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The V410M mutation associated with pyrethroid resistance in

Heliothis virescens reduces the pyrethroid sensitivity of house fly

sodium channels expressed in Xenopus oocytes

Si Hyeock Lee

_{, David M. Soderlund}

Department of Entomology, New York State Agricultural Experiment Station, Cornell University, Geneva, NY 14456, USA Received 22 November 1999; received in revised form 18 April 2000; accepted 19 April 2000

Abstract

Some strains of Heliothis virescens carry a novel sodium channel mutation, corresponding to the replacement of Val410 by Met (designated V410M) in the house fly Vssc1 sodium channel, that is genetically and physiologically associated with pyrethroid resistance. To test the functional significance of this mutation, we created a house fly Vssc1 sodium channel containing the V410M mutation by site-directed mutagenesis, expressed wildtype and specifically mutated sodium channels in Xenopus laevis oocytes, and evaluated the effects of the V410M mutation on the functional and pharmacological properties of the expressed channels by two-electrode voltage clamp. The V410M mutation caused depolarizing shifts of |9 mV and |5 mV in the voltage dependence of activation and steady-state inactivation, respectively, of Vssc1 sodium channels. The V410M mutation also reduced the sensitivity of Vssc1 sodium channels to the pyrethroid cismethrin at least 10-fold and accelerated the decay of cismethrin-induced sodium tail currents. The degree of resistance conferred by the V410M mutation in the present study is sufficient to account for the degree of pyrethroid resistance in H. virescens that is associated with this mutation. Although Val410 is located in a sodium channel segment identified as part of the binding site for batrachotoxin, the V410M mutation did not alter the sensitivity of house fly sodium channels to batrachotoxin. The effects of the V410M mutation on the voltage dependence and cismethrin sensitivity of Vssc1 sodium channels were indistinguishable from those caused by another sodium channel point mutation, replacement of Leu1014 by Phe (L1014F), that is the cause of knockdown resistance to pyrethroids in the house fly. The positions of the V410M and L1014F mutations in models of the tertiary structure of sodium channels suggest that the pyrethroid binding site on the sodium channel α subunit is located at the interface between sodium channel domains I and II.2001 Elsevier Science Ltd. All rights reserved.

Keywords: Insecticide; Resistance; Pyrethroid; Batrachotoxin; Voltage-sensitive sodium channel; Musca domestica

1. Introduction

In the house fly (Musca domestica), the knockdown resistance (kdr) trait confers resistance to pyrethroid and diphenylethane [e.g., dichlorodiphenyltrichloroethane (DDT)] insecticides by a reduction in the sensitivity of the nervous system to these compounds, and resistance traits similar to kdr have also been identified in numer-ous other insect species (Soderlund and Bloomquist, 1990; Soderlund, 1997). In the house fly (Williamson et

* Corresponding author. Tel.:+1-315-787-2364; fax: +1-315-787-2326.

E-mail address: dms6@cornell.edu (D.M. Soderlund).

1 _{Present address: Department of Entomology, Fernald Hall,}

Uni-versity of Massachusetts, Amherst, MA 01003, USA.

al., 1993; Knipple et al., 1994), Heliothis virescens (Taylor et al., 1993), Blattella germanica (Dong and Scott, 1994) and Leptinotarsa decemlineata (Lee et al., 1999b), these resistance traits are genetically linked to sodium channel αsubunit genes that are orthologous to the para sodium channel gene of Drosophila melanogas-ter (Loughney et al., 1989).

Comparison of sodium channel gene sequences from susceptible and knockdown-resistant insects has ident-ified point mutations in sodium channel gene sequences that are associated with knockdown resistance traits. Replacement of Leu1014 by Phe (designated L1014F) in transmembrane segment IIS6 of the house fly sodium channel is associated with the kdr trait of the house fly (Miyazaki et al., 1996; Williamson et al., 1996). The same mutation is also found at the corresponding leucine

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residue of sodium channels from pyrethroid-resistant strains of several other insect species (Miyazaki et al., 1996; Dong, 1997; Guerrero et al., 1997; Martinez-Torres et al., 1998; Schuler et al., 1998; Lee et al., 1999b), whereas some resistant populations of H. vires-cens have a histidine replacing leucine at this position (Park and Taylor, 1997). In the house fly, a second mutation (M918T) in the short cytoplasmic domain between transmembrane segments IIS4 and IIS5 is found together with the L1014F mutation and is correlated with enhanced pyrethroid resistance in strains carrying the super-kdr trait (Williamson et al., 1996). The functional significance of the L1014F and M918T mutations has been confirmed by inserting these mutations, singly or in combination, into the wildtype house fly Vssc1 sodium channel sequence, expressing both wildtype and mutated channels in Xenopus laevis oocytes, and documenting the reduced sensitivity of the mutated channels to pyr-ethroids (Smith et al., 1997; Lee et al., 1999c).

