U.S. Government work not protected by U.S. copyright Mol Pharmacol 67:513–522, 2005 Printed in U.S.A. Identification of Amino Acid Residues in the Insect SodiumChannel Critical for Pyrethroid Binding Jianguo Tan, Zhiqi Liu, Ruiwu Wang,1 Zachary Y. Huang, Andrew C. Chen,Michael Gurevitz, and Ke Dong Departments of Entomology (J.T., Z.L., R.W., Z.Y.H., K.D.), Ecology, Evolutionary Biology, and Behavior (Z.Y.H.) andNeuroscience Programs (K.D.), Michigan State University, East Lansing, Michigan; United States Department of Agriculture,Agricultural Research Service, Knipling-Bushland U.S. Livestock Insects Research Laboratory, Kerrville, Texas (A.C.C.); andDepartment of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Israel (M.G.) Received August 17, 2004; accepted November 2, 2004 ABSTRACT
The voltage-gated sodium channel is the primary target site of
sensitivity to deltamethrin only by 3- to 10-fold, indicating that pyrethroids, which constitute a major class of insecticides used an aromatic residue at this position is critical for the interaction worldwide. Pyrethroids prolong the opening of sodium chan- of pyrethroids with sodium channels. The F1519I mutation, nels by inhibiting deactivation and inactivation. Despite numer- however, did not alter the action of two other classes of sodium ous attempts to characterize pyrethroid binding to sodium channel toxins, batrachotoxin (a site 2 toxin) and Lqh␣-IT (a site channels in the past several decades, the molecular determi- 3 toxin). Schild analysis using competitive interaction of pyre- nants of the pyrethroid binding site on the sodium channel throid-stereospecific isomers demonstrated that the F1519W remain elusive. Here, we show that an F-to-I substitution at mutation and a previously known pyrethroid-resistance muta- 1519 (F1519I) in segment 6 of domain III (IIIS6) abolished the tion, L993F in IIS6, reduced the binding affinity of 1S-cis- sensitivity of the cockroach sodium channel expressed in Xe- permethrin, an inactive isomer that shares the same binding nopus laevis oocytes to all eight structurally diverse pyrethroids site with the active isomer 1R-cis-permethrin. Our results pro- examined, including permethrin and deltamethrin. In contrast, vide the first direct proof that Leu993 and Phe1519 are part of substitution by tyrosine or tryptophan reduced the channel the pyrethroid receptor site on an insect sodium channel.
Voltage-gated sodium channels are essential for the initi- labeling and site-directed mutagenesis approaches have been ation and propagation of action potential in the nervous instrumental in elucidating molecular determinants of these system and other excitable cells (Catterall, 2000). Because of receptor sites (Cestele and Catterall, 2000; Blumenthal and their fundamental role in membrane excitability, sodium Seibert, 2003). Because of the unique pharmacological effects channels are an effective target site for a variety of neuro- of various neurotoxins on channel functional properties, toxins produced by plants and animals for their defense or studies of toxin binding sites have played an important role preying strategies. These sodium channel neurotoxins alter in the understanding of sodium channel functions and mo- various channel properties, including ion conductance, ion lecular bases of neurotoxicity.
selectivity, activation, or inactivation. At least six distinct Pyrethroid insecticides are among the earliest neurotox- receptor sites are recognized (Gordon, 1997; Zlotkin 1999; ins that were identified to act on sodium channels (Nara- Catterall et al., 2003; Wang and Wang, 2003). Photoaffinity hashi, 2000). They are synthetic analogs of the naturallyoccurring pyrethrum from the flower extracts of Chrysan-themum species. With a few exceptions of more recently The study was supported by National Institutes of Health grant GM057440 developed compounds, pyrethroids are typically esters of (to K.D.), Generating Research and Extension to Meet Economic and Environ- chrysanthemic acid (Elliott, 1977; also see pyrethroid mental Needs grant GR99-037 from Michigan State University (to K.D. andZ.Y.H.), and Binational Agricultural Research and Development grant IS- chemical structures in Fig. 1). The commercial develop- 3480-03 (to K.D. and M.G.).
ment of pyrethroids is one of the major success stories in J.T. and Z.L. contributed equally to this project.
