Correspondence to: Charles J. Cohen, Merck Research Laboratories, P.O. Box 2000, Rm. 80N-31C, Rahway, NJ 07065. Fax:732-594-3925 E-mail:cohenc{at}merck.com.
Released online: 28 February 2000
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Abstract |
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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(S4S5) 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.
Key Words: insecticide, pyrethroid, para mutation, voltage gated
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INTRODUCTION |
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Voltage-activated sodium channels provide selective and rapidly activating ion pathways required for action potential generation and propagation. The subunit of these channels contains multiple binding sites for neurotoxins and therapeutically important drugs (
subunit of mammalian sodium channels (
Pyrethroids are commonly used as insecticides in crop protection, animal health, and the control of insects that endanger human health. These insecticides combine high insecticidal activity with low mammalian toxicity and constitute >25% of the world insecticide market. The intensive use of pyrethroids over the last 20 yr has led to the development of resistance in many insect species (
Molecular analysis of the full 6.3-kb coding sequence of the housefly para-type sodium channel identified two key amino acid substitutions in pyrethroid-resistant flies, L1014F in domain IIS6 and M918T in the IIS4S5 linker ( subunit and we have previously reported the expression of this protein in Xenopus oocytes alone and in combination with tipE, a putative Drosophila sodium channel accessory subunit (
-cyano group), is >100-fold more potent for Para than for rat-brain type IIA sodium channels (
-cyano) pyrethroid deltamethrin and show that the kdr and super-kdr mutations alter both the potency and efficacy of this insecticide. The kdr and super-kdr mutations also reduce the potency of cismethrin and cypermethrin to modify housefly sodium channels (
Modification of vertebrate Na channels by pyrethroids and other Na channel activators such as the plant alkaloids and halogenated hydrocarbons (DDT) is enhanced by electrical activity. This modification has generally been described with a "foot-in-the-door" model (
We find that deltamethrin effects on Para/TipE sodium channels are far more potent than those previously reported with vertebrate or marine invertebrate channels. This potency allowed us to examine the mechanism of action of deltamethrin at low concentrations of drug (0.110 nM). In this concentration range, the voltage dependence of sodium channel modification is simpler to describe and is generally consistent with a modified foot-in-the-door model. The kdr and super-kdr mutations reduce Na channel opening in the absence of drug by reducing the fraction of channels that open in response to depolarization (i.e, the mutations enhance closed-state inactivation). In addition, these mutations reduce the affinity of deltamethrin for Na channels and reduce the time that the channel remains open once drug has bound. Our studies suggest that the super-kdr mutations reduce the number of pyrethroid binding sites per channel from two to one. Thus, the mutations reduce both the potency and efficacy of insecticide action. Finally, we present a means of overcoming pyrethroid resistance.
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MATERIALS AND METHODS |
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Plasmid Constructs and Mutagenesis
The para sodium channel cDNA construct (para 13-5) was as described previously (
Materials
The isoleucine isoform of toxin II from Anemonia sulcata (ATX-II) was obtained from Calbiochem Corp. Racemic deltamethrin was obtained from Crescent Chemical (U.S. distributor for Riedel-de Haen); it was dissolved in ethanol and usually diluted 1,000-fold from a stock solution.
Oocyte Expression and Electrophysiological Measurements
Expression of sodium channel cRNAs in Xenopus laevis oocytes was performed as previously described (
Voltage-clamp experiments were performed using a CA-1 amplifier (Dagan Instruments). The bath solution was ND-96 consisting of (mM): 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES, adjusted to pH 7.5 with NaOH. In most experiments, the Na concentration of this solution was reduced by equimolar replacement of NaCl with N-methyl-D-glucamine to limit the maximal peak inward current to <1 µA, and thereby to achieve better voltage control. Experiments were performed at room temperature (2123°C). Sodium currents were measured using a two-microelectrode voltage clamp and the holding potential was usually -90 mV; deltamethrin-induced tail currents were measured at -110 mV unless indicated otherwise. Voltage-measuring electrodes were filled with 1 M KCl and had resistances <1 M. Current-injecting electrodes were filled with 0.7 M KCl plus 1.7 M K3-citrate and had resistances <0.5 M
. Agar bridges to the bath electrodes contained platinized Pt wires and had resistances <7 k
. Data were acquired using the program Pulse (Instrutech Corp.), and most analyses were performed with the companion program Pulsefit. Linear leak and capacitive currents were subtracted with P/5 steps from -120 mV. Data were sampled at 50 kHz and filtered at 10 kHz, unless indicated otherwise.
