Direct evidence for high affinity blockade of Na_V1.6 channel subtype by huwentoxin-IV spider peptide, using...

The Chinese bird spider huwentoxin-IV (HwTx-IV) is well-known to be a highly potent blocker of Na_V1.7 subtype of voltage-gated sodium (Na_V) channels, a genetically validated analgesic target, and thus promising as a potential lead molecule for the development of novel pain therapeutics. In the present study, the interaction between HwTx-IV and Na_V1.6 channel subtype was investigated using multiscale (from in vivo to individual cell) functional approaches. HwTx-IV was approximatively 2 times more efficient than tetrodotoxin (TTX) to inhibit the compound muscle action potential recorded from the mouse skeletal neuromuscular system in vivo, and 30 times more effective to inhibit nerve-evoked than directly-elicited muscle contractile force of isolated mouse hemidiaphragms. These results strongly suggest that the inhibition of nerve-evoked skeletal muscle functioning, produced by HwTx-IV, resulted from a toxin-induced preferential blockade of Na_V1.6, compared to Na_V1.4, channel subtype. This was confirmed by whole-cell automated patch-clamp experiments performed on human embryonic kidney (HEK)-293 cells overexpressing hNa_V1.1-1.8 channel subtypes. HwTx-IV was also approximatively 850 times more efficient to inhibit TTX-sensitive than TTX-resistant sodium currents recorded from mouse dorsal root ganglia neurons. Finally, based on our data, we predict that blockade of the Na_V1.6 channel subtype was involved in the in vivo toxicity of HwTx-IV, although this toxicity was more than 2 times lower than that of TTX. In conclusion, our results provide detailed information regarding the effects of HwTx-IV and allow a better understanding of the side-effect mechanisms involved in vivo and of channel subtype interactions resulting from the toxin activity.