1 the use of natural products as a source of leads for the Current address: Departments of Physiology and Biophysics and Biochem- istry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada.
production of novel insecticidal compounds. Because of the Article, publication date, and citation information can be found at relatively low mammalian toxicity and favorable environ- mental properties, pyrethroids represent a major class of ABBREVIATIONS: BTX, batrachotoxin; kdr, knockdown resistance; G, sodium channel conductance.

Tan et al.
insecticides used to control many agriculturally and med- hydrophobicity of pyrethroids, resulting in extremely high ically important arthropod pests.
nonspecific binding (Rossignol, 1988; Pauron et al., 1989; Pyrethroids are classified into type I and type II, based on Dong, 1993).
their chemical structure: type I pyrethroids lack a cyano Due to intensive use of pyrethroids in arthropod control, group at the phenylbenzyl or other alcohols, whereas type II many arthropod populations have developed resistance to pyrethroids contain an ␣-cyano-3-phenylbenzyl alcohol. De- these compounds. One major mechanism of pyrethroid resis- spite differences in the chemical structure, poisoning syn- tance, known as knockdown resistance (kdr), was first dis- dromes, and their differential effects on the nervous system, covered in house flies and subsequently in many other insect both type I and II pyrethroids prolong sodium channel open- and arachnid species (Soderlund and Bloomquist, 1990). Ex- ing by inhibiting inactivation and deactivation, resulting in a tensive research in the past decade convincingly showed that slowly decaying tail current associated with repolarization kdr and kdr-like mechanisms are caused by mutations in (Narahashi, 2000). Pyrethroids have one to three chiral cen- sodium channels (Dong, 2002, and references therein; Soder- ters, which may be located at C1 and C3 of the cyclopropane lund and Knipple, 2003). Like mammalian sodium channel ring and at the ␣C atom of the alcohol moiety. Type I pyre- ␣-subunits, the primary structure of insect sodium channel throids permethrin and tetramethrin, for example, have four proteins contains four homologous domains (I–IV), each con- stereospecific isomers: 1R-cis-, 1R-trans-, 1S-cis-, and 1S- taining six transmembrane segments (S1–S6) (Loughney et trans-. The stereoisomerism of pyrethroids is critically im- al., 1989). To date, more than half a dozen kdr mutations portant for pyrethroid action. 1R-cis- and 1R-trans-isomers have been demonstrated to reduce channel sensitivity to are active, whereas the other two are not. Furthermore, the pyrethroids in the Xenopus laevis oocyte expression system.
inactive 1S-cis-tetramethrin competes with the active 1R-cis- An F-to-I kdr mutation in domain III segment 6 (IIIS6), at tetramethrin for the same binding site and antagonizes the the position corresponding to Phe1519 in the cockroach so- action of the active isomer (Lund and Narahashi, 1982).