Although mutations at leucine residues aligning with Leu1014 of the house fly sodium channel are most com-monly associated with knockdown resistance, other sodium channel point mutations are also associated with kdr-like resistance in some species (Williamson et al., 1996; Guerrero et al., 1997; Park et al., 1997; Pittendrigh et al., 1997; Head et al., 1998; Schuler et al., 1998). Among these mutations, the V410M2 _{mutation found in} some H. virescens populations (Park et al., 1997) is of particular interest for three reasons. First, this mutation occurs in a position in transmembrane segment IS6 that is similar to the location of the L1014F mutation in transmembrane segment IIS6. Second, neurons from insects carrying this mutation have sodium channels with altered voltage dependence and pharmacological proper-ties (Lee et al., 1999a), but these altered properproper-ties have not been definitively correlated with the V410M mutation. Finally, this mutation occurs in a region of the sodium channel protein where other mutations are already known to alter both the pharmacology and func-tion of sodium channels. As shown in Fig. 1, Val410 of the house fly Vssc1 protein aligns with Ile433 of the rat skeletal muscle sodium channel (rSkM1) protein. Mutagenesis studies with rSkM1 have shown that the I433K, N434K and L437K mutations each render the rSkM1 channel completely insensitive to batrachotoxin (BTX) (Wang and Wang, 1998), whereas the N434A mutation confers partial resistance to BTX (Wang and Wang, 1998), produces depolarizing shifts in the voltage dependence of activation and steady-state inactivation and enhances slow inactivation (Wang and Wang, 1997). Photoaffinity labeling studies with a BTX derivative also

2 _{For clarity and consistency, all mutations in insect sodium channel}

genes are numbered throughout this report according to the amino-acid sequence numbering of the house fly Vssc1 sodium channel (GenBank accession number U38813).

Fig. 1. Alignment of the domain IS6 amino-acid sequences of the house fly (Vssc1) and rat skeletal muscle (rSkM1) sodium channel αsubunits, with identical amino-acid residues indicated in bold face, illustrating the relationship between the resistance-associated mutation in Vssc1 and mutations that alter the properties and BTX sensitivity of rSkM1.

implicate this region of domain IS6 as part of the BTX binding site (Trainer et al., 1996).

In this paper, we describe the introduction of the V410M mutation into the wildtype house fly Vssc1 sodium channel by site-directed mutagenesis, the expression of functional wildtype and mutated channels in Xenopus oocytes, and the biophysical properties and sensitivity to cismethrin and BTX of the expressed chan-nels. Our results document the reduced sensitivity to cis-methrin but not BTX of channels containing the V410M mutation.

2. Materials and methods

2.1. Site-directed mutagenesis and cDNA clone construction

The complete wildtype Vssc1 cDNA sequence, methods for preparing fly head first-strand cDNA, and methods for amplification by the polymerase chain reac-tion (PCR) of segments of the Vssc1 cDNA from this template are described elsewhere (Ingles et al., 1996). The Vssc1 cDNA was amplified as two contiguous frag-ments of 3.7 and 2.7 kb flanking a unique AatII restric-tion site as described previously (Smith et al., 1997). For the present study, the 3.7 kb fragment was mutated to introduce the resistance-associated V410M mutation (GTG to ATG) by using oligonucleotide-mediated mutagenesis (Altered Sites II in vitro mutagenesis sys-tem, Promega, Madison, WI). A full-length cDNA clone containing the V410M mutation was assembled as described previously (Smith et al., 1997) and its integrity was confirmed by DNA sequencing prior to its use as the template for cRNA synthesis. The D. melanogaster tipE cDNA, used as a template for in vitro transcription, was obtained by PCR as described previously (Smith et al., 1997).

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2.2. Expression

Oocytes were obtained surgically from female frogs (Nasco, Ft. Atkinson, WI) and defolliculated by incu-bation with Type 1A collagenase (Sigma, St. Louis, MO) followed by manual removal of remaining follicle cells. Stage IV–VI oocytes isolated by these methods were incubated in ND-96 medium (Goldin, 1992) sup-plemented with 1% sodium pyruvate, 1% (v/v) of peni-cillin (5000 units/ml)/streptomycin (5 mg/ml) solution (Sigma) and 5% horse serum (Sigma) for 24 h at 18°C prior to injection. The cRNA used in expression experi-ments was synthesized from linearized plasmid (Vssc1) or purified PCR fragment (tipE) templates using a com-mercial kit (mMessage mMachine, Ambion, Austin, TX). The integrity and approximate concentration of the RNAs obtained by these methods were determined by electrophoresis in formaldehyde agarose gels. Oocytes were injected with 25–50 nl of an aqueous solution of cRNA (1:1 molar ratio of Vssc1 and tipE cRNAs, |1 ng/nl) and incubated in supplemented ND-96 medium at 18°C for up to 24 days prior to electrophysi-ological analysis of sodium currents.

2.3. Electrophysiology

Electrophysiological recordings of sodium currents were obtained from oocytes in ND-96 at 16–20°C. Rec-ordings were performed in Plexiglas recording chambers (200µl bath volume) with a glass coverslip bottom bonded with cyanoacrylate glue. Membrane currents of oocytes were recorded using a two-microelectrode volt-age clamp with a virtual ground (TEV-200, Dagan Corp., Minneapolis, MN), filtered with a 2 kHz low pass (four-pole Bessel) filter, digitized at 10 kHz (inactivation experiments) or 20 kHz (all other experiments), and stored electronically (MacLab, AD Instruments, Milford, MA). Borosilicate glass recording electrodes (0.3– 2.0 MV) were filled with filtered 3 M KCl, coated with insulating resin, and shielded with grounded aluminum foil. Compensation circuitry (TEV-208, Dagan) was used to remove leakage current. Net sodium currents, with capacitive transients removed, were derived by sub-tracting traces from the same oocyte obtained in the pres-ence of 1µM tetrodotoxin (TTX; Sigma). Cismethrin (M. Elliott, Rothamsted Experimental Station, UK) and BTX (J. Daly, National Institute of Arthritis, Metab-olism, and Digestive Disease, Bethesda, MD) were pre-pared as stock solutions in dimethyl sulfoxide (DMSO) and diluted with ND-96 immediately before bath appli-cation. Oocytes were incubated with cismethrin or BTX in a non-perfused recording bath for 3 min prior to the collection of data. Each recording chamber was used only once to prevent cross-contamination. The final DMSO concentration in the bath did not exceed 1%, a concentration that had no effect on sodium currents.

