Two recombinant depressant scorpion neurotoxins differentially affecting mammalian sodium channels

Abstract

The scorpion depressant toxins are a group of evolutionarily conserved polypeptides targeting sodium channels, which show preferential ability to induce flaccid paralysis in insects, making them attractive candidates for the construction of transgenic plants or viral vectors to control pests. In this study, two new depressant toxin-like peptides (BmKITc and BmKITc2) differing only at position 52 (Lys for Thr) were produced in Escherichia coli. Circular dichroism analysis indicated that these two recombinant peptides display a typical structural feature similar to native scorpion toxins. They both cause a maintained current component at the last phase of inactivation of the insect sodium channel DmNav1/tipE expressed in Xenopus oocytes and interestingly, they do not produce a beta effect despite of their primary structure as beta-toxins. Furthermore, an inhibitory effect with BmKITc but not with BmKITc2 was observed on TTX-R sodium currents in rat DRG neurons. We hypothesize that such differential potency highlights a crucial role of lysine 52 in channel selectivity. Our results therefore indicate that, in spite of the general idea, not all scorpion depressant toxins interact with mammalian and/or insect sodium channels in the same manner.

Introduction

Voltage-gated sodium channels (VGSCs) are complex membrane proteins that regulate and control electrical excitability of insect and mammalian muscles and nerves (Catterall et al., 2007). Modification of the pharmacological activities of these channels by toxins from various venomous animals causes rapid immobilization of their preys. Scorpion venom-derived depressant toxins comprise a subfamily of bioactive polypeptides that contain 61–65 amino acids stabilized by four disulfide bridges (Possani et al., 1999, Rodriguez de la Vega and Possani, 2005, Gurevitz et al., 2007). These β-toxins bind to the receptor site 4 of the VGSCs and affect channel activation in a manner by shifting the voltage dependence of activation in the hyperpolarizing direction. In a recent work, Bosmans et al. have found that β-toxins can interact with multiple Na channel paddle motifs from domains II, III or IV in rNav1.2, but only with domain II paddle in rNav1.4 (Bosmans et al., 2008). Scorpion depressant toxins exhibit preferential ability in induction of a transient contraction paralysis of insect larvae followed by a progressive flaccid paralysis, making them attractive candidates for construction of transgenic plants or viral vectors to control pests. In fact, by combination of viral promoters, LqhIT2 and Lqh-dprIT3 have shown strong insecticidal efficacy (Gurevitz et al., 2007).

So far, more than 24 depressant toxins have been functionally characterized (Fig. 1). Although most of them show high preference for insect VGSCs and modulate their activation (Bosmans et al., 2005, Karbat et al., 2007), some members are active on mammals, e.g. (1) BmKIM, a recombinant peptide toxic to insects and mammals, could inhibit Na⁺ currents in rat DRG neurons and ventricular myocytes (Peng et al., 2002); (2) BmKdITAP3 was reported to have a dual bioactivity, a depressant toxicity on insects and an analgesic effect on mice (Guan et al., 2001); (3) BmKAEP had little toxicity on mice and insects but was found to have an anti-epilepsy effect in rats (Wang et al., 2001); (4) LqhIT2 was demonstrated to bind and affect rat skeletal muscle channels (Cohen et al., 2007).

In this study, we recombinantly produced two new depressant toxins (BmKITc and BmKITc2) in E. coli and characterized their structural and functional features. BmKITc and BmKITc2 differ by only one amino acid subsitution at position 52 (Thr52Lys) and therefore constitute an interesting starting point of investigation. As described below, we have found that this single-residue change resulted in functional diversification of these two toxins, making BmKITc rather than BmKITc2 a weak inhibitor of the TTX-R and TTX-S Na⁺ channels in rat DRG neurons. This observation highlights the crucial role of lysine 52 in interacting with mammalian VGSCs. In addition, although lacking sequence conservation in the equivalent positions corresponding to the ‘pharmacophore’ of scorpion β-toxins (Karbat et al., 2007), these two peptides were able to produce a significant change on the slow phase of inactivation of the Drosophila DmNav1/tipE channels expressed in Xenopus oocytes.