The tarantula toxins ProTx-II and huwentoxin-IV differentially interact with human Nav1.7 voltage sensors to inhibit channel activation and inactivation

Abstract

The voltage-gated sodium channel Na(v)1.7 plays a crucial role in pain, and drugs that inhibit hNa(v)1.7 may have tremendous therapeutic potential. ProTx-II and huwentoxin-IV (HWTX-IV), cystine knot peptides from tarantula venoms, preferentially block hNa(v)1.7. Understanding the interactions of these toxins with sodium channels could aid the development of novel pain therapeutics. Whereas both ProTx-II and HWTX-IV have been proposed to preferentially block hNa(v)1.7 activation by trapping the domain II voltage-sensor in the resting configuration, we show that specific residues in the voltage-sensor paddle of domain II play substantially different roles in determining the affinities of these toxins to hNa(v)1.7. The mutation E818C increases ProTx-II's and HWTX-IV's IC(50) for block of hNa(v)1.7 currents by 4- and 400-fold, respectively. In contrast, the mutation F813G decreases ProTx-II affinity by 9-fold but has no effect on HWTX-IV affinity. It is noteworthy that we also show that ProTx-II, but not HWTX-IV, preferentially interacts with hNa(v)1.7 to impede fast inactivation by trapping the domain IV voltage-sensor in the resting configuration. Mutations E1589Q and T1590K in domain IV each decreased ProTx-II's IC(50) for impairment of fast inactivation by ~6-fold. In contrast mutations D1586A and F1592A in domain-IV increased ProTx-II's IC(50) for impairment of fast inactivation by ~4-fold. Our results show that whereas ProTx-II and HWTX-IV binding determinants on domain-II may overlap, domain II plays a much more crucial role for HWTX-IV, and contrary to what has been proposed to be a guiding principle of sodium channel pharmacology, molecules do not have to exclusively target the domain IV voltage-sensor to influence sodium channel inactivation.

Fig. 1.

Effects of ProTx-II and HWTX-IV on WT Na_v1.7 expressed in HEK293 cells. A, sequence alignment of ProTx-II and HWTX-IV. Six conserved cysteines are identified by the encompassing rectangles in the sequence alignment. B, differential effects of two toxins on the current-voltage relationships of WT Na_v1.7. Cells were held at −100 mV. Na_v1.7 currents were elicited by 50-ms depolarization steps to various voltages ranging from −80 to +100 mV in 5-mV increments. Currents elicited before and after application of 100 nM ProTx-II (left) or 100 nM HWTX-IV (right) were normalized to the maximum amplitude of control peak current. C, effects of the two toxins on normalized steady-state activation and inactivation of WT Na_v1.7. Channel conductances before and after application of 100 nM ProTx-II or 100 nM HWTX-IV were calculated with the equation: G(V) = I/(V − V_rev), in which I, V, and V_rev represented inward current elicited as described in B, test potential, and reversal potential, respectively. Data are plotted as a fraction of the maximum conductance. The voltage-dependence of steady-state inactivation was estimated using a standard double-pulse protocol, in which a 20-ms depolarizing test potential of 0 mV followed a 500-ms prepulse at potentials that ranged from −130 to −10 mV with a 10-mV increment. Cells were held at −100 mV. All curves were fit with the Boltzmann equation as described under Materials and Methods. D, concentration-dependent inhibition of WT Na_v1.7 by two toxins. Data points (mean ± S.E., each from three to four cells) were fit with the Hill equation as described under Materials and Methods. The values of IC₅₀, slope factor (n_H), and f_bottom yielded are shown in Tables 1 and 2.

Fig. 2.

Concentration-response inhibitory curves of ProTx-II (A) and HWTX-IV (B) on DI and DII mutant Na_v1.7 channels. Sodium current was induced at 5-s intervals by a 20-ms depolarization from a holding potential of −100 mV. The test pulse potentials to activate channels were set to −10 mV (WT and L201V/N206D), −5 mV (F204A/F813G, F204A/F813G, F813G, E1589Q), 0 mV (E818C and F813G/E818C), and +10 mV (E203K/E818C), respectively. The residual current after toxin treatment was plotted as fraction of the control current. Data points (mean ± S.E., each from 3 - 7 cells) were fit with a Hill equation as described under Materials and Methods. The values of IC₅₀, slope factor (n_H), and f_bottom yielded are shown in Table 1 and 2.

Fig. 3.

ProTx-II significantly impeded fast inactivation of WT Na_v1.7 expressed in HEK293 cells. Cells were held at −100 mV. Families of current traces before (A) and after application of 1 μM ProTx-II (B) or 200 nM TTX (C) were induced by 50-ms depolarizing steps to various potentials ranging from −100 to +100 mV in 5-mV increments. D and E, effects of 1 μM ProTx-II (D) or 200 nM TTX (E) on the current voltage (I-V) relationship of WT Na_v1.7. All currents induced before and after toxin treatment were plotted as fraction of the maximum amplitude of control peak current. The dotted line indicates the control I-V curve. The red filled circles indicate the peak I-V curves after application of 1 μM ProTx-II. I_10ms (blue open diamond) was shown as the current inactivated at 10 ms after application of 1 μM ProTx-II. E, effects of 200 nM TTX on the current-voltage (I-V) relationship of WT Nav1.7 sustained currents induced by ProTx-II. All currents induced before and after toxin treatment were plotted as fraction of the maximum amplitude of control peak current. The dotted line indicates the control I-V curve. The green open circles indicate the I-V curve after application of 200 nM TTX. I_10ms (blue open diamond) was shown as the current inactivated at 10 ms after application of 1 μM ProTx-II.