dium channel, was previously identified in the sodium chan- The pyrethroid binding site on the sodium channel has not nel of pyrethroid-resistant southern cattle tick (Boophilus been defined at the molecular level and remains a major microplus) (He et al., 1999). Substitution of F with I in a unresolved issue in sodium channel pharmacology. Substan- recombinant rat Na 1.4 sodium channel reduced the channel tial evidence from electrophysiological and pharmacological sensitivity to a pyrethroid insecticide deltamethrin (Wang et studies indicates that the pyrethroid receptor site is distinct from, yet allosterically coupled with, several other receptor In this study, we assessed the role of Phe1519 in pyre- sites, such as site 2 to which batrachotoxin (BTX) binds throid binding and action on an insect sodium channel. Our (Catterall, 1992; Gordon, 1997). Specific binding of radiola- results show that F1519I abolished the channel sensitivity to beled pyrethroids was detected in rat brain membrane prep- pyrethroids and that an aromatic residue (Phe, Trp, or Tyr) arations (Trainer et al., 1997). However, attempts to charac- at position 1519 is essential for the action of pyrethroids. By terize specific binding of pyrethroids to insect nerve using the competitive binding of active and inactive pyre- membrane preparations have failed because of the extreme throid stereospecific isomers to the sodium channel, we dem- Fig. 1. Tail currents induced by bioallethrin, biores-
methrin, tetramethrin, permethrin, NAK5710, fen-
fluthrin, cypermethrin, and deltamethrin in Bg-
Na 1-1 wild-type channel (A–H). No tail current
was induced in the F1519I mutant channel, even at100 ␮M deltamethrin (I). The chemical structure ofthe pyrethroids tested is shown in each inset. Thetail current was elicited by 100-pulse of a 67-Hztrain of 5-ms depolarization from ⫺120 to 0 mV.
The tail current decay was best fitted with oneexponential with the time constant of 268.3 ⫾ 85.4ms for 1R-cis-permethrin, and two exponentialswith time constants, ␶ ⫽ 1.3 ⫾ 0.5 s and ␶ ⫽ 0.3 ⫾ 0.07 s, for deltamethrin.
Molecular Determinants of the Pyrethroid Receptor Site
onstrated that F1519W reduced the pyrethroid binding to the of the prepulse potential. The data were fitted with a two-state cockroach sodium channel. We also show that a kdr mutation Boltzmann equation of the form I/I ⫽ [1 ⫹ (exp(V V )/k)]⫺1, in in IIS6 found in many insect sodium channels (corresponding is the maximal current evoked, V is the potential of the to the L993F mutation in the cockroach sodium channel) also voltage pulse, V is the half-maximal voltage for inactivation, and k reduced pyrethroid binding. Together, these results for the is the slope factor.
first time define specific amino acid residues involved in the To determine recovery from fast inactivation, sodium channels pyrethroid receptor site on an insect sodium channel.
were inactivated by a 200-ms depolarizing pulse to ⫺10 mV and thenrepolarized to ⫺120 mV for an interval of variable durations followedby a 20-ms test pulse to ⫺10 mV. The peak current during the test Materials and Methods
pulse was divided by the peak current during the inactivating pulseand plotted as a function of duration time between the two pulses.
Site-Directed Mutagenesis. A cockroach sodium channel vari-
To determine the development of fast inactivation, prepulse po- ant, BgNa 1-1 (formerly KD1), was subjected to site-directed mu- tentials ranging from ⫺80 to ⫺20 mV in 10-mV increments of vary- tagenesis to generate recombinant constructs containing the ing durations were applied from the holding potential of ⫺120 mV Phe15193Ile1519, 3Ala1519, 3Arg1519, 3Trp1519, or 3Tyr1519 followed by a test pulse at ⫺10 mV for 20 ms to determine the mutation. Briefly, a 1.4-kilobase Eco47III fragment containingPhe1519 was excised from BgNa 1-1 and cloned into the pAlter 1 fraction of current inactivated during the prepulse.
vector of the Altered Sites II in vitro mutagenesis system (Promega, To determine the steady-state slow inactivation, oocytes were held Madison, WI). The 1.4-kilobase mutated Eco47III fragment carrying at prepulse potentials ranging from ⫺120 to ⫹10 mV in 10-mV I1519, A1519, R1519, W1519, or Y1519 was cloned back into Bg- increments for 50 s. A 100-ms recovery pulse to ⫺120 mV and a Na 1-1 to generate mutant channels F1519I, F1519A, F1519R, 20-ms test pulse to ⫺10 mV were given before returning to the F1519W, and F1519Y.
