Effects of JZTX-V on the wild type Kv4.3 Expressed in HEK293T and Molecular Determinants in the Voltage-sensing Domains of Kv4.3 Interacting with JZTX-V

Abstract

JZTX-V is a toxin isolated from the venom of the Chinese spider Chilobrachys jingzhao. Previous studies had shown that JZTX-V could inhibit the transient outward potassium current of Kv4.2 and Kv4.3 expressed in Xenopus oocytes but had no effects on Kv1.2-1.4. However, the underlying action mechanism of JZTX-V on Kv4.3 remains unclear. In our study, JZTX-V could inhibit not only transient outward potassium currents evoked in small-sized DRG neurons but also Kv4.3-encoded currents expressed in HEK293T cells in the concentration and voltage dependence. The half maximal inhibitory concentration of JZTX-V on Kv4.3 was 9.6 ± 1.2 nM. In addition, the time course for JZTX-V inhibition and release of inhibition after washout were 15.8 ± 1.54 s and 58.8 ± 4.35 s. Electrophysiological assays indicated that 25 nM JZTX-V could shift significantly the voltage dependence of steady-state activation and steady-state inactivation to depolarization. Meanwhile, 25 nM JZTX-V decreased markedly the time constant of activation and inactivation but had no effect on the time constant of recovery from inactivation. To study the molecular determinants of Kv4.3, we performed alanine scanning on a conserved motif of Kv4.3 and assayed the affinity between mutants and JZTX-V. The results not only showed that I273, L275, V283, and F287 were molecular determinants in the conserved motif of Kv4.3 for interacting with JZTX-V but also speculated the underlying action mechanism that the hydrophobic interaction and steric effects played key roles in the binding of JZTX-V with Kv4.3. In summary, our studies have laid a scientific theoretical foundation for further research on the interaction mechanism between JZTX-V and Kv4.3.

Keywords: JZTX-V; electrophysiological assays; kv4.3; molecular determinants; mutants.

Figure 1.

Alignment of JZTX-V with five gating-modifier toxins of Kv channels. Strictly conserved cysteines are indicated by deep blue and other less conserved amino acids are marked using different colors. PaTx1(UniProtKB ID = P61230), HpTx2(UniProtKB ID = P58426), HmTx2(UniProtKB ID = P60993), HaTx2(UniProtKB ID = P56853) and SgTx1(UniProtKB ID = P56855) were from Phrixotrichus auratus, Heteropoda venatoria, Heteroscodra maculate, Grammostola spatulate and Scodra griseipes, respectively.

Figure 2.

The effects of JZTX-V and 4-AP on the K⁺ currents in rat small-sized DRG. (a) The inhibition effects of 1 μM JZTX-V. (b) The inhibition effects of 10 mM 4-AP.

Figure 3.

The characteristics of JZTX-V to wild-type Kv4.3 expressed in HEK293T cells. (a)The inhibition effect of JZTX-V on Kv4.3 current. With the increase of JZTX-V concentration, Kv4.3 current gradually decreased. (b) The dose–inhibition curve. It was fitted by the Hill logistic equation: Inhibition% = (A₁-A₂)/ [1 +(x/x₀) ^p] +A₂, in which A₁, A₂, x, x₀ and p was the initial value of inhibition, final value of inhibition, JZTX-V concentration, IC₅₀ and power, respectively. The IC₅₀ is 9.6 ± 1.2 nM. (c) The effect of 100 nM JZTX-V on Kv4.3 current recorded at +10 nM and +50 nM, respectively. The inhibition percentage of 100 nM JZTX-V on wild-type Kv4.3 current at +10 mV (94%) is higher than at +50 mV (57%). (d) The Voltage–inhibition curve. At each potential, the peak amplitude of current in the presence of JZTX-V was normalized to that in the absence of JZTX-V. The inhibition of Kv4.3 current by JZTX-V was voltage dependent (*P < 0.05 or ** P < 0.01 Vs −30 mV). (e) Time course of inhibition of Kv4.3 with 100 nM JZTX-V and recovery from inhibition. The two bars indicate the duration of 100 nM JZTX-V application and washout, respectively. The pulse protocol started from a holding potential of −80 mV followed by a pulse to 10 mV for 150 ms with a 1 s interpulse interval. The data represent the normalized reciprocal current amplitude measured from the maximum of the pulse. The on and off rates were fit with the functions ƒ_on (t) = C× exp(-t/τ_on) and ƒ_off (t) = C×[(1-exp(-t/τ_off)]⁴, respectively. All data were presented as means ±SD, and came from 4 ~ 6 independent cell experiments.