Data analysis was performed in Axograph (Axon Instru-ments, Burlingame, CA). Midpoint potentials for acti-vation and steady-state inactiacti-vation (e.g., test potentials producing half-maximal activation or steady-state inactivation) were determined by least-squares fits of current–voltage data from individual experiments to the Boltzmann distribution (r.0.95). The unpaired Stud-ent’s t-test was used to compare midpoint potentials for activation and steady-state inactivation obtained with different Vssc1 channel variants.

3. Results

In this study, we obtained the functional expression of house fly sodium channels in Xenopus oocytes by the coinjection of cRNAs for wildtype or specifically mutated Vssc1 sodium channel α subunits and the D. melanogaster tipE protein, which is known to enhance the expression of Vssc1 sodium channels in this system (Smith et al., 1997). Expression of such channels (Vssc1/tipE channels) containing the V410M mutation resulted in sodium currents measured by two-electrode voltage clamp analysis under standard assay conditions (50 ms depolarizations from 2100 mV to 0 mV) that were indistinguishable in mean amplitude and fast inacti-vation kinetics from those measured for wildtype Vssc1/tipE channels under the same assay conditions (data not shown).

We assessed the voltage dependence of activation of Vssc1/tipE channels carrying the V410M mutation (V410M channels) by measuring the amplitude of the peak transient sodium currents obtained upon 50 ms depolarizations from a holding potential of2120 mV to test potentials ranging from290 to 90 mV in 5–10 mV increments. Fig. 2 shows a family of sodium current traces obtained under these conditions from a representa-tive oocyte expressing the V410M channel [Fig. 2(a)], plots of the current–voltage relationship for mean nor-malized peak transient currents obtained from multiple data sets [Fig. 2(b)], and a fit of mean conductance values derived from these data to the Boltzmann distri-bution [Fig. 2(c)]. The conductance values in Fig. 2(c) were calculated for each data set using the value for the sodium reversal potential determined in each experi-ment. Table 1 compares the midpoint potential for volt-age-dependent activation of the V410M channel, calcu-lated from individual fits of multiple data sets, with previously published values for the wildtype and L1014F variants of the Vssc1/tipE sodium channel (Lee et al., 1999c). The |9 mV depolarizing shift in the midpoint potential for the activation of the V410M channel com-pared with wildtype channel was statistically significant (P,0.0001, df=15). However, the midpoint potentials for the V410M and L1014F channel variants were not significantly different (P=0.0618, df=14).

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Fig. 2. Voltage dependence of activation of the V410M variant of the Vssc1/tipE sodium channel. (a) Family of sodium currents obtained from a single oocyte by 50 ms depolarizations from2120 mV to test potentials of290 to 90 mV. (b) Current–voltage relationship derived from multiple data set such as that shown in (a). (c) Conductance transformation of the current–voltage data shown in (b).

Table 1

Comparison of the voltage dependence of activation and steady-state inactivation of wildtype and specifically mutated house fly Vssc1 volt-age-sensitive sodium channels expressed in Xenopus oocytes

Vssc1 variant Midpoint potential (mV±standard deviation) (n)

Activation Steady-state inactivation

Wildtype 219.7±1.3 (8)a ₂34.5±3.8 (6)a

V410M 210.6±2.9 (9)b ₂29.5±1.6 (8)c

L1014F 213.8±3.6 (7)a ₂31.1±2.0 (7)a a_{Previously published data (Lee et al., 1999c) used in statistical}

comparisons.

b _{Calculated from data from multiple experiments such as that}

shown in Fig. 2(a).

c_{Calculated from data from multiple experiments such as that}

shown in Fig. 3(a).

We also examined the effects of the V410M mutation on the voltage dependence of steady-state inactivation of Vssc1/tipE sodium channels. The two-pulse protocol employed in these experiments began with a step from a holding potential of2100 mV to a conditioning poten-tial ranging from ₂90 mV to 30 mV (in 5–10 mV increments) for 160 ms, which was followed by a second pulse to ₂10 mV for 50 ms after a brief step (1 ms) to the holding potential. The short step to the holding potential between pulses was inserted to facilitate the subtraction of capacitive transients and did not affect inactivation. Fig. 3(a) shows a family of current traces obtained using this pulse protocol from a single oocyte expressing the V410M channel, and Fig. 3(b) shows a plot of mean normalized peak current against condition-ing prepulse potential derived from the multiple data sets such as that shown in Fig. 3(a). Table 1 compares the midpoint potential for steady-state activation of the V410M channel, calculated from individual fits of

mul-tiple data sets, with previously published values for the wildtype and L1014F variants of the Vssc1/tipE sodium channel (Lee et al., 1999c). The |5 mV depolarizing shift in the midpoint potential for steady-state inacti-vation of the V410M channel compared with wildtype channel was statistically significant (P=0.0054, df=12) but the midpoint potentials for the V410M and L1014F channel variants were not significantly different (P=0.1097, df=13).