holding potential of ⫺120 mV. The peak current amplitude during Expression of BgNa 1-1 Sodium Channels in X. laevis Oo-
the test depolarization was normalized to the maximum current cytes. Oocyte preparation and cRNA injection was performed as
amplitude and plotted as a function of the prepulse potential. The described previously (Tan et al., 2002b). For robust expression of the data were fitted with a two-state Boltzmann equation of the form BgNa 1-1 channel, BgNa 1-1 cRNA was coinjected into oocytes with ⫽ [1 ⫹ (exp(V V )/k)]⫺1, in which I Drosophila melanogaster tipE cRNA (2:1 ratio), which enhances the current evoked, V is the potential of the voltage pulse, V expression of insect sodium channels in oocytes (Feng et al., 1995; half-maximal voltage for inactivation, and k is the slope factor.
Warmke et al., 1997).
Measurement of Tail Currents Induced by Pyrethroids. The
Electrophysiological Recording and Analysis. Sodium cur-
method for application of pyrethroids in the recording system was rents were recorded using standard two-electrode voltage clamping.
identical to that described by Tan et al. (2002a). A disposable perfu- The borosilicate glass electrodes were filled with filtered 3 M KCl in sion system developed by Tatebayashi and Narahashi (1994) was 0.5% agarose and had a resistance of 0.5 to 1.0 M⍀. The recording used, which contained a petri dish placed on an adjustable support solution was ND-96, consisting of 96 mM NaCl, 2.0 mM KCl, 1.0 mM stand, a recording chamber built with glue, and Tygon tubing con- MgCl , 1.8 mM CaCl , and 10 mM HEPES, pH adjusted to 7.5 with necting the Petri dish and the recoding chamber. The solution was NaOH. Stock solutions of BTX (1 mM) and pyrethroids (100 mM) delivered by hydrostatic force by adjusting the level of the petri dish were dissolved in dimethyl sulfoxide. BTX and pyrethroids were relative to the recording chamber. The pyrethroid-induced tail cur- generous gifts from John Daly (National Institutes of Health, Be-thesda, MD), and Klaus Naumann and Ralf Nauen (Bayer Crop- rent was recorded during a 100-pulse train of 5-ms depolarization Science, Research Triangle Park, NC), respectively. Stock solution of from ⫺120 to 0 mV with a 5-ms interpulse interval (Vais et al., 2000).
␣-scorpion toxin Lqh␣IT (100 ␮M) was dissolved in distilled water The percentage of channels modified by pyrethroids was calculated containing 10% bovine serum albumin to prevent adherence of toxin using the equation M ⫽ {[I /(E E )]/[I /(E E )]} ⫻ 100 to the vials. Sodium currents were measured using the oocyte clamp (Tatebayashi and Narahashi, 1994), where I is the maximal tail instrument OC725C (Warner Instrument, Hamden, CT), Digidata current amplitude, E is the potential to which the membrane is 1200A, and pCLAMP 6 software interface (Axon Instruments Inc., repolarized, E is the reversal potential for sodium current deter- Foster City, CA). All experiments were performed at room tempera- mined from the current-voltage curve, I is the amplitude of the ture (20–22°C). Capacitive transient and linear leak currents were peak current during depolarization before pyrethroid exposure, and corrected using P/N subtraction or by subtraction of records obtained E is the potential of step depolarization. The concentration–re- in the presence of 20 nM tetrodotoxin, which completely blocks the sponse data were fitted to the Hill equation: M M /{1 ⫹ (K / BgNa 1-1 sodium channel (Tan et al., 2002a). The maximal peak [P])nH}, where [P] represents the concentration of pyrethroid and Kd sodium current was limited to ⬍2.0 ␮A to achieve better voltage represents the concentration of pyrethroid that produced the half- control by adjusting the amount of cRNA and the incubation time maximal effect, n represents the Hill coefficient, and the M after injection. The effects of pyrethroids, BTX, and Lqh␣IT were the maximal percentage of sodium channels modified.
measured 10 min after toxin application.
