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

Abstract

Peptide toxins with high affinity, divergent pharmacological functions, and isoform-specific selectivity are powerful tools for investigating the structure-function relationships of voltagegated sodium channels (VGSCs). Although a number of interesting inhibitors have been reported from tarantula venoms, little is known about the mechanism for their interaction with VGSCs. We show that huwentoxin-IV (HWTX-IV), a 35-residue peptide from tarantula Ornithoctonus huwena venom, preferentially inhibits neuronal VGSC subtypes rNav1.2, rNav1.3, and hNav1.7 compared with muscle subtypes rNav1.4 and hNav1.5. Of the five VGSCs examined, hNav1.7 was most sensitive to HWTX-IV (IC 50 ϳ 26 nM). Following application of 1 M HWTX-IV, hNav1.7 currents could only be elicited with extreme depolarizations (>؉100 mV). Recovery of hNav1.7 channels from HWTX-IV inhibition could be induced by extreme depolarizations or moderate depolarizations lasting several minutes. Site-directed mutagenesis analysis indicated that the toxin docked at neurotoxin receptor site 4 located at the extracellular S3-S4 linker of domain II. Mutations E818Q and D816N in hNav1.7 decreased toxin affinity for hNav1.7 by ϳ300-fold, whereas the reverse mutations in rNav1.4 (N655D/ Q657E) and the corresponding mutations in hNav1.5 (R812D/ S814E) greatly increased the sensitivity of the muscle VGSCs to HWTX-IV. Our data identify a novel mechanism for sodium channel inhibition by tarantula toxins involving binding to neurotoxin receptor site 4. In contrast to scorpion ␤-toxins that trap the IIS4 voltage sensor in an outward configuration, we propose that HWTX-IV traps the voltage sensor of domain II in the inward, closed configuration.

Analysis of the Structural and Molecular Basis of Voltage-sensitive Sodium Channel Inhibition by the Spider Toxin Huwentoxin-IV (μ-TRTX-Hh2a)

“…S4 was manually moved down into a resting state configuration, the S1-S2 and S3-S4 loops were regenerated, and the entire model was energy-minimized. Native HwTX-IV was manually docked into the Nav1.7 homology model based on the results of the alanine scan of HwTX-IV inhibition against Nav1.7 and on published Nav1.7 mutations that affect HwTX-IV binding (8,18,19). Following the manual docking, the entire Nav1.7 DII S1-S4 with docked HwTX-IV system was minimized, and an implicit membrane molecular dynamics simulation was performed using the CHARMm force field with Generalized Born Implicit Membrane (Discovery Studio (36)) to further refine the docked structure.…”

“…2 Because the majority of spiders prey primarily on invertebrates, it is likely that these toxins target VGSCs in the insect nervous system, providing a highly efficient mechanism for spiders to incapacitate their prey. However, given the structural similarity between many insect and vertebrate sodium channels, it is not surprising that many spider venom peptides also modulate the activity of vertebrate VGSCs (5)(6)(7)(8)(9). In vertebrates, there are nine VGSC isoforms, Nav1.1-Nav1.9, with each isoform exhibiting a unique pattern of tissue distribution.…”

“…However, very little is known about the channel residues involved in these interactions. The only studies addressing this question are from Cummins and colleagues (8,18,19), who have identified several residues in the S3-S4 linker region of domain II that are crucial for huwentoxin-IV inhibition of Nav1.7. Even less is known about the toxin residues that interact with VGSCs.…”

“…Peptides from family 1 include huwentoxin-1 (/-TRTX-Hh1a) (16), hainantoxin-IV (-TRTXHhn1b) (9), phrixotoxin-3 (␤-TRTX-Ps1a) (5), ceratotoxin-1 (␤-TRTX-Cm1a) (5), and huwentoxin-4 (-TRTXHh2a, HwTX-IV) (8,17). All of these peptides are inhibitors of vertebrate VGSCs, and their mechanism of action probably involves a specific interaction with the voltage sensor region(s), impeding its movement and inhibiting channel activation (5,8). However, very little is known about the channel residues involved in these interactions.…”

“…All peptides from this family were isolated from the venom of tarantulas (Theraphosidae), and they consist of [33][34][35] dues with three disulfide bridges that form an inhibitory cysteine knot (ICK) motif (14). Peptides from family 1 include huwentoxin-1 (/-TRTX-Hh1a) (16), hainantoxin-IV (-TRTXHhn1b) (9), phrixotoxin-3 (␤-TRTX-Ps1a) (5), ceratotoxin-1 (␤-TRTX-Cm1a) (5), and huwentoxin-4 (-TRTXHh2a, HwTX-IV) (8,17). All of these peptides are inhibitors of vertebrate VGSCs, and their mechanism of action probably involves a specific interaction with the voltage sensor region(s), impeding its movement and inhibiting channel activation (5,8).…”