We evaluated the effect of the V410M mutation on the sensitivity of Vssc1/tipE sodium channels to pyr-ethroids in assays comparing the effects of cismethrin on the wildtype and V410M channel variants. Fig. 4(a) shows sodium current traces obtained from an oocyte expressing the wildtype channel in the presence or absence of cismethrin (0.3–600µM). As documented in previous reports (Smith et al. 1997, 1998; Lee et al., 1999c), cismethrin produced both a prolonged sodium current (“late current”) during a 50 ms depolarization and a slowly decaying biphasic tail current following repolarization. The amplitudes of both the late current and the tail current were concentration-dependent. The decay of the falling phase of the tail current in the pres-ence of 30µM cismethrin was fitted to a single-exponential curve with a time constant of 465±262 ms (n=5). Fig. 4(b) shows traces obtained from an oocyte expressing the V410M channel in the presence or absence of the same concentrations of cismethrin. In contrast to wildtype channel, the amplitudes of the late and tail currents were markedly reduced relative to the amplitude of the peak current obtained prior to cis-methrin treatment. In addition, the tail current observed in assays with the V410M variant decayed _|10 times more rapidly (time constant=47.2±38 ms; n=4) than that obtained with wildtype channels.

We quantified the extent of modification of the wild-type and V410M sodium channel variants by different

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Fig. 3. Voltage dependence of steady-state inactivation of the V410M variant of the Vssc1/tipE sodium channel. (a) Family of sodium currents obtained from a single oocyte by 50 ms depolarizations from2100 mV to210 mV following 160 ms conditioning prepulses from2100 mV to test potentials of290 to 30 mV. (b) Steady-state inactivation curve derived from multiple data set such as that shown in (a).

Fig. 4. Effects of the V410M mutation on the response of the Vssc1/tipE sodium channel to cismethrin. Sodium currents were measured from oocytes expressing wildtype (a) or V410M (b) channels using the indicated pulse protocols before and after bath application of cismethrin a the concentrations shown. (c) Comparison of the normalized amplitudes of cismethrin-induced late currents from the wildtype and channels (n=2-4 independent experiments per data point). The late currents were measured at the end of a 50 ms depolarization.

cismethrin concentrations by determining the normalized amplitude of the cismethrin-induced late current (measured at the end of a 50 ms depolarization). Plots of normalized late current amplitudes against cismethrin concentration [Fig. 4(c)] identified a threshold for detect-able cismethrin modification of the wildtype channel of 0.3µM, whereas the threshold for detectable modifi-cation of the V410M channel was 10 times higher (3µM). In these experiments the responses to cismethrin were not saturated at the highest concentration attain-able, thus preventing the calculation of cismethrin

con-centrations causing half-maximal modification. As an alternative, we determined the concentrations of cis-methrin that produced the same normalized late current amplitude in both channels. As shown in Fig. 4(c), 600µM cismethrin was required to produce late currents from V410M that were of the same amplitude as currents produced by 30µM cismethrin from wildtype channels. By this criterion, the V410M mutation reduces the sensi-tivity of Vssc1/tipE sodium channels to cismethrin by approximately 20-fold.

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V410M sodium channels to BTX. Fig. 5 shows typical sodium current traces obtained from oocytes expressing wildtype Vssc1/tipE sodium channels upon 50 ms depol-arizations from 2100 mV to25 mV in the presence of 2µM BTX after the indicated number of 30 ms depolar-izing prepulses from2100 mV to25 mV at a pulse fre-quence of 25 Hz. The extent of sodium channel modifi-cation by BTX was strongly use-dependent. There was little or no detectable modified current after 10 prepul-ses, but further stimulation produced a sustained late cur-rent without inducing a tail curcur-rent following repolariz-ation. To compare the sensitivities of the wildtype and V410M chanel variants to BTX, we determined the con-centration-dependent modification of sodium currents measured after 1000 depolarizing 30 ms prepulses deliv-ered at a frequency of 25 Hz. Fig. 6(a) and (b) shows typical sodium currents obtained from oocytes express-ing either the wildtype or V410M channel, respectively, following exposure to BTX concentrations ranging from 0.08µM to 4.2µM. The responses of both channel vari-ants to BTX in these assays was very similar. Compari-son of the normalized amplitudes of the BTX-induced late currents obtained in such assays [Fig. 6(c)] showed that the BTX sensitivity of the V410M channel appeared to be slightly less than that of the wildtype channel, but the mean amplitudes of the normalized late currents obtained at each BTX concentration did not differ sig-nificantly between the two channel variants.

4. Discussion

Previous studies in this laboratory have employed site-directed mutagenesis and heterologous functional expression in Xenopus oocytes to document the reduced pyrethroid sensitivity of house fly Vssc1/tipE sodium

Fig. 5. Use-dependent action of BTX on the Vssc1/tipE sodium chan-nel. Sodium currents were measured by 50 ms depolarization from

2100 mV to25 mV in the presence of 2µM BTX with the indicated number of 30 ms depolarizing prepulses from2100 mV to25 mV.

channels containing the L1014F and M918T mutations that are associated with the kdr and super-kdr resistance traits in the house fly (Smith et al., 1997; Lee et al., 1999c). The present study extends this work by taking advantage of the house fly sodium channel expression system to assess the effects of the V410M mutation, which is associated by both genetic and physiological criteria with kdr-like resistance in some populations of H. virescens (Park et al., 1997; Lee et al., 1999a). The V410M mutation, located in transmembrane segment IS6 of the sodium channelαsubunit, identifies a domain of the sodium channel that has not previously been recognized as a determinant of pyrethroid sensitivity.

