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 K_V4 family potassium channels. κ-LhTx-1 is a gating-modifier toxin impeded K_V4 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 K_V4 channels, as revealed by toxin inhibiting K_V4.2/4.3 with much more stronger voltage-dependence than that for K_V4.1. We proposed that κ-LhTx-1 trapped the voltage sensor of K_V4.1 in a much more stable resting state than that for K_V4.2/4.3 and further explored the underlying mechanism. Swapping the non-conserved S3b segments between K_V4.1(₂₈₀FVPK₂₈₃) and K_V4.3(₂₇₅VMTN₂₇₈) fully reversed their voltage-dependence phenotypes in inhibition by κ-LhTx-1, and intensive mutation analysis has identified P282 in K_V4.1, D281 in K_V4.2 and N278 in K_V4.3 being the key residues. Furthermore, the last two residues in this segment of each K_V4 channel (P282/K283 in K_V4.1, T280/D281 in K_V4.2 and T277/N278 in K_V4.3) likely worked synergistically as revealed by our combinatorial mutations analysis. The present study has clarified the molecular basis in K_V4 channels for their different modulations by κ-LhTx-1, which have advanced our understanding on K_V4 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 K_V4.2/4.3 channels.

Keywords: KV4 channels; anti-arrhythmic drugs; molecular basis; spider toxin; voltage-dependent inhibition.

FIGURE 1

Purification and characterization of κ-LhTx-1. (A), RP-HPLC profile of Pandercetes sp (inset) venom, the red asterisk labeled peak contains κ-LhTx-1. (B), κ-LhTx-1 was purified to homogeneity by analytical RP-HPLC (red asterisk labeled peak). (C), MALDI-TOF MS analysis of purified κ-LhTx-1, the inset shows its single isotopic molecular weight. (D), cDNA and protein sequence of κ-LhTx-1. The signal peptide, propeptide and mature peptide was shown in black bold, green bold and red bold, respectively. N-terminal sequence of κ-LhTx-1 was determined by Edman degradation and highlighted in yellow. (E), Representative traces showing K_V4.1 was concentration-dependently inhibited by κ-nLhTx-1 (n = 5). Currents were elicited by a 300 ms depolarization to +30 mV from −80 mV holding. Scale bars, 0.5 nA × 50 ms. (F), The concentration-response curves of κ-nLhTx-1 and κ-sLhTx-1 inhibiting K_V4.1 at + 30 mV, the IC₅₀ values were determined as 1.36 ± 0.38 μM and 0.87 ± 0.15 μM for κ-nLhTx-1 and κ-sLhTx-1, respectively (n = 5). (G), Sequence alignment of κ-LhTx-1 with several toxins in the database using MEGA8.0.

FIGURE 2

κ-LhTx-1 inhibits K_V4 channels with different voltage-dependence. (A), Representative traces showing the inhibition of K_V4 channels by κ-LhTx-1 at different depolarizing voltages (Upper panel: test at V_a voltage for K_V4.1, K_V4.2 and K_V4.3, respectively; Lower panel: test at +30 mV; n = 5–6). Holding potential was set to -80 mV, the V_a value for each K_V4 subtype was as shown in (B). Scale bars, 0.5 nA × 50 ms (Upper panel), 2 nA × 50 ms (Lower panel). (B, C), The concentration-response curves of κ-LhTx-1 inhibiting K_V4 channels at their V_a depolarization voltages (B) or +30 mV (C). The IC₅₀ values in K_V4.1, K_V4.2 and K_V4.3 were determined as 0.51 ± 0.11 μM and 0.87 ± 0.15 μM, 0.03 ± 0.01 μM and 0.14 ± 0.05 μM, 0.06 ± 0.02 μM and 0.16 ± 0.04 μM, at V_a voltage and +30 mV, respectively (n = 5–6). (D), The I-V relationships of K_V4.1, K_V4.2 and K_V4.3 channels before (black) and after (red) 10 μM κ-LhTx-1 treatment, currents at all voltages were normalized to the control current (before toxin treatment) at +100 mV in each group (n = 5–8). (E), The steady-state activation/inactivation curves of K_V4.1, K_V4.2 and K_V4.3 channels before (black) and after (red) 10 μM κ-LhTx-1 treatment (n = 5–10). (F), The inhibition ratio of 10 µM κ-LhTx-1 on the currents mediated by K_V4.1 (black), K_V4.2 (blue) and K_V4.3 (red) at different depolarizing voltages (n = 5–8). a, p < 0.05 when comparing the ratio in K_V4.2 with that in K_V4.1; b, p < 0.05 when comparing the ratio in K_V4.3 with that in K_V4.1(ONE-WAY ANOVA). (G), Representative traces showing κ-LhTx-1 inhibited the gating currents of K_V4.1–4.3 channels. Cells were held at -100 mV, currents were elicited by a 20 ms depolarization to +30 mV, note the ON gating current (I_{g ON}) was larger than the OFF gating current (I_g OFF) due to gating charge immobilization; Scale bar: 100 pA × 5 ms; n = 5–6. (H), Statistics of inhibition of gating charge (Q; integral of I_g ON) movement by application of 500 nM and 10 µM κ-LhTx-1, note 500 nM and 10 µM toxin inhibited essentially the same proportion of gating charge movement in K_V4.2/4.3 channels but not in K_V4.1 (n = 5–6).

