Variation of Two S3b Residues in KV4.1–4.3 Channels Underlies Their Different Modulations by Spider Toxin κ-LhTx-1

Abstract

The naturally occurred peptide toxins from animal venoms are valuable pharmacological tools in exploring the structure-function relationships of ion channels. Herein we have identified the peptide toxin κ-LhTx-1 from the venom of spider Pandercetes sp (the Lichen huntsman spider) as a novel selective antagonist of the KV4 family potassium channels. κ-LhTx-1 is a gating-modifier toxin impeded KV4 channels’ voltage sensor activation, and mutation analysis has confirmed its binding site on channels’ S3b region. Interestingly, κ-LhTx-1 differently modulated the gating of KV4 channels, as revealed by toxin inhibiting KV4.2/4.3 with much more stronger voltage-dependence than that for KV4.1. We proposed that κ-LhTx-1 trapped the voltage sensor of KV4.1 in a much more stable resting state than that for KV4.2/4.3 and further explored the underlying mechanism. Swapping the non-conserved S3b segments between KV4.1(280FVPK283) and KV4.3(275VMTN278) fully reversed their voltage-dependence phenotypes in inhibition by κ-LhTx-1, and intensive mutation analysis has identified P282 in KV4.1, D281 in KV4.2 and N278 in KV4.3 being the key residues. Furthermore, the last two residues in this segment of each KV4 channel (P282/K283 in KV4.1, T280/D281 in KV4.2 and T277/N278 in KV4.3) likely worked synergistically as revealed by our combinatorial mutations analysis. The present study has clarified the molecular basis in KV4 channels for their different modulations by κ-LhTx-1, which have advanced our understanding on KV4 channels’ structure features. Moreover, κ-LhTx-1 might be useful in developing anti-arrhythmic drugs given its high affinity, high selectivity and unique action mode in interacting with the KV4.2/4.3 channels.

Molecular mechanism of the spider toxin κ-LhTx-I acting on the bacterial voltage-gated sodium channel NaChBac

“…We screened RP-HPLC fractions of the venom of spider Pandercetes sp (the lichen huntsman spider) for potential modulators of NaChBac. We identified κ-LhTx-1, an antagonist of K V 4 family potassium channels, as a potent inhibitor of NaChBac ( Figure 1B ) ( Xiao et al, 2021 ). The amino acid sequence of κ-LhTx-1 shows relatively low homology to the other known NaChBac peptide inhibitors, including GrTx1 (45%), GsAF-I (42%), JZTx-27 (29%), and JZTx-14 (20%).…”

“…Chemical synthesis and refolding of the linear κ-LhTx-1 was conducted as previously described ( Xiao et al, 2021 ). The purity of synthesized toxins was determined to be >98% by analytic HPLC and MALDI-TOF MS analysis.…”

“…In the present study, we identified the spider toxin κ-LhTx-1, which was previously reported as an antagonist of the K V 4 family voltage-gated potassium channel channels ( Xiao et al, 2021 ), as a novel antagonist of the NaChBac channel. Compared with the other known NaChBac peptide inhibitors, κ - LhTx-1 selectively inhibited NaChBac but greatly spared the mammalian Na V channels.…”

Molecular Diversity of Peptide Toxins in the Venom of Spider Heteropoda pingtungensis as Revealed by cDNA Library and Transcriptome Sequencing Analysis

“…Blasting analysis revealed that most toxins, except for HptTx-273, in this family are best aligned with kappa-LhTx-1 from the spider Pandercetes sp, with cluster 2 toxins showing slightly higher sequence homology (65-70% identity and 75-80% similarity) than that of cluster 1 toxins (57-61% identity and 72-81% similarity). This spider toxin was proved in our previous study to be a Kv4 channels' specific antagonist, which inhibits Kv4.1-4.3 subtypes with different voltage dependence [28]. HptTx-273, however, has the highest homology with U19-sparatoxin-Hv1a from the spider Heteropoda venatoria (57% identity and 69% similarity), whose biological function is not experimentally determined yet.…”

“…Briefly, the coverslip with NaV1.7-transfected cells seeded on it was put in the perfusion chamber with 100 µL bath solution, and 100 µL 2-folds concentrated toxin was quickly added using a microsyringe connected with tubing of a small dead volume. The full sequences of µ-Sparatoxin-Hp1 and µ-Sparatoxin-Hp2 were determined by combining EDMAN degradation and cDNA library analysis, as described in our previous studies [28]. Briefly, the N-terminal sequence of the toxin was determined by EDMAN degradation and blasting the resulted sequence against the toxin sequence database as presented in this study, determined the full sequence, and the toxin's identity was further cross-checked by matching its experimentally determined MW with the theoretical one.…”