Elucidation of the Molecular Basis of Selective Recognition Uncovers the Interaction Site for the Core Domain of Scorpion α-Toxins on Sodium Channels

Abstract

Neurotoxin receptor site-3 at voltage-gated Na(+) channels is recognized by various peptide toxin inhibitors of channel inactivation. Despite extensive studies of the effects of these toxins, their mode of interaction with the channel remained to be described at the molecular level. To identify channel constituents that interact with the toxins, we exploited the opposing preferences of LqhαIT and Lqh2 scorpion α-toxins for insect and mammalian brain Na(+) channels. Construction of the DIV/S1-S2, DIV/S3-S4, DI/S5-SS1, and DI/SS2-S6 external loops of the rat brain rNa(v)1.2a channel (highly sensitive to Lqh2) in the background of the Drosophila DmNa(v)1 channel (highly sensitive to LqhαIT), and examination of toxin activity on the channel chimera expressed in Xenopus oocytes revealed a substantial decrease in LqhαIT effect, whereas Lqh2 was as effective as at rNa(v)1.2a. Further substitutions of individual loops and specific residues followed by examination of gain or loss in Lqh2 and LqhαIT activities highlighted the importance of DI/S5-S6 (pore module) and the C-terminal region of DIV/S3 (gating module) of rNa(v)1.2a for Lqh2 action and selectivity. In contrast, a single substitution of Glu-1613 to Asp at DIV/S3-S4 converted rNa(v)1.2a to high sensitivity toward LqhαIT. Comparison of depolarization-driven dissociation of Lqh2 and mutant derivatives off their binding site at rNa(v)1.2a mutant channels has suggested that the toxin core domain interacts with the gating module of DIV. These results constitute the first step in better understanding of the way scorpion α-toxins interact with voltage-gated Na(+)-channels at the molecular level.

Structural Insights into Antibody Sequestering and Neutralizing of Na+ Channel α-Type Modulator from Old World Scorpion Venom

“…Recent initial modeling of the interaction of ␣-toxin LqhII, a close homologue of AahII, with the Na v channel has led to suggest that residues Phe-15, Arg-18, Trp-38, and Asn-44 at the side of the toxin core would recognize the voltage-sensing (gating) module in domain IV of the channel, whereas residues Lys-2, Thr-57, and Lys-58 in the toxin N-and C-terminal region would recognize the pore module in domain I of the channel (73). In fact, of these residues only AahII Phe-15 is buried at the Fab4C1 complex interface, whereas in AahI, His-15, Pro-18, and Phe-36 contribute interactions with Fab9C2 (Figs.…”

The scorpion toxin Bot IX is a potent member of the α‐like family and has a unique N‐terminal sequence extension

“…Bot IX was initially purified from whole scorpion venom [24] and partial amino acid sequencing revealed that the toxin possesses a unique N-terminal sequence extension of four amino acids. Hereafter, Bot IX was found homologous to the classical a-toxin AaH I (residue [5][6][7][8][9][10][11][12][13][14][15][16], and the a-like toxin Bot I (residue 17-33).…”

“…However, accumulating reports on the characterization of more than one hundred scorpion toxins able to modulate Na v channel function led to a revision of their classification, one that primarily takes into account their pharmacological response in electrophysiological experiments with voltage-gated ion channels. Specifically, a-toxins interact with the voltage-sensor domain (VSD) in domain IV of the channel [6][7][8][9][10][11][12] to induce a prolongation of the action potential by blocking channel fast inactivation, with toxin binding being dependent on the membrane potential [1]. In contrast, b-toxins bind primarily to the VSD in domain II and act on channel activation by shifting its voltage-dependency toward more negative potentials [3,5].…”

Mapping the Interaction Site for a β-Scorpion Toxin in the Pore Module of Domain III of Voltage-gated Na+ Channels

“…Detailed mapping of the amino acid residues in the ISS2-S6, IVS1-S2, and IVS3-S4 segments that are required for high-affinity binding of ␣-scorpion toxins has led to the conclusion that these three extracellular loops form the receptor site for ␣-scorpion toxins with a similar conformation as we have proposed here for the receptor site for ␤-scorpion toxins (29). Moreover, single amino acid mutations in the IVS3-S4 loop reduce binding of ␣-scorpion toxins by nearly 100-fold (13,29,30). These results argue strongly that the highaffinity receptor site for ␣-scorpion toxins on the voltage sensor in domain IV is unique and cannot be substituted by the other three voltage sensors in Na V channels.…”