Molecular analysis of the sea anemone toxin Av3 reveals selectivity to insects and demonstrates the heterogeneity of receptor site-3 on voltage-gated Na+ channels

Abstract

Av3 is a short peptide toxin from the sea anemone Anemonia viridis shown to be active on crustaceans and inactive on mammals. It inhibits inactivation of Na(v)s (voltage-gated Na+ channels) like the structurally dissimilar scorpion alpha-toxins and type I sea anemone toxins that bind to receptor site-3. To examine the potency and mode of interaction of Av3 with insect Na(v)s, we established a system for its expression, mutagenized it throughout, and analysed it in toxicity, binding and electrophysiological assays. The recombinant Av3 was found to be highly toxic to blowfly larvae (ED50=2.65+/-0.46 pmol/100 mg), to compete well with the site-3 toxin LqhalphaIT (from the scorpion Leiurus quinquestriatus) on binding to cockroach neuronal membranes (K(i)=21.4+/-7.1 nM), and to inhibit the inactivation of Drosophila melanogaster channel, DmNa(v)1, but not that of mammalian Na(v)s expressed in Xenopus oocytes. Moreover, like other site-3 toxins, the activity of Av3 was synergically enhanced by ligands of receptor site-4 (e.g. scorpion beta-toxins). The bioactive surface of Av3 was found to consist mainly of aromatic residues and did not resemble any of the bioactive surfaces of other site-3 toxins. These analyses have portrayed a toxin that might interact with receptor site-3 in a different fashion compared with other ligands of this site. This assumption was corroborated by a D1701R mutation in DmNa(v)1, which has been shown to abolish the activity of all other site-3 ligands, except Av3. All in all, the present study provides further evidence for the heterogeneity of receptor site-3, and raises Av3 as a unique model for design of selective anti-insect compounds.

Figure 1. Characterization of the recombinant Av3

(A) Amino acid sequence and disulfide bonds (broken lines) of Av3 [22]. (B) Effect of the recombinant Av3 on DmNa_v1 inactivation. Oocytes expressing DmNa_v1/TipE were clamped at −80 mV, and currents were elicited by step depolarization to −10 mV in the absence (control) and presence of the indicated recombinant Av3 concentrations. (C) Competition of the recombinant Av3 with ¹²⁵I-LqhαIT on binding to cockroach neuronal membranes. The membranes (7 μg/ml) were incubated for 60 min at 22°C in the presence of 0.1 nM ¹²⁵I-LqhαIT and increasing concentrations of toxin. Non-specific binding determined in the presence of 1 μM LqhαIT was subtracted.

Figure 2. Analysis of the recombinant Av3 activity on mammalian Na_vs expressed in Xenopus oocytes

Oocytes expressing rNa_v1.2a, rNa_v1.4, hNa_v1.5 and rNa_v1.6 were clamped at −80 mV, and currents were elicited by step depolarization to −10 mV. Av3 was applied at a concentration of 10 μM, and a minor effect was observed only in the cardiac channel. To examine the extent of inhibition of inactivation this channel undergoes in the presence of a cardio-active site-3 toxin [3,23], 1 μM Av2 was applied after 20 min when the maximal effect of Av3 was reached. The strong inhibitory effect on channel inactivation induced by Av2 emphasized the negligible effect of Av3 at the cardiac channel. The y-axis scale bar is 400 nA for rNa_v1.2, 500 nA for rNa_v1.4, 1.6 μA for hNa_v1.5 and 1.1 μA for rNa_v1.6.

Figure 3. The toxicity of site-3 toxins is enhanced by Bj-xtrIT^E15R

Dose–response curves for the toxicity in blowfly larvae of the site-3 toxins LqhαIT, Av2 and Av3 in the presence of increasing concentrations of the non-toxic site-4 ligand Bj-xtrIT^E15R. Each data point represents the ED₅₀ equivalent of the site-3 toxin in the presence of the indicated dose of Bj-xtrIT^E15R.

Figure 4. Effects of substitutions on the binding affinity and CD spectrum portray the bioactive surface of Av3

(A) Competition curves of Av3 and its mutants with ¹²⁵I-LqhαIT on binding to cockroach neuronal membranes. Membranes (7 μg/ml) were incubated at 22°C for 60 min with 0.1 nM ¹²⁵I-LqhαIT and increasing concentrations of the various mutants. Non-specific binding, determined in the presence of 1 μM LqhαIT, was subtracted. The K_i values are Av3, 21.4±7.1 nM; R1A, 127±38 nM; Y7A, 6081±3703 nM; and P25A, 5.8±1.5 nM. (B) Far-UV CD spectra of recombinant Av3 and representative mutants. (C) Space-filled presentation of the Av3 structure based on PDB code 1ANS [17]. Residues whose substitution affected the binding affinity and activity of the toxin are coloured as follows: amino acids carrying aromatic side chains are magenta; those with aliphatic side chains are green; those with polar side chains are orange; positively charged residues are blue; and negatively charged residues are red.

Figure 5. Effects of Av3, LqhαιT and Av2 on DmNa_v1 and DmNa_v1^D1701R

(A) Sequence alignment of the S3-S4 extracellular loop of domain 4 in various Na_vs. rNa_v1.2 and rNa_v1.6, rat neuronal Na⁺ channel subtypes; rNa_v1.4, rat skeletal muscle Na⁺ channel; hNa_v1.5, human cardiac muscle channel; DmNa_v1, Drosophila melanogaster Na⁺ channel. The conserved negatively charged residue, whose substitution affects the activity of site-3 toxins is indicated in bold. (B) Xenopus oocytes expressing DmNa_v1 or DmNa_v1^D1701R were clamped at −80 mV, and currents were elicited by step depolarization to −10 mV. The effects on inactivation were monitored on DmNa_v1 (upper panels) in the presence of 1 μM Av3 (y-axis scale bar=3 μA), 20 nM LqhαIT (y-axis scale bar=450 nA) or 1 μM Av2 (y-axis scale bar=3 μA). The effects on DmNa_v1^D1701R (lower panels) were measured in the presence of 1 or 5 μM Av3 (y-axis scale bar=1.3 μA), 1 μM LqhαIT (y-axis scale bar=400 nA) or 1 μM Av2 (y-axis scale bar=400 nA).

Figure 6. Comparison of the bioactive surfaces of toxins that bind receptor site-3

The residues of the bioactive surfaces toward insects of the scorpion α-toxin LqhαIT (based on [25,40]), the type I sea anemone toxin Av2 (based on [23]) and the type III sea anemone toxin Av3 (the present study) are presented as ‘sticks’ on a Cα ribbon structure. The PDB codes for the LqhαIT and Av3 structures are 1LQI [41] and 1ANS [17] respectively. The residues are coloured as described in Figure 4(C).