Solution Structure and Alanine Scan of a Spider Toxin That Affects the Activation of Mammalian Voltage-gated Sodium Channels

Abstract

Magi 5, from the hexathelid spider Macrothele gigas, is a 29-residue polypeptide containing three disulfide bridges. It binds specifically to receptor site 4 on mammalian voltage-gated sodium channels and competes with scorpion ␤-toxins, such as Css IV from Centruroides suffusus suffusus. As a consequence, Magi 5 shifts the activation voltage of the mammalian rNav1.2a channel to more hyperpolarized voltages, whereas the insect channel, DmNav1, is not affected. To gain insight into toxinchannel interactions, Magi 5 and 23 analogues were synthesized. The three-dimensional structure of Magi 5 in aqueous solution was determined, and its voltage-gated sodium channel-binding surfaces were mapped onto this structure using data from electrophysiological measurements on a series of Ala-substituted analogues. The structure clearly resembles the inhibitor cystine knot structural motif, although the triple-stranded ␤-sheet typically found in that motif is partially distorted in Magi 5. The interactive surface of Magi 5 toward voltage-gated sodium channels resembles in some respects the Janus-faced atracotoxins, with functionally important charged residues on one face of the toxin and hydrophobic residues on the other. Magi 5 also resembles the scorpion ␤-toxin Css IV, which has distinct nonpolar and charged surfaces that are critical for channel binding and has a key Glu involved in voltage sensor trapping. These two distinct classes of toxin, with different amino acid sequences and different structures, may utilize similar groups of residues on their surface to achieve the common end of modifying voltage-gated sodium channel function.

Tarantula Huwentoxin-IV Inhibits Neuronal Sodium Channels by Binding to Receptor Site 4 and Trapping the Domain II Voltage Sensor in the Closed Configuration

“…2, C and D). In contrast to ProTx-II and Magi5 (21,24), HWTX-IV did not modify the threshold of initial channel activation on the five tested VGSC subtypes. Because the toxin had relatively little effect on Nav1.4 and Nav1.5 channels (Figs.…”

“…Many toxins from spiders, such as ␦-atracotoxins; Magi5; CcoTx1, CcoTx2, and CcoTx3; PaurTx3; and ProTx-II, are able to shift the threshold of sodium channel activation to more negative or positive potentials (21)(22)(23)(24)(25)(26). Therefore we investigated the effects of subsaturating toxin concentrations on the voltage dependence of current activation.…”

Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells

“…Probes for different channel activities could be developed from toxins that stabilize different conformations of ion channels. For example, the Magi5 peptide stabilizes activated Na + channels, whereas ProTx-II stabilizes resting conformations (65,66). Similarly, GxTX inhibits K + channels that are activated by the related VST, hanatoxin (35).…”

Use of Venom Peptides to Probe Ion Channel Structure and Function

“…Magi 5 binds to receptor site 4 on mammalian Na v 1.2 and competes with scorpion ␤-toxins such as CssIV. A complete alanine scan revealed nine mutants lost biological activity, with an NMR structure attributing these residues to distinct polar and non-polar surfaces (48). Thus, spider toxins provide opportunities for development of subtype-specific Na ϩ channel inhibitors.…”