Miniaturization of Scorpion β-Toxins Uncovers a Putative Ancestral Surface of Interaction with Voltage-gated Sodium Channels

Abstract

The bioactive surface of scorpion ␤-toxins that interact with receptor site-4 at voltage-gated sodium channels is constituted of residues of the conserved ␤␣␤␤ core and the C-tail. In an attempt to evaluate the extent by which residues of the toxin core contribute to bioactivity, the anti-insect and anti-mammalian ␤-toxins Bj-xtrIT and Css4 were truncated at their N and C termini, resulting in miniature peptides composed essentially of the core secondary structure motives. The truncated ␤-toxins (⌬⌬Bj-xtrIT and ⌬⌬Css4) were non-toxic and did not compete with the parental toxins on binding at receptor site-4. Surprisingly, ⌬⌬Bj-xtrIT and ⌬⌬Css4 were capable of modulating in an allosteric manner the binding and effects of site-3 scorpion ␣-toxins in a way reminiscent of that of brevetoxins, which bind at receptor site-5. While reducing the binding and effect of the scorpion ␣-toxin Lqh2 at mammalian sodium channels, they enhanced the binding and effect of Lqh␣IT at insect sodium channels. Co-application of ⌬⌬Bj-xtrIT or ⌬⌬Css4 with brevetoxin abolished the brevetoxin effect, although they did not compete in binding. These results denote a novel surface at ⌬⌬Bj-xtrIT and ⌬⌬Css4 capable of interaction with sodium channels at a site other than sites 3, 4, or 5, which prior to the truncation was masked by the bioactive surface that interacts with receptor site-4. The disclosure of this hidden surface at both ␤-toxins may be viewed as an exercise in "reverse evolution," providing a clue as to their evolution from a smaller ancestor of similar scaffold.Voltage-gated sodium channels (Na v s) 3 are critical for generation and propagation of action potentials in excitable cells and are targeted by a large variety of ligands that bind at distinct receptor sites on the pore-forming ␣-subunit (1). Most lipidsoluble Na v activators, including pyrethroid insecticides, toxic alkaloids (e.g. veratridine, from the plant family of Liliaceae, and batrachotoxin, from the skin of the Colombian frog Phyllobates aurotaenia), and marine cyclic polyether toxins (e.g. brevetoxins produced by "red tide" dinoflagellates), are effective in invertebrates as well as vertebrates. In addition, a wide array of proteinaceous Na v modifiers exists in the venom of scorpions, spiders, cone snails, and sea anemones, which are utilized for prey and defense (1, 2). Na v gating modifiers from scorpion venom are divided into two classes, ␣ and ␤, according to their mode of action and binding properties (3-5). ␣-Toxins prolong the action potential by inhibiting the fast inactivation of the Na v upon binding to receptor site-3 (4, 6), which involves extracellular loops in domains 1 and 4 (6). These toxins are divided into three groups (7): (i) anti-mammalian ␣-toxins (e.g. Lqh2 from Leiurus quinquestriatus hebraeus), which are highly toxic in mammalian brain and bind with high affinity to rat brain Na v s; (ii) antiinsect ␣-toxins (e.g. Lqh␣IT), which are highly toxic to insects and show weak activity in mammalian brain; and (iii) ␣-like toxins (e.g. Lqh3), ...

Drosomycin, an Innate Immunity Peptide of Drosophila melanogaster, Interacts with the Fly Voltage-gated Sodium Channel

“…Although by themselves, the ⌬⌬␤-toxins (⌬⌬Css4 and ⌬⌬Bj-xtrIT) were nontoxic and did not bind at the receptor sites of the parental toxins, they exhibited an unexpected ability to allosterically facilitate the activity of a scorpion ␣-toxin (inhibition of Na v fast inactivation), which binds at receptor site-3 on insect Na v s (10), and the effect of the marine polyether toxin brevetoxin-2 (PbTx-2, facilitator of Na v activation), which binds to receptor site-5 (11). However, a short chain potassium channel blocker (charybdotoxin) with a CS␣␤ structural fold did not exert any of these effects (9). These results indicated that it is not only the CS␣␤ motif but that specific amino acids at key sites on the protein exterior that can interact with ion channels and either block voltage-gated potassium channels or induce conformational alteration of voltage-gated sodium channels.…”

“…3). However, as we have recently demonstrated, the interaction of various peptides with Na v s can be monitored indirectly by the allosteric effects imposed upon the activity of another channel ligand (9,15). Using this sensitive assay, we examined whether the inhibitory effect of the scorpion ␣-toxin Lqh␣IT (a site-3 Na v ligand) on DmNa v 1 inactivation would be modulated by DRS.…”

“…The truncated scorpion ␤-toxins ⌬⌬Css4 and ⌬⌬Bj-xtrIT were produced as described previously (9). The scorpion ␣-toxin Lqh␣IT was produced in recombinant form as was described previously (14).…”

“…8). We have recently reported that truncated scorpion ␤-toxins, lacking the N-and C-terminal regions of the parental peptides but maintaining the CS␣␤ motif (⌬⌬␤-toxins), are able to interact at high affinity with Na v s (9). Although by themselves, the ⌬⌬␤-toxins (⌬⌬Css4 and ⌬⌬Bj-xtrIT) were nontoxic and did not bind at the receptor sites of the parental toxins, they exhibited an unexpected ability to allosterically facilitate the activity of a scorpion ␣-toxin (inhibition of Na v fast inactivation), which binds at receptor site-3 on insect Na v s (10), and the effect of the marine polyether toxin brevetoxin-2 (PbTx-2, facilitator of Na v activation), which binds to receptor site-5 (11).…”

Molecular Description of Scorpion Toxin Interaction with Voltage-Gated Sodium Channels

“…The structural models of Css4 (based on the NMR structure of Cn2; PDB code 1CN2), Bj-xtrIT (PDB code 1BCG), and charybdotoxin (potassium channel blocker; PDB code 2CRD) at the lower right-hand side are covered by semitransparent molecular surfaces and are spatially aligned. The moieties representing DDBj-xtrIT and DDCss4 are colored in orange on the ribbon structure of the parent toxins, and the cyan ribbons represent deleted N-and C-termini (note the resemblance of the core in the three toxins suggesting common ancestry Froy et al 1999;Cohen et al 2008). The models were prepared using PyMOL.…”