The principal finding of the present study is that the V410M mutation, when inserted into the wildtype Vssc1 sodium channel, altered the properties and reduced the cismethrin sensitivity of Vssc1/tipE sodium channels expressed in Xenopus oocytes in a manner virtually identical to the effects of the L1014F mutation described previously (Smith et al., 1997). Both mutations produced statistically significant depolarizing shifts in both the activation and the steady-state inactivation of Vssc1/tipE channels. However, the midpoint potentials for acti-vation and steady-state inactiacti-vation of the V410M and L1014F channel variants were not statistically dis-tinguishable from each other. Both mutations decreased the threshold sensitivity of expressed channels approxi-mately 10-fold and increased the concentration of cis-methrin required to achieve an equivalent extent of chan-nel modification at least 20-fold. Finally, both mutations altered the deactivation kinetics of cismethrin-modified channels, so that the decay of the tail current was accel-erated. The effects of the V410M mutation in these assays are sufficient to account for the degree of resist-ance observed in toxicity bioassays with H. virescens larvae carrying this mutation (Lee et al., 1999a).

The biophysical properties of the V410M variant of the house fly Vssc1/tipE sodium channel determined in the present study are in substantial agreement with the properties of H. virescens sodium channels assayed in neurons from insects known to carry this mutation (Lee et al., 1999a). The depolarizing shifts in activation and steady-state inactivation observed in our assays with expressed channels were slightly smaller in magnitude (_|9 mV versus _|13 mV for activation, _|5 mV versus |7 mV for steady-state inactivation) but otherwise simi-lar to the deposimi-larizing shifts observed for H. virescens sodium channels containing the mutation corresponding to V410M. However, all of the midpoint potentials in our assays were shifted 10–15 mV in the direction of depolarization compared with those measured in H. vire-scens neurons. This discrepancy may reflect the different properties of the house fly and H. virescens sodium channels, the impact of heterologous expression of insect sodium channels in a vertebrate cell of non-neuronal ori-gin, or the absence of an auxiliary subunit other than tipE

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Fig. 6. Effects of the V410M mutation on the response of the Vssc1/tipE sodium channel to BTX. Sodium currents were measured from oocytes expressing wildtype (a) or V410M (b) channels using the indicated pulse protocols following 1000 prepulses (30 ms from2100 mV to25 mV) in the presence or absence of 0.08–4.2µM BTX. (c) Comparison of the normalized amplitudes of BTX-induced late currents from the wildtype and V410M channels (n=3–6 independent experiments per data point). The late currents were measured at the end of 50 ms depolarization following 1000 prepulses.

that could modify the voltage dependence of expressed channels. The discrepancy in voltage dependence is not due, however, to the use of the D. melanogaster tipE protein in our expression assays, because coexpression of the cloned house fly ortholog of tipE (designated Vsscβ) with the Vssc1 α subunit gave channels with midpoint potentials statistically indistinguishable from those reported for Vssc1/tipE channels (Lee et al., 2000). The pharmacological properties of the V410M variant of the Vssc1 sodium channel in our assays were also in substantial agreement with the properties of sodium channels in neurons from resistant H. virescens (Lee et al., 1999a). We observed _|10- to _|20-fold resistance to cismethrin in oocytes expressing the V410M sodium channel variant depending on the criterion used for com-parison, whereas channels in neurons isolated from resistant H. virescens were |21-fold resistant to per-methrin, a pyrethroid with effects on D. melanogaster para/tipE sodium channels expressed in oocytes that are comparable to the effects of cismethrin in our assays (Warmke et al., 1997). However, the acceleration of tail current decay observed in our studies was apparently not found in assays comparing sodium channels in neurons from resistant and susceptible H. virescens (Lee et al., 1999a). The coupling of pyrethroid sensitivity and changes in tail current kinetics, which we also observed in assays with oocytes expressing Vssc1/tipE channels carrying the L1014F mutation (Smith et al., 1997), is not a feature unique to the Vssc1 channel. We have observed a similar relationship between changes in pyrethroid sen-sitivity and deactivation kinetics in assays of sodium cur-rents from Xenopus oocytes expressing rat TTX-resistant peripheral nerve sodium channels that contain mutations that alter pyrethroid sensitivity (Lee and Soderlund,

1999). However, it is possible that the coupling of changes in pyrethroid sensitivity and changes in deacti-vation kinetics in mutated channels is unique to channels expressed in oocytes.

