Abstract
Scorpion α-toxins are neurotoxins that target the fast inactivation mechanism of voltage-gated sodium (NaV) channels leading to several neuro- and cardiotoxic effects in mammals. The toxin AahII is the most active α-toxin from the North African scorpion Androctonus australis Hector that slows the fast inactivation of NaV channels. To fight scorpion envenomation, an anti-AahII nanobody named NbAahII10 (Nb10) was developed. The efficiency of this nanobody has been evaluated in vivo on mice, but its mechanism of action at the cellular level remains unknown. Here we have shown that AahII toxin slows the fast inactivation of the adult cardiac NaV1.5 channels, expressed in HEK293 cells, in a dose-dependent manner, while current amplitude was not affected. The inactivation of NaV1.5 is slower by a factor of 4, 7, and 35 in the presence of [AahII] at 75, 150, and 300 nM, respectively. The washout partially reversed the toxin effect on inactivation from 8.3 ± 0.9 ms to 5.2 ± 1.2 ms at 75 nM. We have also demonstrated that the highly neutralizing Nb10 can fully reverse the effect of AahII toxin on the channel inactivation kinetics even at the 1:1 M ratio. However, the 1:0.5 M ratio is not able to neutralize completely the AahII effect. Therefore, the application of Nb10 promotes a partial abolishment of AahII action. Bioinformatic analysis and prediction of NaV1.5-driven docking with AahII show that Ala39 and Arg62 of AahII play a crucial role to establish a stable interaction through H-bound interactions with Gln1615 and Lys1616 (S3–S4 extracellular loop) and Asp1553 (S1–S2 loop) from the voltage-sensing domain IV (VSD4) of NaV1.5, respectively. From this, we notice that AahII shares the same contact surface with Nb10. This strongly suggests that Nb10 dynamically replaces AahII toxin from its binding site on the NaV1.5 channel. At the physiopathological level, Nb10 completely neutralized the enhancement of breast cancer cell invasion induced by AahII. In summary, for the first time, we made an electrophysiological and structural characterization of the neutralization potent of Nb10 against the α-scorpion toxin AahII in a cellular model overexpressing NaV1.5 channels.
Highlights
IntroductionVoltage-gated sodium (NaV) channels are large transmembrane proteins responsible for the initiation and propagation of action potentials in excitable cells (Ahern et al, 2016)
We investigated, by patch-clamp technique, the effect of AahII toxin purified from the venom of Androctonus australis Hector scorpion on a cardiac sodium channel NaV1.5 subunit using the human embryonic kidney cell line HEK293 stably transfected with α subunit of NaV1.5 of human origin
All data are presented as mean ± SE (***p < 0.001). (D) Mean of kinetics of the inactivation time constant of the NaV1.5 channel following perfusion of AahII toxin at 75 nM from 1 to 7 min followed by the perfusion of a mixture of AahII and Nb10 at a 1:1 ratio up to 14 min
Summary
Voltage-gated sodium (NaV) channels are large transmembrane proteins responsible for the initiation and propagation of action potentials in excitable cells (Ahern et al, 2016). Eukaryotic NaV channels count 9 isoforms (NaV1.1–NaV1.9), encoded by 9 distinct genes (SCN1–5A and SCN8–11A) with almost 50% of homology in the amino acid sequences (Goldin et al, 2000; Catterall et al, 2005). These channels consist of the heteromeric assembly of an α-subunit which forms the channel pore and provides its function with two auxiliary β-subunits. The S4 segments known as voltage sensors modules are positively charged due to four to eight arginine/lysine residues flanked by two hydrophobic residues (Ahern et al, 2016; Catterall et al, 2017) This positively charged motif serves as a gating charge and moves outward upon depolarization to initiate the channel activation
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