Fig. 4.

ProTx-II differentially inhibited both activation and inactivation of sodium channel subtypes expressed in HEK293 cells. Cells were held at −100 mV. A, currents through WT Na_v1.2, Na_v1.3, Na_v1.4 and Na_v1.7 were induced by a 20-ms depolarizing potential of −10 mV. Na_v1.5 current was elicited at −30 mV. The dotted lines show the residual current in the presence of 1 μM ProTx-II after normalization to the maximum amplitude of control current. B, concentration-response inhibitory curves of ProTx-II on the activation of five sodium channel subtypes (Na_v1.2, Na_v1.3, Na_v1.4, Na_v1.5, and Na_v1.7). The residual current after ProTx-II treatment was plotted as a fraction of control current. Data points (mean ± S.E., each from three to seven cells) were fit with Hill equation as described under Materials and Methods. The IC₅₀ values were estimated to be 52.9 ± 1.1 (Na_v1.2a), 109.9 ± 7.1 (Na_v1.3), 107.6 ± 7.7 (Na_v1.4), and 79.4 ± 40.7 (Na_v1.5) nM, respectively. The slope factor (n_H) ranged from 0.9 to 1.1. C, concentration-response inhibitory curves of ProTx-II on the fast inactivation of five sodium channel subtypes. The I_10ms was plotted as a fraction of the residual current after ProTx-II treatment. Inhibition of fast-inactivation increases the ratio of I_10ms/I_peak. Data points (mean ± S.E., each from three to seven cells) were fit with Hill equation as described under Materials and Methods. The IC₅₀ values are shown in Supplementary Table 2.

Fig. 5.

Amino acid sequence alignment of the DIV S3–S4 linkers of seven α subunit isoforms from human. A, crucial determinants of neurotoxin receptor 3 are located in the S3–S4 linker of sodium channel domain II. The positions of amino acid residues of interest are shaded in gray. B, schematic diagram of sodium channel α subunit. The voltage sensor (the fourth segment) of each domain is shaded in gray and marked with “++.” The amino acid sequence of DIV S3–S4 linker is shown in the square frame of A as indicated by arrows.

Fig. 6.

Mutations in DIV S3–S4 linker differentially alter the effect of ProTx-II on fast-inactivation of hNa_v1.7 channels expressed in HEK293 cells. A, representative current traces for five mutant (D1586A, D1586E, E1589Q, T1590K, and F1592A) hNa_v1.7 channels. The test pulse potential was −10 mV (D1586A and D1586E) and −5 mV (E1589Q, T1590K and F1592A), respectively. Cells were held at −100 mV. The dotted line shows the residual current in the presence of 0.1 or 1 μM ProTx-II after normalization to the maximum amplitude of control current. B, concentration-response inhibitory curves of ProTx-II on the activation of WT and five mutant (D1586A, D1586E, E1589Q, T1590K, and F1592A) Na_v1.7 channels. Residual current after toxin treatment was plotted as a fraction of control peak current amplitude. Data points (mean ± S.E., each from three to six cells) were fitted with Hill equation as described under Materials and Methods. The calculated values of IC₅₀, slope factor (n_H), and f_bottom are shown in Table 1. C, concentration-response inhibitory curves of ProTx-II on fast inactivation of WT and five mutant (D1586A, D1586E, E1589Q, T1590K, and F1592A) Na_v1.7 channels. The I_10ms was plotted as a fraction of the residual current after ProTx-II treatment. Data points (mean ± S.E., each from three to six cells) were fitted with the Hill equation as described under Materials and Methods.

Fig. 7.

Two mutations (D1586A and F1592A) enhanced the ProTx-II slowing fast inactivation of hNa_v1.7 in the presence of K1590. A, representative current traces for two double mutant (D1586A/T1590K and T1590K/F1592A) hNa_v1.7 channels. The test pulse potential was −5 mV for D1586A/T1590K and 0 mV for T1590K/F1592A, respectively. Cells were held at −100 mV. The dotted lines show the residual current in the presence of 1 μM ProTx-II after normalization to the maximum amplitude of control current. B, concentration-response inhibitory curves of ProTx-II on the activation of three mutant (T1590K, D1586A/T1590K, and T1590K/F1592A) Na_v1.7 channels. Residual current after toxin treatment was plotted as a fraction of control peak current amplitude. The calculated values of IC₅₀, slope factor (n_H), and f_bottom are shown in Table 1. C, concentration-response inhibitory curves of ProTx-II on fast-inactivation of three mutant (T1590K, D1586A/T1590K, and T1590K/F1592A) Na_v1.7 channels. The I_10ms was plotted as a fraction of the residual current after ProTx-II treatment. The IC₅₀ values are shown in Supplementary Table 2. In B and C, data points (mean ± S.E., each from three to four cells) were fitted with a Hill equation as described under Materials and Methods.