Unique bell-shaped voltage-dependent modulation of Na+ channel gating by novel insect-selective toxins from the spider Agelena orientalis

Abstract

Spider venoms provide a highly valuable source of peptide toxins that act on a wide diversity of membrane-bound receptors and ion channels. In this work, we report isolation, biochemical analysis, and pharmacological characterization of a novel family of spider peptide toxins, designated beta/delta-agatoxins. These toxins consist of 36-38 amino acid residues and originate from the venom of the agelenid funnel-web spider Agelena orientalis. The presented toxins show considerable amino acid sequence similarity to other known toxins such as mu-agatoxins, curtatoxins, and delta-palutoxins-IT from the related spiders Agelenopsis aperta, Hololena curta, and Paracoelotes luctuosus. beta/delta-Agatoxins modulate the insect Na(V) channel (DmNa(V)1/tipE) in a unique manner, with both the activation and inactivation processes being affected. The voltage dependence of activation is shifted toward more hyperpolarized potentials (analogous to site 4 toxins) and a non-inactivating persistent Na(+) current is induced (site 3-like action). Interestingly, both effects take place in a voltage-dependent manner, producing a bell-shaped curve between -80 and 0 mV, and they are absent in mammalian Na(V) channels. To the best of our knowledge, this is the first detailed report of peptide toxins with such a peculiar pharmacological behavior, clearly indicating that traditional classification of toxins according to their binding sites may not be as exclusive as previously assumed.

FIGURE 1.

Purification of β/δ-agatoxins. RP-HPLC of A. orientalis crude venom on a Jupiter C₅ column (4.6 × 150 mm) using a linear gradient of acetonitrile concentration in 0.1% triethylamine (shown with a line). Numbered fractions contain toxins active on DmNa_V1/tipE channels: 1, β/δ-Aga-5; 2, β/δ-Aga-4; 3, β/δ-Aga-6; 4, β/δ-Aga-7; 5, β/δ-Aga-1; 6, β/δ-Aga-2; and 7, β/δ-Aga-3.

FIGURE 2.

Activity of β/δ-Aga-1 on Na_V channels. Typical effects induced by β/δ-Aga-1 on currents from the cloned insect Na⁺ channel DmNa_V1/tipE from D. melanogaster (A–C) and the cloned mammalian Na⁺ channel rNa_V1.2/β₁ from R. norvegicus (D–F), expressed in X. laevis oocytes. A and D, representative whole cell current traces in control (black trace) and in the presence of 1 μm β/δ-Aga-1 (gray trace). Arrows indicate the toxin-induced effect on the current of DmNa_V1/tipE: an increase in peak amplitude, a decrease in time-to-peak, and the generation of a persistent current. B and E, superimposed representative whole cell current traces elicited by 5-mV step depolarizations between −90 and 10 mV in control (left) and in the presence of 1 μm β/δ-Aga-1 (right). The dotted line indicates the zero current level. C and F, normalized current-voltage relationships (n = 6 for DmNa_V1/tipE and n = 3 for rNa_V1.2/β₁) of transient peak currents (I_Na, black circles) and persistent currents measured after 50 ms (I_NaP, gray triangles) in control (closed symbols) and in the presence of 1 μm β/δ-Aga-1 (open symbols).

FIGURE 3.

Bell-shaped voltage dependence of the effects of β/δ-Aga-1 on the activation of the insect Na⁺ channel DmNa_V1/tipE. A, upper panels show the steady-state activation curves (n = 3–6) in control (closed circles) and in the presence of varying concentrations of β/δ-Aga-1 (open circles). All steady-state activation curves are fit with the Boltzmann equation. The bottom panels display the subtraction (toxin minus control) of the normalized g_Na values (Δg_Na), revealing the voltage range wherein the toxin-induced effects on the activation occur. B, superimposed curves of the Δg_Na at varying concentrations of β/δ-Aga-1 (see inset at upper right), showing the concentration dependence of the toxin-induced effects on the activation. C, dose-response curve of the Δg_Na measured at −35 mV (i.e. the voltage at which the effect of toxin on g_Na is maximal). The data are fit with the Hill equation, yielding an EC₅₀ value of 288.2 ± 116.4 nm (n = 3–6; Hill coefficient is 1.5 ± 0.7).