Schild Analysis. K values were determined from the dose-re-
The voltage dependence of sodium channel conductance (G) was sponse curves of the active 1R-cis-isomer on wild-type and mutant calculated by measuring the peak current at test potentials ranging sodium channels by measuring the amplitude of 1R-cis-isomer-in- from ⫺80 to ⫹65 mV in 5-mV increments and divided by (V V duced tail current and calculating percentage of channel modifica- where V is the test potential and V is the reversal potential for tion as described above. A series of K values (denoted as K ⬘) of the sodium ion. Peak conductance values were normalized to the maxi- active 1R-cis-isomer were determined in the presence of increasing mal peak conductance (G ) and fitted with a two-state Boltzmann equation of the form G/G ⫽ [1 ⫹ exp(V V )/k]⫺1, in which V is concentrations of the inactive 1S-cis-isomer. Schild analysis was the potential of the voltage pulse, V is the half-maximal voltage for used to determine the affinity of the inactive isomer, calculated from activation, and k is the slope factor.
the equation log(dose ratio ⫺ 1) ⫽ logK ⫺ log[B], where dose ratio ⫽ The voltage dependence of fast inactivation was determined using K ⬘/K , [B] is the molar concentration of the inactive 1S-cis-isomer, 200-ms inactivating prepulses from a holding potential of ⫺120 to 40 is the dissociation constant of the inactive 1S-cis-isomer.
mV in 5-mV increments, followed by test pulses to ⫺10 mV for 20 ms.
⫺Log(dose ratio ⫺ 1) was plotted as a function of ⫺log[B]. The data The peak current amplitude during the test depolarization was nor- were fitted with a linear regression, generating the Schild plot slope malized to the maximum current amplitude and plotted as a function and the x-intercept, pA , which equals ⫺logK .
Tan et al.
mV (Fig. 2B; Table 1). Although the voltage of half steady-state inactivation was not affected by F1519I (Fig. 2C; Table Tail Currents Induced by Type I and Type II Pyre-
1), the voltage dependence of inactivation curve showed in- throids. The amplitude and decay kinetics of the tail current
complete inactivation at positive potentials for the mutant are commonly used to quantify the effects of pyrethroids channel, consistent with the detection of the sustained cur- (Tatebayashi and Narahashi, 1994; Vais et al., 2000). For rent shown in Fig. 2A.
type I pyrethroids, such as bioallethrin, a single, long (50-ms) F1519I Does Not Alter the Action of BTX or LqhIT
depolarization produced a detectable tail current from wild- on Sodium Channel Function. Substantial evidence indi-
type BgNa 1-1 channel. However, no tail current was elicited cates that amino acid residues in the middle portion of mul- by a type II pyrethroid, deltamethrin, using the same record- tiple S6 segments are critical components of the BTX recep- ing protocol. Detection of deltamethrin-induced tail currents tor site (Blumenthal and Seibert, 2003; Wang and Wang, requires a 100-pulse train of 5-ms depolarization from ⫺120 2003, and references therein). Interestingly, Phe1519 is only to 0 mV with a 5-ms interpulse interval (Vais et al., 2000; one amino acid residue apart from Ser1276 in Na 1.4, which Tan et al., 2002a). For direct comparison, we used the latter is part of the BTX receptor site (Wang et al., 2000). BTX protocol in this study to elicit tail currents by both type I and causes persistent activation of sodium channels at the rest- type II pyrethroids (Fig. 1). Tail currents with amplitudes ing membrane potential by blocking sodium channel inacti- in the microampere range were induced in the wild-type vation and shifting the voltage dependence of channel acti- BgNa 1-1 channel by 1 ␮M deltamethrin or 3 ␮M of the other vation to more negative membrane potentials (Catterall, six pyrethroids. The type II pyrethroids cypermethrin and 1988; Hille, 1992). Previously, Wang et al. (2001) reported deltamethrin induced tail currents decay rather slowly with that the rat Na 1.4 mutant channel carrying F1278I (equiv- a biphasic decay (␶ ⫽ 1.3 ⫾ 0.5 s and ␶ ⫽ 0.3 ⫾ 0.07 s for alent to F1519I) remained sensitive to BTX. To evaluate deltamethrin), whereas most type I pyrethroid-induced tail whether the F1519I mutation alters the action of BTX on an currents returned to the baseline within 1 s, exhibiting a insect sodium channel, we examined the BTX effects on both monophasic decay (␶ ⫽ 268.3 ⫾ 85.4 ms for 1R-cis-per- wild-type and F1519I channels. At 100 nM, BTX inhibited methrin). These results are consistent with earlier findings the inactivation and reduced the amplitude of the peak cur- that both type I and type II pyrethroids preferably bind to rent of both wild-type and mutant channels, as indicated by open sodium channels, although type I pyrethroids can also the sodium current recording traces (Fig. 3A). Furthermore, bind to closed sodium channels (Vais et al., 2000, 2003).