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RESULTS |
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Effects of kdr and super-kdr Mutations on Channel Gating
Previous studies with pyrethroids suggested that channel modification is promoted by opening (
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Fig 1D and Fig G, shows the effects of ATX-II for the kdr and super-kdr mutant channels, respectively. The effects of toxin are similar to those with wild-type channels, but the increase in peak inward current is greater. This indicates that ATX-II causes even larger increases in GNa,max for channels with the kdr and super-kdr mutations (Fig 1C, Fig F, and Fig I). The effects of toxin on the voltage dependence of channel activation are shown in Fig 1E and Fig H. The solid curves in Fig 1 D indicate the best fit by a Boltzmann distribution assuming a linear single-channel currentvoltage relationship. These fits indicate that the toxin increases GNa,max with little effect on the voltage dependence of channel activation. This toxin effect can be seen more clearly by converting the currentvoltage relationship into conductance measurements (Fig 1 H). Similar results were obtained in six other experiments with the L1014F mutation and three other experiments with the super-kdr double mutation (see Table 1 for a summary).
As for wild-type channels, the actions of ATX-II can be accounted for solely by slowing of inactivation and suggest that ATX-II inhibits inactivation from both open and closed states (
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Modification by Deltamethrin Is Promoted by Channel Opening
Previous studies with vertebrate and marine invertebrate Na channels have shown that pyrethroids slow inactivation and deactivation and induce channel opening at more negative potentials than normal (
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Likewise, modification by deltamethrin is promoted by ATX-II, which increases channel opening by slowing inactivation (Fig 3 B). The upper record shows the current during a 1-s depolarization with and without 100 nM deltamethrin. Even with this relatively high concentration, there is no channel modification. The lower record shows the same pulse protocol after adding 100 nM ATX-II. There is a small component of rapidly activating Na current also seen with ATX-II alone (see Fig 1). The slow increase in current during the depolarization and large tail current are due to deltamethrin and indicate greatly potentiated modification. Thus, modification by deltamethrin is enhanced by an agent that increases occupancy of the open state at the expense of the inactivated state.
Fig 3 C shows a detailed examination of the voltage dependence of modification by deltamethrin. The voltage dependence of channel opening was determined in 1 µM ATX-II (). This toxin simplifies measurements of channel opening because it eliminates rapid inactivation and thereby facilitates measurements of activation over an extended voltage range. The oocyte was then exposed to deltamethrin and modification was induced with trains of depolarizations. The amplitude of the tail current measured at the end of the pulse train is plotted as a function of the conditioning voltage (Vc) used during the train (
and right-hand ordinate). The ordinates have been adjusted so that the maximal tail current coincides with maximal Na conductance. The close correlation between the voltage dependence of channel opening and that of pyrethroid modification indicates that channels must open before modification by deltamethrin can occur. The experiment shown is with Na channels containing the super-kdr mutations; these were easier than wild type to study because the deltamethrin-induced tail currents decay more rapidly. Similar results were obtained with wild-type channels.
Quantifying the Modification of Sodium Channels by Deltamethrin
Thus far we have used tail current analysis to measure relative levels of channel modification by pyrethroids. To quantify the effects of kdr and super-kdr mutations on the affinity of deltamethrin, it is necessary to have a measure of the fraction of Na channels modified by this ligand. If we assume that the single channel conductance is unchanged by deltamethrin, as is the case for mammalian sodium channels (m/GNa,max, where GNa,
m is the conductance of deltamethrin-modified channels. If one also assumes a linear currentvoltage relationship, then:
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(1) |
where Vtail is the voltage of the tail current measurement (typically -110 mV in our studies) and Vrev is the reversal potential. This equation is similar to that used for studies of mammalian sodium channels (
In addition, Equation 1 assumes that the currentvoltage relationship is linear at all voltages, but this has only been validated for Vt > -40 mV (Fig 1). This assumption was tested by measuring the currentvoltage relationship of tail currents induced by deltamethrin (Fig 4). The voltage was ramped at 1 V/s before and after a train of test pulses in 1 nM deltamethrin. The plot shows the difference current from -120 to -50 mV; at more positive potentials the currentvoltage relationship is linear (Fig 1). Since the deltamethrin-induced tail current decays very slowly, this measurement indicates the single-channel slope conductance. The currentvoltage relationship is highly nonlinear, perhaps due to voltage-dependent block by extracellular calcium (
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(2) |
where the ATX-II availability factor is the fold increase in GNa,max indicated in the last row of Table 1 and Vtail,eff = -70 mV.
