Activation of Drosophila sodium channels promotes modification by deltamethrin. Reductions in affinity caused by knock-down resistance mutations

Abstract

kdr and super-kdr are mutations in houseflies and other insects that confer 30- and 500-fold resistance to the pyrethroid deltamethrin. They correspond to single (L1014F) and double (L1014F+M918T) mutations in segment IIS6 and linker II(S4-S5) of Na channels. We expressed Drosophila para Na channels with and without these mutations and characterized their modification by deltamethrin. All wild-type channels can be modified by <10 nM deltamethrin, but high affinity binding requires channel opening: (a) modification is promoted more by trains of brief depolarizations than by a single long depolarization, (b) the voltage dependence of modification parallels that of channel opening, and (c) modification is promoted by toxin II from Anemonia sulcata, which slows inactivation. The mutations reduce channel opening by enhancing closed-state inactivation. In addition, these mutations reduce the affinity for open channels by 20- and 100-fold, respectively. Deltamethrin inhibits channel closing and the mutations reduce the time that channels remain open once drug has bound. The super-kdr mutations effectively reduce the number of deltamethrin binding sites per channel from two to one. Thus, the mutations reduce both the potency and efficacy of insecticide action.

Figure 1

The kdr and super-kdr mutations reduce the availability of Na channels during a step depolarization. (A) Sodium currents measured at a test potential (V_t) of −10 mV with and without 0.5 μM ATX-II; blanking interval, 740 μs. (B) Sodium current measured in 0.5 μM ATX-II for the same preparation as for A. The test depolarization lasted 600 ms. (C) Relative G _Na plotted as a function of V_t with and without 100 nM ATX-II. In C, F, H, and I, G_Na,max is calculated from the peak Na current assuming a linear current–voltage relationship and the curves are the best-fit Boltzmann distribution. Control: slope factor (k) = 9.03 mV, midpoint potential (V_1/2) = −19.1 mV; maximal sodium conductance (G_Na,max) = 5.33 μS; reversal potential = +44.0 mV. +ATX-II: k = 8.55 mV, V_1/2 = −19.4 mV, G_Na,max = 9.71; reversal potential = +37.3 mV. In C, F, and I, the data are normalized by G_Na,max in ATX-II to emphasize that toxin causes a greater percent change in G_Na,max for kdr and super-kdr mutants than for wild-type channels. (D) Superimposed current records from an oocyte expressing the kdr Na channel with and without 1 μM ATX-II; V_t = 0 mV; blanking interval, 400 μs. (E) Peak sodium current plotted as a function of V_t with and without 1 μM ATX-II (same experiment as for A). Both curves assume a linear current–voltage relationship and are the best-fit Boltzmann distribution. Control: k = 6.58 mV, V_1/2 = −12.7 mV, G_Na,max = 12.8 μS. It is assumed that the reversal potential for the Na current is the same as in ATX-II (+48 mV). +ATX-II: k = 6.74 mV, V_1/2 = −13.3 mV, G_Na,max = 50.3 μS, reversal potential = 48. mV. (F) Relative G _Na with and without 1 μM ATX-II for a kdr mutant Na channel. Control: k = 6.58 mV, V_1/2 = −12.7 mV, G_Na,max = 12.8 μS, reversal potential = +48.0 mV. +ATX-II: k = 6.74 mV, V_1/2 = −13.3 mV, G_Na,max = 50.3 μS, reversal potential = +48.0 mV. (G) Sodium currents recorded at 0 mV with and without 1 μM ATX-II from an oocyte expressing super-kdr Na channels; blanking interval, 400 μs. (H) Peak G _Na plotted as a function of V_t with and without 1 μM ATX-II for a super-kdr Na channel. Control: k = 6.21 mV, V_1/2 = −17.3 mV, G_Na,max = 29.4 μS, reversal potential = +47.0 mV. +ATX-II: k = 8.58 mV, V_1/2 = −17.9 mV, G_Na,max = 161.8 μS, reversal potential = +47.7 mV. (I) Same data as in H normalized by G_Na,max in ATX-II.

Figure 3

Modification by deltamethrin requires Na channel opening. (A) Trains of brief depolarizations are more effective than one long depolarization at producing modification by deltamethrin. Tail currents were measured at −110 mV after depolarizations to 0 mV using the indicated pulse protocol. The train of depolarizations was delivered at 66 Hz; 10 nM deltamethrin. (B) Deltamethrin induces large tail currents after a single long depolarization if inactivation is first slowed by ATX-II. The upper record shows superimposed current measurements with and without 100 nM deltamethrin alone. The voltage was stepped from −90 to −40 mV for 1 s and repolarized to −130 mV. Note that these records are at higher gain than below. The lower current record is offset. The pulse protocol is shown at the bottom. ATX-II causes a small rapidly activating current that was also seen with ATX-II alone. This is followed by a slowly activating current and, subsequently, a large tail current. The latter is not obtained with ATX-II alone (Fig. 1). (C) Tail current induced in 500 nM deltamethrin by trains of pulses increases with stronger depolarizations and the increase parallels the voltage dependence of channel opening. □ (left ordinate), peak G _Na for super-kdr mutant in 1 μM ATX-II. This toxin eliminates current decay during the trains of pulses so that peak current is proportional to the time integral of current. ▴ (right ordinate), amplitude of tail currents elicited after application of 100 conditioning pulses (5 ms at 66 Hz) for each conditioning voltage (V_c; see top inset for the tail current pulse protocol). The ordinates have been adjusted so that the maximal tail current amplitude coincides with maximal Na conductance.

