Charge substitutions at the voltage-sensing module of domain III enhance actions of site-3 and site-4 toxins on an insect sodium channel

Abstract

Scorpion α-toxins bind at the pharmacologically-defined site-3 on the sodium channel and inhibit channel inactivation by preventing the outward movement of the voltage sensor in domain IV (IVS4), whereas scorpion β-toxins bind at site-4 on the sodium channel and enhance channel activation by trapping the voltage sensor of domain II (IIS4) in its outward position. However, limited information is available on the role of the voltage-sensing modules (VSM, comprising S1-S4) of domains I and III in toxin actions. We have previously shown that charge reversing substitutions of the innermost positively-charged residues in IIIS4 (R4E, R5E) increase the activity of an insect-selective site-4 scorpion toxin, Lqh-dprIT_3-c, on BgNa_v1-1a, a cockroach sodium channel. Here we show that substitutions R4E and R5E in IIIS4 also increase the activity of two site-3 toxins, LqhαIT from Leiurusquinquestriatus hebraeus and insect-selective Av3 from Anemonia viridis. Furthermore, charge reversal of either of two conserved negatively-charged residues, D1K and E2K, in IIIS2 also increase the action of the site-3 and site-4 toxins. Homology modeling suggests that S2-D1 and S2-E2 interact with S4-R4 and S4-R5 in the VSM of domain III (III-VSM), respectively, in the activated state of the channel. However, charge swapping between S2-D1 and S4-R4 had no compensatory effects on gating or toxin actions, suggesting that charged residue interactions are complex. Collectively, our results highlight the involvement of III-VSM in the actions of both site 3 and site 4 toxins, suggesting that charge reversing substitutions in III-VSM allosterically facilitate IIS4 or IVS4 voltage sensor trapping by these toxins.

Keywords: Electrophysiology; Homology modeling; Insect sodium channel; Mutagenesis; Scorpion α-toxin; Scorpion β-toxin.

Fig. 1.

Substitutions R4E and R5E in IIIS4 increase the action of Av3 and LqhαIT. A-B, Representative sodium current traces in the absence or presence of Av3 (A) or LqhαIT (B). The protocols are presented above the current traces: the currents were elicited by a 20 ms test pulse to −10 mV, from a holding potential of −120 mV. C-D, Effects of charge reversal of the five positively-charged residues in IIIS4 on BgNa_v1–1a sensitivity to Av3 (C) or LqhαIT (D). The inhibitory effect on fast inactivation by the toxins was calculated by measuring the current that remained at 20 ms (I₂₀) divided by the peak current (I_peak). * denotes significant difference compared to WT BgNa_v1–1a channels (p˂ 0.05).

Fig. 2.

Voltage dependence of activation and inactivation of four charge reversal channel mutants at IIIS1 and IIIS2. A, Schematic diagram of a sodium channel indicating the positions of the charge reversing substitutions of four highly conserved positively-charged residues and negatively-charged residues in domain III. B–C, Voltage dependence of activation (B) and inactivation (C). Data are presented as mean ± SEM for 10–20 oocytes. * indicates a significant difference compared to that of BgNav1–1a channels only using a one-way ANOVA with Scheffe’s post hoc analysis (p < 0.05).

Fig. 3.

Charge reversal of two negatively-charged residues in IIIS2 increase the effect of site-3 toxins. A-B, Representative sodium current traces of channel mutants S2-D1K and S2-E2K in the absence or presence of Av3 (A) or LqhαIT (B). C-D, Effects of LqhαIT (C) and Av3 (D) on fast inactivation of the four charge reversal channel mutants. The toxin inhibitory effects on fast inactivation were calculated by measuring the current that remained after 20 ms (I₂₀) divided by the peak current (I_peak) for S1-E1K, S1-E2K and S2-E2K channel mutants. Since after 20 ms, the inactivation of S2-D1K was not complete, the remaining current of this channel mutant was determined only after 50 ms, and so the value measured was I₅₀/I_peak. *denotes significant difference compared to WT BgNa_v1–1a channels determined by one-way ANOVA with Scheffe’s post hoc analysis (p˂ 0.05).

Fig. 4.

Charge reversal of two negatively-charged residues in IIIS2 increase the effect of Lqh-dprIT₃. A-E, Conductance-voltage relations in WT BgNa_v1–1a channels (A) or the four channel mutants substituted in domain III (B-E) in the absence (●) or presence (○) of 300 nMLqh-dprIT₃. To measure the toxin effect, a 20 Hz train (50) of 5 ms depolarizing prepulses to 50 mV from a holding potential of −120 mV was followed by a series of 20 ms depolarizing test pulses between −80 and −65 mV. F, Percentage of modification of the WT and four mutant channels elicited by Lqh-dprIT₃. * denotes a significant difference compared to WT BgNa_v1–1a channels, as determined by one-way ANOVA with Scheffe’s post hoc analysis (p˂ 0.05).

Fig. 5.

The sensitivity of S2-D1K/R4E double mutant channels to Av3 and LqhαIT compared to channel mutants S2-D1K and R4E. A-B, Representative sodium current traces of R4E and IIIS2-D1K/R4E channels in the absence or presence of Av3 (A) and LqhαIT (B). C-D, Effect of S2-D1K/R4E double substitution on channel sensitivity to Av3 (C) and LqhαIT (D) in comparison to the single channel substitution. * denotes significant differences compared to WT BgNa_v1–1a channels as determined by one-way ANOVA with Scheffe’s post hoc analysis (p˂ 0.05).