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The kdr and super-kdr Mutations Reduce the Affinity of Deltamethrin for Para Sodium Channels
Fig 5 shows two protocols used to vary the amount of modification by deltamethrin. Fig 5 (top) shows deltamethrin-induced tail currents measured after a train of brief test pulses of constant duration and with increasing concentrations of deltamethrin. The lower panels show tail currents in a fixed concentration of deltamethrin and for pulse trains of increasing duration. The tail currents are easiest to understand for the super-kdr mutant (Fig 5E and Fig F). For this mutant, the time course of the tail current is independent of the amount of modification and only the amplitude of the tail current increases with higher concentration of insecticide or longer pulse trains. This is the result expected for a 1:1 ligandreceptor binding reaction and was observed in studies of vertebrate sodium channels modified by pyrethroids or alkaloids such as veratridine (
The time course of modification by deltamethrin is determined by applying trains of brief depolarizations of varying duration. The peak amplitude of the deltamethrin-induced tail current is converted into fractional modification using Equation 2 and the percent modification is plotted as a function of pulse train length (Fig 6). For the wild-type channel, modification by deltamethrin is effectively irreversible so that all channels can be modified by 3 nM insecticide after a long pulse train (Fig 5 B and 6 A). In contrast, modification of the super-kdr mutant quickly saturates as the pulse train is prolonged, and even 5 µM deltamethrin modifies only 6% of the channels (Fig 6 B). The solid curves are the best fit to an exponential rate of onset of modification. For both the wild-type and super-kdr channels, the onset of modification is well described by first-order kinetics. For the wild-type channel, we carefully looked for a sigmoidal onset rate predicted by a higher-order binding process, but there was no consistent evidence for this at 15 nM deltamethrin.
Although there are clear differences in the sensitivity to deltamethrin among the three constructs, it is difficult to assess the relative affinity for open channels by applying trains of test pulses. A larger fraction of the channels open for the wild type (as shown by studies with ATX-II described above) and the slower decay of wild-type tail currents allows for greater levels of modification as the pulse train is prolonged (compare Fig 5B with F, and 6, A with B). To distinguish between effects of mutations on binding affinity and those on channel gating, we determined modification by deltamethrin in the presence of maximally effective ATX-II during a single long depolarization (320 ms to 0 mV; Fig 7). This protocol is an attempt to produce equivalent amounts of channel opening for all three constructs. The tail currents that are induced are qualitatively similar to those obtained in the absence of ATX-II: tail currents through the super-kdr channels decay relatively rapidly and the time course is little effected by the concentration of deltamethrin; tail currents through wild-type channels decay much more slowly, the time course slows as the concentration of deltamethrin increases, and there is a very steep concentrationresponse relationship.
The concentrationresponse relationships for all three constructs are shown in Fig 7 D. The maximal tail current amplitude has been converted to percent modification using Equation 2. Note that the affinity of deltamethrin is reduced almost 20-fold by the kdr mutation and 100-fold by the super-kdr double mutation. In addition, the apparent binding kinetics are reduced from 2:1 for the wild-type and kdr constructs to 1:1 for the super-kdr construct. This analysis is imperfect because the maximal amount of modification calculated with Equation 2 is >100% for the wild-type and kdr constructs. This probably occurs because the peak conductance in ATX-II is an underestimate of GNa,max. Furthermore, the amount of modification produced by >1 µM deltamethrin was greater than predicted by the curve fitting. This discrepancy may be due to nonspecific or secondary effects of deltamethrin that occur at very high concentrations (
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DISCUSSION |
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We find that deltamethrin effects on Para/TipE Na channels are far more potent than those previously reported for insect, vertebrate, or marine-invertebrate channels. All wild-type channels can be modified by <10 nM deltamethrin (Fig 6). The mutations L1014F and L1014F + M918T confer 30- and 500-fold resistance to deltamethrin, respectively (
A Model that Describes the Mechanism of Action of Deltamethrin and of the kdr Mutations
Most of the effects of deltamethrin and the kdr mutations can be accounted for by the model presented in Fig 8. The model for the effects of deltamethrin on the wild-type channel (Fig 8 A) is very similar to that used to describe modification of vertebrate sodium channels by the alkaloids batrachotoxin and veratridine (
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The kdr and super-kdr mutations reduce sodium channel opening and thereby reduce the occupancy of the high affinity state for deltamethrin. These mutations also increase the rate of dissociation of deltamethrin from open channels (Fig 8 B). The faster rate of dissociation is inferred because tail currents through kdr and super-kdr channels decay faster than those for wild type. If the rate of decay of tail current indicates the rate of dissociation of insecticide from the channel, then the super-kdr mutations speed the rate of dissociation ~100-fold and this would account for the 100-fold increase in dissociation constant due to these mutations (Fig 7). While the decay of the tail current probably represents the rate of dissociation of deltamethrin from the open channel for the super-kdr mutant, the decay of wild-type tail currents is so slow that transitions from O* R* may be significant.