The location of the V410M mutation in a domain of the sodium channel that is thought to form part of the BTX binding site led us to investigate effects of the V410M mutation on BTX sensitivity. The use-dependent modification of Vssc1/tipE sodium channels by BTX observed in our experiments (see Fig. 5) is typical of the actions of BTX on sodium channels in a variety of neu-ronal preparations and is consistent with the finding that BTX binds with highest affinity to the open state of the sodium channel (Brown, 1988). The effects of BTX on Vssc1 sodium channels were also qualitatively similar to the effects of this compound on rSkM1 sodium channels expressed in oocytes (Wang and Wang, 1998). The V410M mutation, unlike the substitution of lysine at the corresponding position of the rSkM1 sodium channel (Wang and Wang, 1998), did not significantly affect the sensitivity of Vssc1/tipE sodium channels to BTX. Sub-stitution of methionine for either valine or isoleucine is conservative in some sequence contexts, whereas substi-tution of lysine for either of these residues is a non-conservative substitution (Bordo and Argos, 1991). In the present study, the V410M mutation appears to be a conservative substitution with respect to the action of BTX but not pyrethroids on sodium channels.

The identification of mutations that modify pyrethroid sensitivity in both the IS6 (this study) and IIS6 (Smith et al., 1997) transmembrane segments of the sodium channel suggests that both of these sodium channel domains may participate in pyrethroid binding. To inter-pret the possible role of residues in the IS6 and IIS6

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helices as components of the pyrethroid binding site, it is first necessary to know the spatial relationship between these segments in the tertiary structure of the sodium channel. In the absence of crystallographic data defining the tertiary structure of the sodium channels, models of sodium channel structure must be inferred from the properties of the primary amino-acid sequence. These models (Guy and Seetharamulu, 1986; Sato and Matsumoto, 1992; Kallen et al., 1993; Fozzard and Hanck, 1996) propose a consensus transmembrane top-ology for each of the four repeated domains [Fig. 7(a)] and suggest that each domain forms a subunit-like struc-ture, analogous to a potassium channel subunit, which assemble into a radially symmetrical pseudotetramer sur-rounding the ion pore. Some models (Guy and Seethara-mulu, 1986; Sato and Matsumoto, 1992) place the S4 helices at the channel pore and the S6 helices on the periphery of the channel protein, whereas other models (Kallen et al., 1993; Fozzard and Hanck, 1996) place the S6 helices in close proximity to each other at the center of the channel [Fig. 7(b)]. The proposed central location of the S6 helices in the latter models is supported by studies of the structural determinants for the action of local anesthetics, which bind within the sodium channel pore and block sodium transport (Hille, 1992). Mutagenesis of residues within segments IS6 and IVS5 disrupt local anesthetic action (Ragsdale et al., 1994; Nau et al., 1999), thereby locating the S6 segments at or very near the pore.

Additional experimental evidence for the close physi-cal proximity of the IS6 and IVS6 helices, as shown in Fig. 7(b), is provided by the identification of residues in domains I and IV that determine the binding of BTX and brevetoxin to sodium channels. The importance of the IS6 helix in the binding and action of BTX is evident from photoaffinity labeling (Trainer et al., 1996) and site-directed mutagenesis (Wang and Wang, 1998) experiments (see Fig. 1). The involvement of the IVS6 helix in BTX binding was recently demonstrated in stud-ies of specifically mutated rat brain IIa and skeletal mus-cle sodium channels, which showed that amino-acid sub-stitutions in that segment known to affect local anesthetic binding also affected BTX binding (Linford et al., 1998; Wang and Wang, 1999). The latter result suggested that different faces of the same amino-acid residues may contribute to the receptor sites for these two agents, thereby mediating the well-characterized negative allosteric interaction between BTX and ligands that bind to the local anesthetic site (Linford et al., 1998; Wang and Wang, 1999). Brevetoxin is a lipophilic poly-ether toxin that binds to a site on the sodium channel that is physically distinct from and allosterically coupled to the BTX site (Trainer et al., 1993). Photoaffinity labe-ling studies with a brevetoxin derivative documented the labeling of sodium channel peptides derived from seg-ments IS6 and IVSS (Trainer et al. 1991, 1994). These

results are consistent with the hypothesis that the BTX and brevetoxin binding sites are located at the boundary of homology domains I and IV [Fig. 7(c)] and exert their effects on sodium channel gating and the binding of ligands to other sites by modifying conformational inter-actions between domains (Linford et al., 1998).

By analogy, our results documenting the effects of mutations in segments IS6 and IIS6 on the pyrethroid sensitivity of insect sodium channels suggest that pyr-ethroids may bind to sodium channels at a site located at the interface between homology domains I and II that is formed, at least in part, by Val410 in IS6 and Leu1014 in IIS6 [Fig. 7(c)]. Resistance-causing mutations at either of these residues alter both the binding of pyr-ethroids, inferred from the reduction of the threshold sensitivity to cismethrin, and the effects of bound pyr-ethroid on sodium channel gating, inferred from the acceleration of sodium tail current kinetics. The fact that mutations at Val410 or corresponding amino-acid resi-dues affect the sensitivity to both pyrethroids and BTX suggests that opposite faces of the Val410 sidechain may contribute to the pyrethroid and BTX binding sites. The close physical proximity of the pyrethroid and BTX binding sites predicted by this model is consistent with the allosteric enhancement of BTX binding by pyrethro-ids (Brown et al., 1988; Lombet et al., 1988; Rubin et al., 1993; Trainer et al., 1993). This allosteric mechanism is analogous to that proposed for the BTX and local anes-thetic sites mediated through amino-acid residues in IVS6 (Linford et al., 1998; Wang and Wang, 1999), except that the allosteric interaction between pyrethroid and BTX binding results in an increase rather than a decrease in the affinity of the BTX binding site. More-over, the additive effects of pyrethroids and brevetoxin as allosteric enhancers of BTX binding (Trainer et al., 1993) could be mediated by conformational changes in transmembrane segment IS6 caused by the binding of pyrethroids and brevetoxins that increase the affinity of the BTX site.