Figure 2

Onset of and recovery from inactivation is faster for the kdr and super-kdr Na channels. Three different pulse protocols were used. For V ≤ −50 mV, symbols indicate the time constant for recovery from inactivation. For −50 < V ≤ −35 mV, symbols indicate the time constant for onset of inactivation. Both sets of data were obtained by applying conditioning pulses of varying duration before the test pulse; in the case of recovery from inactivation, conditioning pulses were preceded by a preconditioning pulse to induce inactivation. For V > −35 mV, symbols indicate the time constant for onset of inactivation determined by fitting a single exponential to the decay of the current during a test pulse. Each data point represents the mean of at least four experiments and the error bars indicate SEM.

Figure 5

Deltamethrin-induced tail currents measured after trains of 5-ms depolarizations to 0 mV at 66 Hz. The left, center, and right columns show results for wild type, kdr, and super-kdr mutants, respectively. Note the expanded time scale in E and F. A, C, and E each show tail currents produced by identical pulse trains (50 pulses for A and 100 for C and E), but with increasing concentrations of deltamethrin. The concentrations of deltamethrin are indicated at the left of each set of traces. B, D, and F show tail currents produced by pulse trains of variable duration in the presence of 1 nM, 100 nM, or 5 μM deltamethrin for Na channels of wild type, kdr and super-kdr, respectively. The number of pulses in each train is indicated at the left of each set of traces. The decay phase of the largest tail currents in A and C–F was fit by the sum of one or two exponentials plus a constant. The fits are superimposed on the tail currents. The time constants (τ₁ and τ₂) for the fits were: A: τ₁ = 0.71 s, τ₂ = 17.7 s; C: τ₁ = 1.87 s; D: τ₁ = 0.92 s, τ₂ = 8.7 s; E: τ₁ = 0.17 s; F: τ₁ = 0.19 s.

Figure 4

Instantaneous current–voltage relationship of wild–type sodium channels modified by 1 nM deltamethrin. The voltage was ramped at 1 V/s before and after a train of 200 pulses of 5-ms duration at 66 Hz. The record shows the difference between measurements made before and after the pulse train, and thus indicates the current induced by deltamethrin. A linear extrapolation from the data goes through the reversal potential determined in this oocyte before adding deltamethrin (V_rev is approximately +10 mV in saline with 20 mM Na). This extrapolation was necessary because at voltages more positive than −50 mV channels not modified by deltamethrin were also opened. The dotted vertical line indicates that the driving force on the sodium current at −110 mV is equivalent to that obtained with a linear I-V at about −70 mV.

Figure 7

The kdr and super-kdr mutations reduce the potency of deltamethrin for sodium channels modified with ATX-II. All panels show Na currents measured in ≥500 nM ATX-II during a 320-ms depolarization to 0 mV and after repolarization to −110 mV. A–C show results for wild-type, kdr, and super-kdr mutants, respectively. The pulse protocol is shown below B. Note that C is shown on an expanded time scale. (D) Percent modification plotted versus deltamethrin concentration for wild-type, kdr, and super-kdr constructs (▪, ▴, and •, respectively). The fraction of modified channels was calculated using ; the ATX-II factor was made equal to one since all experiments were conducted in maximally effective ATX-II. The solid curves through the data indicate the best fits to the equation: percent modification = M _max/{1 + (K _d/[δM])ⁿ}, where [δM] is the concentration of deltamethrin. Wild type (▪): M _max = 187%, K _d = 4.71 nM, n = 2 · L1014F mutant; (▴): M _max = 87.6%, K _d = 82 nM, n = 2. Data for concentrations of deltamethrin >1 μM were excluded from the curve fitting; L1014F + M918T double mutant (•): M _max = 99.8%, K _d = 478 nM, n = 1. The datum point for 10 μM deltamethrin was excluded from the curve fitting. Each set of data was obtained with a single preparation.

Figure 6

Super-kdr mutations reduce the maximal amount of modification obtainable with trains of brief depolarizations. Deltamethrin-induced tail currents were measured after trains of 5-ms depolarizations to 0 mV at 66 Hz (see inset in A for pulse protocol). The percent modification was calculated using and the ATX-II factors are given in Table . The solid curves through the data indicate the best fits to the equation: percent modification = M _max [1 − exp(−n/τ)], where M _max is the maximal percentage of modification, n is the number of pulses, and τ is an effective time constant with units of number of pulses. (A) Percent modification as a function of pulse train length for wild-type channels. ▪, ▴, and • indicate results with 1, 3, and 5 nM deltamethrin, respectively. For 1 nM, M _max = 48.8%, τ = 3,730 pulses. For 3 and 5 nM, the fitting assumes M _max = 100%; τ = 1,810 pulses for 3 nM and 873 pulses for 5 nM. (B) Modification of super-kdr channels. The ▪ and ▴ indicate results with 0.5 and 5 μM deltamethrin, respectively. For 500 nM, M _max = 6.3% and τ = 135 pulses. For 5 μM, M _max = 6.7% and τ = 43.6 pulses.

Figure 8

A model accounting for the modification of gating by deltamethrin and the super-kdr mutations. (A) According to voltage, the drug-free channel (top) switches between multiple states (R, resting; O, open; I, inactivated). Deltamethrin binds and unbinds only to and from the open conformation and drug binding dramatically slows transitions to closed states. The drug-bound “open” channel is conducting. For simplicity, only one rested, open, or inactivated state is shown. (B) The super-kdr mutations have two effects: (a) closed-state inactivation is favored so that most channels do not open before inactivating, thereby reducing the fraction of channels that enter the high affinity state for deltamethrin and reducing channel opening near threshold; and (b) the rate of dissociation of deltamethrin from open channels is faster—this accounts for the lower affinity and faster decay of insecticide-induced tail currents.