Scorpion Neurotoxin BeM9 Derivative Uncovers Unique Interaction Mode with Nav1.5 Sodium Channel Isoform

Objective: Scorpion α-neurotoxins (ɑ-NaTx) inhibit the inactivation of voltage-gated sodium channels (Na_v) with variable efficiency between organisms and channel isoforms. Based on our previous results, we hypothesized that the derivative of the ɑ-NaTx called BeM9 with two amino acid residues substituted with glycine (A4G and Y17G; BeM9^GG) should be more selective for the mammalian channels. Surprisingly, BeM9^GG lost its activity on the cardiac isoform Na_v1.5. We provide a possible explanation of this effect, taking into account the published structures of ɑ-NaTx–Na_v complexes. Methods: We produced BeM9^GG in Escherichia coli as a fusion protein with thioredoxin, which was cleaved by cyanogen bromide. We purified BeM9^GG using chromatography and measured its activity on Na_v isoforms expressed in Xenopus laevis oocytes by the two-electrode voltage clamp technique. We performed computer modeling of ɑ-NaTx–Na_v complexes to figure out the peculiarities of toxin interactions with Na_v1.5. Results and Discussion: Using electrophysiology, we tested BeM9^GG on Na_v, and unexpectedly observed compromised activity on Na_v1.5. We compared the structures of available ɑ-NaTx–Na_v complexes to explain this effect. In case of Na_v1.5 the toxin-binding site is immersed deeper in the membrane. We explain this by sequence variations in two positions of the voltage-sensing domain IV of Na_v: in Na_v1.5 corresponding residues have shorter side chains, permitting the toxin to sit deeper. At the same time, residue Y17 in BeM9, which is missing in BeM9^GG, interacts with the hydrophobic core of the membrane and may play a significant role in its activity against Na_v1.5. Conclusions: The role of the membrane in ɑ-NaTx interactions with Na_v has been overlooked. It is especially important in case of Na_v1.5, where the tripartite toxin–ion channel– membrane complex is formed. Our study will be of help for the future design of selective Na_v ligands.