Although this model of pyrethroid binding identifies a plausible mechanism underlying the resistance-confer-ring mutations in IS6 and IIS6 and is consistent with other independent lines of experimental evidence, it does not specifically address the mechanisms by which mutations in other regions of the sodium channel cause pyrethroid resistance. The M918T mutation associated with super-kdr resistance in the house fly (Williamson et al., 1996) lies in the center of the IIS4–5 linker, which forms part of the cytoplasmic face of the channel [see Fig. 7(a)] and does not appear to lie close to the putative pyrethroid binding site at the domain I–II interface in the model shown in Fig. 7(c). Interestingly, a screen for pyrethroid resistance in strains of D. melanogaster hav-ing temperature-sensitive paralytic phenotypes that map to the para sodium channel identified two resistance-associated mutations [I253N and A1410V; Fig. 7(a)] in

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Fig. 7. Conceptual models of sodium channel structure. (a) Extended topological model of the sodium channelαsubunit showing the locations of mutations associated with pyrethroid resistance in H. virescens, M. domestica and D. melanogaster. (b) Proposed orientation of transmembrane helical segments around the ion pore (Fozzard and Hanck, 1996). (c) Enlarged view of the S5 and S6 segments from (b) showing the proposed locations of binding sites for local anesthetics (LA), BTX, brevetoxin (PbTx) and pyrethroids (Py) based on the results of mutagenesis and photoaf-finity labeling experiments.

the IS4–5 and IIIS4–5 linkers corresponding to the M918T mutation (Pittendrigh et al., 1997). The repeated occurrence of resistance-associated mutations in the S4– 5 segments of the sodium channel suggest that such mutations may affect pyrethroid sensitivity by a mech-anism involving the effects of these segments on sodium channel conformation or gating, rather than by a direct alteration of residues that participate in pyrethroid bind-ing. In contrast, pyrethroid resistance in two other mutant D. melanogaster strains from this study was

cor-related with mutations in the pore-forming segment between IIIS5 and IIIS6 [V1494A; Fig. 7(a)] and at the extracellular terminus of transmembrane segment IIIS6 [M1524I; Fig. 7(a)] (Pittendrigh et al., 1997). These mutations lie close to the ion pore and could therefore identify residues that either participate directly in pyr-ethroid binding or exert local conformational effects on a pyrethroid binding site located between domains I and II as shown in Fig. 7(c). Additional mutagenesis experi-ments are required to test rigorously the domain interface

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model of pyrethroid binding proposed in this study and to elucidate the mechanisms by which other resistance-conferring mutations alter the pyrethroid sensitivity of sodium channels.

Acknowledgements

We thank J. Daly and M. Elliott for their generous gifts of the samples of BTX and cismethrin used in this study, P. Adams and K. Nelson for technical assistance, and J. Bloomquist, M. Kirby and T. Smith for critical reviews of the manuscript. This work was supported in part by grant number 97-35302-4323 from the United States Department of Agriculture.

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ered at a frequency of 25 Hz. Fig. 6(a) and (b) shows typical sodium currents obtained from oocytes express-ing either the wildtype or V410M channel, respectively, following exposure to BTX concentrations ranging from 0.08µM to 4.2µM. The responses of both channel vari-ants to BTX in these assays was very similar. Compari-son of the normalized amplitudes of the BTX-induced late currents obtained in such assays [Fig. 6(c)] showed that the BTX sensitivity of the V410M channel appeared to be slightly less than that of the wildtype channel, but the mean amplitudes of the normalized late currents obtained at each BTX concentration did not differ sig-nificantly between the two channel variants.

4. Discussion

Fig. 5. Use-dependent action of BTX on the Vssc1/tipE sodium chan-nel. Sodium currents were measured by 50 ms depolarization from 2100 mV to25 mV in the presence of 2µM BTX with the indicated number of 30 ms depolarizing prepulses from2100 mV to25 mV.

sodium channel, altered the properties and reduced the cismethrin sensitivity of Vssc1/tipE sodium channels expressed in Xenopus oocytes in a manner virtually identical to the effects of the L1014F mutation described previously (Smith et al., 1997). Both mutations produced statistically significant depolarizing shifts in both the activation and the steady-state inactivation of Vssc1/tipE channels. However, the midpoint potentials for acti-vation and steady-state inactiacti-vation of the V410M and L1014F channel variants were not statistically dis-tinguishable from each other. Both mutations decreased the threshold sensitivity of expressed channels approxi-mately 10-fold and increased the concentration of cis-methrin required to achieve an equivalent extent of chan-nel modification at least 20-fold. Finally, both mutations altered the deactivation kinetics of cismethrin-modified channels, so that the decay of the tail current was accel-erated. The effects of the V410M mutation in these assays are sufficient to account for the degree of resist-ance observed in toxicity bioassays with H. virescens larvae carrying this mutation (Lee et al., 1999a).

