Effects of HNTX-VII on Kv4.2 and Kv4.3 and Molecular Determinants of Kv4.3 Interacting with HNTX-VII

Abstract HNTX-VII is a novel peptide isolated and purified from the venom of the Chinese spider Ornithoctonus hainana, with a relative molecular mass of 3830.973 Da. Electrophysiological experiments have demonstrated that HNTX-VII exhibits minimal effects on TTX-S, TTX-R, and delayed rectifier potassium channels on dorsal root ganglia (DRG), as well as on Kv1.4 and Kv4.1. However, it significantly inhibits Kv4.2 and Kv4.3 currents, with IC50 values of 299.6 ± 6.48 nM and 114.5 ± 5.36 nM, respectively, for Kv4.2 and Kv4.3. The sequence of HNTX-VII, determined by Edman degradation, is ECRYWLGTCSKTGDCCSHLSCSPKHGWCVWDWT. Composed of 33 amino acids and containing 3 pairs of disulfide bonds, this molecule represents a typical inhibitor cystine knot (ICK) motif. Furthermore, HNTX-VII alters the kinetic properties of Kv4.2 and Kv4.3 channels by causing corresponding shifts in their steady-state activation, steady-state inactivation, and inactivation recovery curves. To further investigate the molecular mechanism underlying the interaction between HNTX-VII and Kv4.3 channels, 19 mutants in the extracellular loops of the S1-S2 and S3b-S4 segments of the Kv4.3 channel were designed and constructed using site-directed mutagenesis. Electrophysiological techniques were then employed to assess the inhibitory activity of HNTX-VII against these mutants. Notably, the V282A mutant in the S3b-S4 loop exhibited the most significant reduction in sensitivity to HNTX-VII, with an IC50 value 5.37 times that of the wild-type channel. Therefore, it is inferred that the 282nd amino acid in the extracellular loop of S3b-S4 serves as a crucial site for the interaction between HNTX-VII and Kv4.3.