A Novel Spider Toxin Inhibits Fast Inactivation of the Na(v)1.9 Channel by Binding to Domain III and Domain IV Voltage Sensors

Venomous animals have evolved to produce peptide toxins that modulate the activity of voltage-gated sodium (Na(v)) channels. These specific modulators are powerful probes for investigating the structural and functional features of Na(v) channels. Here, we report the isolation and characterization of δ-theraphotoxin-Gr4b (Gr4b), a novel peptide toxin from the venom of the spider Grammostola rosea. Gr4b contains 37-amino acid residues with six cysteines forming three disulfide bonds. Patch-clamp analysis confirmed that Gr4b markedly slows the fast inactivation of Na(v)1.9 and inhibits the currents of Na(v)1.4 and Na(v)1.7, but does not affect Na(v)1.8. It was also found that Gr4b significantly shifts the steady-state activation and inactivation curves of Na(v)1.9 to the depolarization direction and increases the window current, which is consistent with the change in the ramp current. Furthermore, analysis of Na(v)1.9/Na(v)1.8 chimeric channels revealed that Gr4b preferentially binds to the voltage-sensor of domain III (DIII VSD) and has additional interactions with the DIV VSD. The site-directed mutagenesis analysis indicated that N1139 and L1143 in DIII S3-S4 linker participate in toxin binding. In sum, this study reports a novel spider peptide toxin that may slow the fast inactivation of Na(v)1.9 by binding to the new neurotoxin receptor site-DIII VSD. Taken together, these findings provide insight into the functional role of the Na(v) channel DIII VSD in fast inactivation and activation.