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Fig. 6. Effects of the V410M mutation on the response of the Vssc1/tipE sodium channel to BTX. Sodium currents were measured from oocytes expressing wildtype (a) or V410M (b) channels using the indicated pulse protocols following 1000 prepulses (30 ms from2100 mV to25 mV) in the presence or absence of 0.08–4.2µM BTX. (c) Comparison of the normalized amplitudes of BTX-induced late currents from the wildtype and V410M channels (n=3–6 independent experiments per data point). The late currents were measured at the end of 50 ms depolarization following 1000 prepulses.

1999). However, it is possible that the coupling of changes in pyrethroid sensitivity and changes in deacti-vation kinetics in mutated channels is unique to channels expressed in oocytes.

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mulu, 1986; Sato and Matsumoto, 1992) place the S4 helices at the channel pore and the S6 helices on the periphery of the channel protein, whereas other models (Kallen et al., 1993; Fozzard and Hanck, 1996) place the S6 helices in close proximity to each other at the center of the channel [Fig. 7(b)]. The proposed central location of the S6 helices in the latter models is supported by studies of the structural determinants for the action of local anesthetics, which bind within the sodium channel pore and block sodium transport (Hille, 1992). Mutagenesis of residues within segments IS6 and IVS5 disrupt local anesthetic action (Ragsdale et al., 1994; Nau et al., 1999), thereby locating the S6 segments at or very near the pore.

sensitivity to cismethrin, and the effects of bound pyr-ethroid on sodium channel gating, inferred from the acceleration of sodium tail current kinetics. The fact that mutations at Val410 or corresponding amino-acid resi-dues affect the sensitivity to both pyrethroids and BTX suggests that opposite faces of the Val410 sidechain may contribute to the pyrethroid and BTX binding sites. The close physical proximity of the pyrethroid and BTX binding sites predicted by this model is consistent with the allosteric enhancement of BTX binding by pyrethro-ids (Brown et al., 1988; Lombet et al., 1988; Rubin et al., 1993; Trainer et al., 1993). This allosteric mechanism is analogous to that proposed for the BTX and local anes-thetic sites mediated through amino-acid residues in IVS6 (Linford et al., 1998; Wang and Wang, 1999), except that the allosteric interaction between pyrethroid and BTX binding results in an increase rather than a decrease in the affinity of the BTX binding site. More-over, the additive effects of pyrethroids and brevetoxin as allosteric enhancers of BTX binding (Trainer et al., 1993) could be mediated by conformational changes in transmembrane segment IS6 caused by the binding of pyrethroids and brevetoxins that increase the affinity of the BTX site.

(4)

(5)

References

of the voltage-sensitive sodium channel. Int. Rev. Neurobiol. 29, 77–116.

voltage-dependent sodium channels: comparison of brain II and cardiac isoforms. Physiol. Rev. 76, 887–926.

Goldin, A.L., 1992. Maintenance of Xenopus laevis and oocyte injec-tion. Meth. Enzymol. 207, 266–297.

Hille, B., 1992. Ionic Channels of Excitable Membranes. Sinauer, Sun-derland, MA.

expression of voltage-dependent sodium channels. Mol. Neurobiol. 7, 383–428.

Loughney, K., Kreber, R., Ganetzky, B., 1989. Molecular analysis of the para locus, a sodium channel gene in Drosophila. Cell 58, 1143–1154.

Ragsdale, D.S., McPhee, J.C., Scheuer, T., Catterall, W.A., 1994. Mol-ecular determinants of state-dependent block of Na+channels by local anesthetics. Science 265, 1724–1728.

Rubin, J.G., Payne, G.T., Soderlund, D.M., 1993. Structure–activity relationships for pyrethroids and DDT analogs as modifiers of [3_{H]batrachotoxinin A 20-}α_{-benzoate binding to mouse brain}

sodium channels. Pestic. Biochem. Physiol. 45, 130–140. Sato, C., Matsumoto, G., 1992. Proposed tertiary structure of the

sodium channel. Biochem. Biophys. Res. Commun. 186, 1158– 1167.

Schuler, T.H., Martinez-Torres, D., Thompson, A.J., Denholm, I., Devonshire, A.L., Duce, I.R., Williamson, M.S., 1998.

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1997. The L1014F point mutation in the house fly Vssc1 sodium channel confers knockdown resistance to pyrethroids. Insect Biochem. Mol. Biol. 27, 807–812.

Soderlund, D.M., 1997. Molecular mechanisms of insecticide resist-ance. In: Sjut, V. (Ed.), Molecular Mechanisms of Resistance to Agrochemicals. Springer, Berlin, pp. 21–56.

Neuro-toxin binding and allosteric modulation at receptor sites 2 and 5 on purified and reconstituted rat brain sodium channels. J. Biol. Chem. 268, 17114–17119.

Trainer, V.L., Baden, D.G., Catterall, W.A., 1994. Identification of the peptide components of the brevetoxin receptor site of rat brain sodium channels. J. Biol. Chem. 269, 19904–19909.

Wang, S.-Y., Wang, G.K., 1997. A mutation in I-S6 alters slow inacti-vation of sodium channels. Biophys. J. 72, 1633–1640.

Wang, S.Y., Wang, G.K., 1998. Point mutations in segment I-S6 render voltage-gated Na+channels resistant to batrachotoxin. Proc. Natl. Acad. Sci. USA 95, 2653–2658.

Wang, S.-Y., Wang, G.K., 1999. Batrachotoxin-resistant Na+channels derived from point mutations in transmembrane segment D4-S6. Biophys. J. 76, 3141–3149.

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