Compound-specific effects of mutations at Val787 in DII-S6 of Nav 1.4 sodium channels on the action of sodium channel inhibitor insecticides

Abstract

Sodium channel inhibitor (SCI) insecticides are hypothesized to inhibit voltage-gated sodium channels by binding selectively to the slow-inactivated state. Replacement of valine at position 787 in the S6 segment of homology domain II of the rat Na(v)1.4 sodium channel by lysine (V787K) enchances slow inactivation of this channel whereas replacement by alanine or cysteine (V787A and V787C) inhibits slow inactivation. To test the hypothesis that SCI insecticides bind selectively to the slow-inactivated state, we constructed mutated Na(v)1.4/V787A, Na(v)1.4/V787C, and Na(v)1.4/V787K cDNAs, expressed wildtype and mutated channels with the auxiliary β1 subunit in Xenopus oocytes, and used the two-electrode voltage clamp technique to examine the effects of these mutations on channel inhibition by four SCI insecticides (indoxacarb, its bioactivated metabolite DCJW, metaflumizone, and RH3421). Mutations at Val787 affected SCI insecticide sensitivity in a manner that was independent of mutation-induced changes in slow inactivation gating. Sensitivity to inhibition by 10 μM indoxacarb was significantly increased in all three mutated channels, whereas sensitivity to inhibition by 10 μM metaflumizone was significantly reduced in Na(v)1.4/V787A channels and completely abolished in Na(v)1.4/V787K channels. The effects of Val787 mutations on metaflumizone were correlated with the hydrophobicity of the substituted amino acid rather than the extent of slow inactivation. None of the mutations at Val787 significantly affected the sensitivity to inhibition by DCJW or RH3421. These results demonstrate that the impact of mutations at Val787 on sodium channel inhibition by SCI insecticides depend on the specific insecticide examined and is independent of mutation-induced changes in slow inactivation gating. We propose that Val787 may be a unique determinant of metaflumizone binding.

Figure 1. Structures of SCI insecticides

(A) Indoxacarb and DCJW. (B) Metaflumizone. (C) RH-3421 and RH-4841.

Figure 2. Voltage-dependent activation and steady-state fast inactivation of Na_v1.4 and V787-mutated sodium channels

(A) Conductance-voltage plots for sodium channel activation; peak sodium currents measured on depolarization from −120 mV to test potentials ranging from − 80 mV to 30 mV were transformed to conductances (G) using the equation G = I/(V_t-V_rev), where I is the peak current, V_t is the voltage of the test potential, and V_rev is the reversal potential; conductances were normalized to the maximal conductance (G_max) for that oocyte; values are means ± S.E. of 15 (Na_v1.4), 9 (Na_v1.4/V787A), 7 (Na_v1.4/V787C), or 11 (Na_v1.4/V787K) separate experiments with different oocytes. (B) Voltage dependence of steady-state fast inactivation; conditioning pulses (200 ms) from −120 mV to potentials ranging from −100 mV to 0 mV (V_p) were followed immediately by 20-ms test pulses to either −10 mV (Na_v1.4, Na_v1.4/V787C, Na_v1.4/V787K) or 0 mV (Na_v1.4/V787A); peak currents were normalized to the maximal current measured during the inactivation protocol for that oocyte; values are means ± S.E. of 11 (Na_v1.4), 9 (Na_v1.4/V787A), 5 (Na_v1.4/V787C), or 4 (Na_v1.4/V787K) separate experiments with different oocytes; curves were fitted to the mean values using the Boltzmann equation.

Figure 3. Voltage dependence of slow inactivation of Na_v1.4 and Val787 mutated sodium channels

Oocytes were clamped at a holding potential (V_h) of either −120 mV (Na_v1.4, Na_v1.4/V787A, Na_v1.4/V787C channels) or −140 mV (Na_v1.4/V787K channels) and stimulated with either a 100-s (Na_v1.4, Na_v1.4/V787A, Na_v1.4/V787C) or 10-s (Na_v1.4/V787K) conditioning pulse (V_p) to potentials ranging from V_h to 0 mV followed by a 50-ms hyperpolarization to V_h and a test pulse to either −10 mV (Na_v1.4, Na_v1.4/V787C, Na_v1.4/V787K) or 0 mV (Na_v1.4/V787A); peak currents were normalized to the maximal current measured during the inactivation protocol for that oocyte; values are means ± S.E. of 7 (Na_v1.4, 4 (Na_v1.4/V787A), 5 (Na_v1.4/V787C), or 6 (Na_v1.4/V787K) separate experiments with different oocytes; curves were fitted to the mean values using the Boltzmann equation.

Figure 4. Time course of inhibition of (A) Na_v1.4, (B) Na_v1.4/V787A, (C) Na_v1.4/V787C, or (D) Na_v1.4/V787K sodium channels by SCI insecticides

Oocytes were clamped at a holding potential (V_h) of either −30 mV (Na_v1.4, Na_v1.4/V787A, Na_v1.4/V787C) or −140 mV (Na_v1.4/V787K); sodium currents were measured once every minute with a test pulse (20 ms) preceded by a hyperpolarization (2 s) to either −120 mV (Na_v1.4, Na_v1.4/V787A, Na_v1.4/V787C) or −140 mV (Na_v1.4/V787K); currents measured in the presence of an insecticide were normalized to the peak current recorded in that oocyte prior to insecticide perfusion; values are means ± S.E. of 3-5 experiments (Na_v1.4), 3-7 (Na_v1.4/V787A, Na_v1.4/V787C), or 4-5 (Na_v1.4/V787K) separate experiments with different oocytes; curves were fitted to the mean values using a single-exponential decay equation.

Figure 5. Effect of hyperpolarization on Na_v1.4/V787K currents and their inhibition by indoxacarb

Oocytes were clamped at a holding potential (V_h) of −140 mV for 5 min followed by 5 min at a V_h of −150 mV while stimulated at a rate of 0.05 Hz with a test pulse (20 ms) to −10 mV; sodium currents were normalized to the maximum current recorded in the same oocyte at − 140 mV prior to indoxacarb perfusion; values are means ± S.E. of 7 separate experiments in different oocytes; curves were fitted to the mean values using a single-exponential decay equation.

Figure 6. Comparative inhibition of Na_v1.4 and Na_v1.4/V787K sodium channels by SCI insecticides

(A) Representative sodium current traces from an oocyte expressing Na_v1.4/V787K at a holding potential (V_h) of −140 mV and −110 mV in the absence of insecticides. (B) Representative sodium current traces from an oocyte expressing Na_v1.4 sodium channels at a V_h of −120 mV and −30 mV in the absence of SCI insecticides. (C) Time course of inhibition of Na_v1.4/V787K by SCI insecticides; oocytes were clamped at a V_h of −110 mV and stimulated once every minute with a test pulse (20 ms) to −10 mV preceded by a hyperpolarization (2 s) to − 140 mV; currents measured in the presence of an insecticide were normalized to the peak sodium current recorded in that oocyte prior to insecticide perfusion; values are means ± S.E. of 5-7 separate experiments with different oocytes; curves were fitted to the mean values using a single-exponential decay equation. (D) Comparison of the fractional inhibition of Na_v1.4 (Fig. 4A) and Na_v1.4/V787K channels by SCI insecticides after 15 min of insecticide perfusion at a V_h of either −30 (Na_v1.4) or −110 mV (Na_v1.4/V787K).