FIGURE 4.

Bell-shaped voltage dependence of the persistent currents (I_NaP) induced by β/δ-Aga-1 on the insect Na⁺ channel DmNa_V1/tipE. To quantify I_NaP, the current amplitude after 50 ms was normalized to peak current amplitude. A, upper panels show the normalized I_NaP (n = 3–6) in control (closed circles) and in the presence of varying concentrations of β/δ-Aga-1 (open circles). The bottom panels display the subtraction (toxin minus control) of the normalized I_NaP values (ΔI_NaP), revealing the voltage range wherein the toxin-induced persistent currents occur. B, superimposed curves of the ΔI_NaP at varying concentrations of β/δ-Aga-1 (see inset at upper right), showing the concentration dependence of the toxin-induced persistent currents. C, dose-response curve of the ΔI_NaP at −45 mV (i.e. the voltage at which the toxin-induced persistent currents are maximal). The data are fit with the Hill equation, yielding an EC₅₀ value of 220.2 ± 32.9 nm (n = 3–6, Hill coefficient 1.5 ± 0.4).

FIGURE 5.

Effect of β/δ-Aga-1 on the inactivation process of the insect Na⁺ channel DmNa_V1/tipE. A, steady-state inactivation curves (n = 3–6) in control (closed squares) and in the presence of 1 μm β/δ-Aga-1 (open squares). Test voltage is set at −10 mV in the left graph and −30 mV in the right graph. B, recovery from inactivation (n = 4) in control (closed circles) and in the presence of 1 μm β/δ-Aga-1 (open circles). Test voltage is set at −10 mV in the left graph and −30 mV in the right graph. C, voltage dependence of the time constants of fast inactivation of DmNa_V1/tipE (n = 4) in control (closed diamonds) and in the presence of 1 μm β/δ-Aga-1 (open diamonds). On the right, superimposed representative current traces in control (black traces) and in the presence of 1 μm β/δ-Aga-1 (gray traces) at different test potentials.

FIGURE 6.

Comparison of the effects of the β/δ-agatoxin homologues on the insect Na⁺ channel DmNa_V1/tipE. To facilitate a proper comparison, all data represent experiments conducted at a toxin concentration of 1 μm (n = 3–6). A, superimposed bell-shaped curves showing the amplitude of the effects of β/δ-agatoxin homologues on the activation (left graph) and the persistent currents (right graph). Analysis of the toxin-induced effects on activation (Δg_Na) and persistent currents (ΔI_NaP) were done in the same manner as in Figs. 3 and 4, respectively. B, bar diagrams showing the relative amplitudes of the effects on the activation (represented by Δg_Na measured at −35 mV) and the persistent currents (represented by ΔI_NaP measured at −45 mV). Amplitudes were normalized by setting the β/δ-agatoxin homologue with the highest effect as 100%.

FIGURE 7.

Comparison of the amino acid sequences of β/δ-agatoxins and similar spider toxins. Sequence identities are shown relative to β/δ-Aga-1, β/δ-Aga-6, and β/δ-Aga-7. Amino acid residues identical with β/δ-Aga-1 are shown on a black background, conservative substitutions are shown on a gray background, and cysteine residues are printed in bold. Breaks (dashes) have been introduced to maximize alignment. All the aligned toxins except for δ-Palu-IT3 are C-terminally amidated, as indicated by asterisks.

FIGURE 8.

Spatial structure model of β/δ-Aga-1. Model was built on the basis of the structure of the homologous δ-palutoxin-IT2 (PDB accession code: 1V91). Main-chain atoms are hidden, side chains are shown as lines, N and C termini are labeled, and β-strands are colored yellow and numbered. Presumed functionally important residues are indicated, the corresponding residues of δ-Palu-IT2 are given in parenthesis; the putative pharmacophore of δ-Palu-IT2 (23) is colored red; and residues believed to conform the scorpion β-toxin-like activity to β/δ-Aga-1 are marked cyan.