Produção da toxina recombinante PnTx4(5-5) e caracterização de sua ação farmacológica sobre canais para sódio de inseto e de mamífero

Abstract

The venom from the spider Phoneutria nigriventer contains several neurotoxic polypeptides, which act on ion channels and other receptors of insects and mammals. The ability of such toxins to discriminate between different ion channels renders them potential models for the development of new biologically active tools of medical or biotechnological applications. Identifying their molecular binding targets and elucidating their mechanism of action are the first challenges to overcome, before considering toxins as potential lead compounds for drug discovery. The toxin PnTx4(5-5) have high insecticidal activity and no macroscopic behavioral effects when injected by intracerebral via in mice. Nevertheless, it can inhibit the NMDA - subtype of glutamate receptors of cultured rat hippocampal neurons. Furthermore, it was demonstrated to have peripheral and central antinociceptive effect in mice and rats. PnTx4(5-5) shares 63% sequence identity with toxin PnTx4(6-1), that was previously shown to act on the insect sodium channel. Thus, we proposed to investigate if the PnTx4(5-5) could have effects both on insect and mammalian sodium channels. However, the difficulty in obtaining large amounts of venom and isolating its toxins represent a limiting factor for carrying out studies on P. nigriventer toxins. To overcome this problem, in this study we have cloned and expressed the toxin PnTx4(5-5) in E. coli. The recombinant toxin, rPnTx4 (5-5), demonstrated to have secondary structure and biological activity similar to the native one. Results from electrophysiological experiments showed that rPnTx4(5-5) is a sodium channel gating modifier and has different affinity and mode of action on insect and mammalian sodium channels. The toxin strongly slowed down the inactivation of the cockroach sodium channel, increasing the peak current. Besides that, the toxin inhibited the sodium current on mammalian sodium channels, which is probably due to the small, but significant shift in the activation potential to more depolarized potentials, as observed on the channels Nav1.3 and Nav1.5. In addition, we produced some mutants of PnTx4(5-5) for structure-function relationship studies. The mutant R32A demonstrated to have lower activity on insect sodium channels than the original toxin, suggesting that this amino acid can be involved in the affinity of the toxin for sodium channels. This study can contribute to better understanding the structure/function relationship of mammal and insect toxins on sodium channels.