Figure 4.

The effects of JZTX-V on activation, inactivation and recovery kinetics of wild-type Kv4.3 current. (a)Current diagram of steady-state activation. In the absence of JZTX-V, K⁺ current was elicited by depolarization ranging from −80 mV to +60 mV in +10 mV increments. (b)Steady-state activation curves. It was fitted by the equation: I/I_max = 1/ [1+ exp (V_0.5-V)/k], in which V_0.5 was half-maximal active potential, k was the slope factor and I_max was the maximal current generated in the absence of JZTX-V and at +60 mV pulse. (c)Current graph of steady-state inactivation. In the absence of JZTX-V, double-currents were evoked by double-pulses including conditioning prepulse ranging from −120 mV to +60 mV and depolarizing pulse at +60 mV. (d) Steady-state inactivation curves. It was fitted by the equation: I/I_max = 1/ [1+ exp -(V_0.5-V)/k], in which V_0.5 is half-maximal inactive potential, k is the slope factor and I_max is the maximal current evoked by P2. (e) τ_a in absence and presence of JZTX-V at different potential ranging from +10 mV to +60 mV. The rising phase of the wild-type Kv4.3 current was fitted by the equation: I(t) = I₀+ A₁[1-exp(-t/τ_a)], in which τ_a was the activation time constant (*P < 0.05 or ** P < 0.01 Vs absence of JZTX-V at each potential). The inset shows the effect of JZTX-V at +20 mV. (f) τ_i in absence and presence of JZTX-V at different potential ranging from +10 mV to +60 mV. The decay phase of the wild-type Kv4.3 current was fitted by the equation: I(t) = I₀+ A₁exp [-(t-t₀)/τ_i], in which τ_i was the inactivation time constant (*P < 0.05 or ** P < 0.01 Vs absence of JZTX-V at each potential). (g) Current traces of Kv4.3 recovery from inactivation recorded by the single cell. The HEK293T cell was depolarized by double-pulse (P1 to +10 mV, with holding potential = −80 mV, followed by P2 to +10 mV), while interpulse interval between them was allowed to vary from 0 to 500 ms in 20 ms increments. (h) τ_r in absence and presence of JZTX-V at different recovery intervals. Peak currents elicited by test pulse(P2) were plotted as a function of time and data points were fitted by equation: I(t) = I₀+ A₁[1-exp(-t/τ_r)], in which τ_r was the recovery time constant. All data(b, d~f, and h) were presented as means ±SD, and came from 6 independent cell experiments.

Figure 5.

Inhibitory Effect of JZTX-V on Alanine mutants of S3b-S4 region in Kv4.3 voltage-sensing domain. (a) Alanine scanning on voltage-sensing domains of Kv4.3. Amino acid residues marked in red were replaced by alanine. (b) The dose–inhibition curves of V283A and F287A. Mutation of V283A and F287A reduced sensitivity to the JZTX-V by ~10-and~ 6-fold with IC₅₀ of 91.3 ± 7.0 and 62.2 ± 7.4 nM, respectively. (c) The dose–inhibition curves of I273A and L275A. The IC₅₀ of I273A and L275A were 5.1 ± 1.1 nM and 6.3 ± 0.2 nM, respectively. (d) Histogram of IC₅₀ value for each mutant. The affinity of mutants, V282A and F286A, to JZTX-V decreased significantly, while the affinity of I273A and L275A to JZTX-V increased significantly (*P < 0.05 or ** P < 0.01 Vs wild-type Kv4.3). All data were presented as means ±SD, and came from 4 ~ 6 independent cell experiments.

Figure 6.

Three-dimensional structure of JZTX-V, PaTx1 (1V7F) and HpTx2(1EMX). (a), (b) and (c) are JZTX-V (PDB code 6CHC), PaTx1 (PDB code 1V7F) and HpTx2(PDB code 1EMX), respectively. In (a), (b) and (c), the left is the side containing the hydrophobic patch, and the right is the 180° rotation side containing the charged residues and surrounding the hydropathic patch. Residues are colored according to their properties. Blue, basic (Arg, Lys, His); Red, acidic (Glu, Asp); Green, Polarity uncharged (Ser, Thr, Tyr, Gln, Asn, Cys and Gly); White, hydrophobic (Ala, Ile, Leu, Met, Phe, Pro, Trp and Val).