FIGURE 3

Characterizing the non-conserved S3b segments in K_V4.1 and K_V4.3 as the key molecular determinants. (A), Left: sequence alignment of the K_V4 channels’ S3b-S4 segments, the non-conserved S3b regions are underlined, and the number below the residue indicates its location in the sequence (in mK_V4.1, rK_V4.2 and rK_V4.3 numbering); Right: Locations of the non-conserved S3b segments in the simulated structures of K_V4.1–4.3 channels as determined by SWISS-MODEL using 5WIE (PDB ID) as the template (https://swissmodel.expasy.org/), note only one subunit for each channel was shown for clarity. (B), The concentration-response curves of κ-LhTx-1 inhibiting the K_V4.1/279LF/AA, K_V4.2/277LV/AA and K_V4.3/274LV/AA mutants at their respective V_a depolarizing voltages (n = 5–7). For comparison, the curves for wild-type K_V4.1, K_V4.2 and K_V4.3 were shown in dashed lines. K_V4.1/279LF/AA, K_V4.2/277LV/AA and K_V4.3/274LV/AA were made by mutating L279/F280 in K_V4.1, L277/V278 in K_V4.2 and L274/V275 in K_V4.3, to alanines, respectively. (C) and (D), The I-V curve of K_V4.1/280VMTN (C) and K_V4.3/275FVPK (D) before (black) and after (red) 10 μM κ-LhTx-1 treatment, currents at all voltages were normalized to the control current (before toxin treatment) at +100 mV in each group (n = 5). K_V4.1/280VMTN and K_V4.3/275FVPK chimeras were made by replacing the ₂₈₀FVPK₂₈₃ segment in K_V4.1 with VMTN and the ₂₇₅VMTN₂₇₈ segment in K_V4.3 with FVPK, respectively. (E), The statistical diagram of the ∆V_a and inhi%_(min) values for wild-type K_V4.1, K_V4.3, K_V4.1/280VMTN and K_V4.3/275FVPK, showing swapping the non-conserved S3b segments between K_V4.1 and K_V4.3 exchanged their voltage-dependence phenotypes in inhibition by κ-LhTx-1 (***, p < 0.001; NS, not significantly different; ONE-WAY ANOVA; n = 5–13).

FIGURE 4

Characterizing the key residues in the non-conserved S3b segment. (A), Representative traces of κ-LhTx-1 inhibiting K_V4.1/F280V, K_V4.1/V281M, K_V4.1/P282T and Kv4.1/K283D mutants (n = 5). (B–D), The I-V curves of K_V4.1/P282T (B), K_V4.2/T280P (C) and K_V4.3/T277P (D) mutants before (black) and after (red) 10 μM κ-LhTx-1 treatment (n = 5–6). Currents at all voltages were normalized to the control current (before toxin treatment) at +100 mV in each group. (E), The concentration-response curves of κ-LhTx-1 inhibiting K_V4.1/F280V, K_V4.1/V281M, K_V4.1/P282T and K_V4.1/K283D mutants at +30 mV (n = 5). The curve for K_V4.1 was also included for comparison. (F–H), The I-V curves of K_V4.1/K283D (F), K_V4.2/D281K (G) and K_V4.3/N278K (H) mutants before (black) and after (red) 10 μM κ-LhTx-1 treatment (n = 5–6). Currents at all voltages were normalized to the control current (before toxin treatment) at +100 mV in each group. (I) and (J), The statistical diagram of the ∆V_a and inhi%_(min) values of wild-type K_V4 channels and mutants referred in this Figure, showing that P282T mutation in K_V4.1, D281K mutation in K_V4.2, as well as N278K mutation in K_V4.3 dramatically changed channel’s voltage-dependence phenotype in inhibition by κ-LhTx-1 (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significantly different; ONE-WAY ANOVA; n = 5–8).

FIGURE 5

The synergistic action of involved key residues. (A–C), The I-V curves of K_V4.1/282TD (A), K_V4.2/280PK (B) and K_V4.3/277PK (C) before and after 10 μM κ-LhTx-1 treatment (n = 5–6). Currents at all voltages were normalized to the control current (before toxin treatment) at +100 mV in each group. K_V4.1/282TD, K_V4.2/280PK and K_V4.3/277PK mutants were made by mutating P282/K283 to T282/D283 in K_V4.1, T280/D281 to P280/K281 in K_V4.2 and T277/N278 to P277/K278 in K_V4.3, respectively. (D), The statistical diagram of the ∆V_a and inhi%_(min) values of wild-type K_V4 channels and mutants referred in this Figure, showing K_V4.1/282TD has the same voltage-dependence phenotype as K_V4.2/4.3, while K_V4.2/280PK and K_V4.3/277PK have the same voltage-dependence phenotype as K_V4.1, as regard to their inhibition by κ-LhTx-1 (***, p < 0.001; NS, not significantly different; ONE-WAY ANOVA).