa_V1 before (black) and after (grey) addition of 1 µM rAps III. Currents were elicited by 5-mV step depolarizations from a holding voltage of −90 mV. (C) Comparison of the gating properties of BgNa_V1 before (black) and after (grey) addition of 1 µM rAps III. Shown are the deduced conductance (G)–voltage (filled circles) and steady-state inactivation (open circles) relationships. Error bars denote SEM, with n = 3 cells. (D) Onset of BgNa_V1-mediated I_Na inhibition by 1 µM rAps III at depolarizations to −20 mV (holding voltage was −90 mV) followed by a complete recovery after toxin washout.

Fig. 7. Three-dimensional structure of rAps III

(A) Stereoview of an overlay of the ensemble of 20 rAps III structures. Disulfide bonds are highlighted in red and the N- and C-termini are labelled. The structures are overlaid over the backbone atoms of residues 2–27 and 32–38 in order to highlight the disordered nature of the triglycine loop (residues 27–32) relative to the well-structured ICK region of the toxin. (B) Ribbon representation of the rAps III structure highlighting the various secondary structure elements and disulfide bonds. The views in panels (B) and (C) are related by a ~180° rotation around the long axis of the molecule. (C) Topology map of the secondary structure of rAps III. The N- and C-termini are labelled.