The foot-in-the-door model used to describe the mechanism of action of veratridine cannot be applied to deltamethrin without some modification. Tail currents due to modification by veratridine decay exponentially, the amplitude increases with drug concentration as expected for 1:1 binding, and the time course is independent of the amount of modification ( R* may be significant and this could account for the complex change in kinetics as modification increases. Tail currents after extensive modification by deltamethrin are hooked or have a sigmoidal onset of decay, both suggestive of transitions between multiple open states (Fig 5 and Fig 7). Previous studies with other sodium channels provide evidence for multiple open states (
The suggestion that there are multiple pyrethroid binding sites per channel is surprising because previous studies with pyrethroids and other sodium channel ligands gave no indication of more than one binding site. Although the subunit of sodium channels has at least six distinct ligand binding sites, previous ligand binding studies suggest that there is only one site of each type per channel (
Our results and the model that we propose for the mechanism of action of deltamethrin differ substantially from most previous studies with pyrethroids. Indeed, for squid axon sodium channels, the time and concentration dependence of modification by deltamethrin and other pyrethroids led to the conclusion that deltamethrin is always bound to the channels and modification occurs because the insecticide slows all gating transitions (
The Insecticidal Activity of Pyrethroids
Previous electrophysiological studies of deltamethrin and related pyrethroids have produced conflicting viewpoints on the mechanism and potency of sodium channel modification and, consequently, on whether sodium channels are indeed the site of action; our results provide a means of reconciling these seemingly disparate results. First, studies of the super-kdr mutations provide compelling evidence that the site of action of deltamethrin is the para sodium channel. The L1014F and M918T double mutations confer 500-fold resistance to deltamethrin and we find correlate changes in the affinity of this drug for para sodium channels (Fig 7). However, our studies also indicate that the amount of modification is exquisitely sensitive to the pattern of electrical activity. For example, Fig 6 A shows that most channels can be modified by 1 nM deltamethrin after long trains of brief depolarizations, whereas Fig 3 B shows no modification by 100 nM deltamethrin during a single long depolarization. Some studies that showed relatively weak effects of deltamethrin used conditions that did not elicit sodium channel opening. One of the most influential studies established two categories of pyrethroids based on their electrophysiological effects (-cyano group), typified by permethrin, there is a good correlation between insecticidal activity and the ability to induce electrical spiking activity in neurons after brief exposure. However, type II (
-cyano) pyrethroids, typified by deltamethrin, are disproportionately weak at inducing spiking activity. This led to the suggestion that type II pyrethroids act at sites other than insect Na channels. This study applied deltamethrin without eliciting channel opening and therefore deltamethrin binding was weak. The enhancement of pyrethroid binding by channel opening is not as great for permethrin. For permethrin at 100 nM, a single long depolarization produces substantial modification (
Common Patterns of Naturally Occurring Resistance to Insecticides
The modification of sodium channels by deltamethrin can account for the high potency of this compound as an insecticide. Once modified, sodium channels remain open after repolarization. This is lethal because, after an action potential, the cell cannot repolarize completely. This is a very efficacious mechanism because modification of a few channels is adequate to trigger spontaneous electrical activity via the Hodgkin cycle; that is, sodium channel opening increases sodium influx, which in turn depolarizes the cell and causes still more sodium channel opening. Deltamethrin augments this positive feedback loop because channel opening enhances deltamethrin modification, which in turn further stimulates channel opening. The kdr and super-kdr mutations, which occur naturally and so represent changes that preserve viability in the field, defeat this toxicity by a combination of effects. First, 7080% of the sodium channels never open due to enhanced closed-state inactivation. In well-studied cases of axonal conduction, there are "extra" sodium channels that provide a substantial safety factor for rapid conduction (
The effect of super-kdr mutations on Na channels is analogous to the effect of the resistance to dieldrin (Rdl) mutation on -aminobutyric acidgated channels (
Finally, our study indicates a possible means of overcoming pyrethroid resistance in the field. Fig 1 indicates that the effect of the super-kdr mutation on Na channel opening can be overcome with an agent that acts like ATX-II because this toxin apparently inhibits closed-state inactivation. A comparison of Fig 6 B with 7 indicates that the ATX-IIinduced slowing of inactivation can result in increased modification of super-kdr channels by deltamethrin. Thus, small molecules that mimic the action of ATX-II might be combined with pyrethroids to sensitize pyrethoid-resistant insects. Small molecules mimics of ATX-II are known (
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Footnotes |
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1 Abbreviations used in this paper: ATX-II, isoleucine isoform of toxin II from Anemonia sulcata; kdr mutation, knockdown resistance mutation.
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Acknowledgements |
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This work was supported by grants to P.N.R. Usherwood (42/INS2999, 42/S10233) and A.L. Devonshire (42/INS2999) from the British Biotechnology and Biological Sciences Research Council.
Submitted: 3 September 1999
Revised: 24 January 2000
Accepted: 24 January 2000
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References |
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