two voltage-dependent components were evident from the F1519I Abolishes the Sensitivity of the Cockroach
current-voltage relationship (Fig. 3B), with one similar to Sodium Channel to Eight Structurally Diverse Pyre-
that of the unmodified channel and the other exhibiting a throids. To date, more than half a dozen kdr mutations have
40-mV hyperpolarizing shift, which represents the voltage been demonstrated to reduce the sensitivity of insect sodium dependence of the BTX-modified channels. These effects were channels to pyrethroids in X. laevis oocytes. The Phe-to-Ile very similar to those observed on rat sodium channels (Cat- mutation was identified in pyrethroid-resistant cattle ticks terall, 1988). There was no difference in the effects of BTX on (He et al., 1999). However, the effect of this mutation on the wild-type and mutant channels (Fig. 3, A–C), suggesting that sensitivity of insect sodium channels to pyrethroids has not the F1519I mutation did not alter the action of BTX on the been examined. Here, we examined the sensitivity of wild- insect sodium channel.
type BgNa 1-1 and the F1519I mutant channel to the eight Previously, positive allosteric interactions between ␣-scor- pyrethroids. We found that in contrast to wild-type BgNa 1-1 pion toxins and pyrethroids have been reported (Trainer et channel, no tail current was detected in oocytes expressing al., 1997; Vais et al., 2000; Gilles et al., 2003). Furthermore, the F1519I channel when exposed to any of these eight py- a kdr mutation in IS6 enhanced the sensitivity of tobacco rethroids, even at the highest concentrations used (shown in budworm sodium channels to Lqh␣IT, an ␣-scorpion toxin Fig. 1I for deltamethrin). These results indicate that the acting on site 3 (Lee et al., 1999). Lqh␣IT shifts the voltage F1519I mutant channel is completely insensitive to the eight dependence of activation of sodium channels in the depolar- structurally diverse pyrethroids and that the Phe1519 resi- izing direction in house fly neurons (Lee et al., 2000). We due is critical for pyrethroid action.
examined the effect of F1519I on the action of Lqh␣IT. At 10 The F1519I Mutation Disrupts Fast Inactivation and
nM, Lqh␣IT nearly abolished channel inactivation during a Alters the Voltage Dependence of Activation. To exam-
20-ms depolarization to ⫺10 mV after 10-min preincubation ine whether the F1519I mutation alters the channel gating with the toxin (Fig. 4, A and C). This toxin also increased the properties, sodium current was recorded from a 20-ms depo- amplitude of peak current by 2-fold (Fig. 4A). These effects larization to ⫺10 mV from the holding potential of ⫺120 mV are typical of site 3 sodium channel toxins (Lee et al., 1999; for both the wild-type and mutant channels. The F1519I Lee et al., 2000; Vais et al., 2000). Lqh␣IT produced a similar mutation did not alter recovery from fast inactivation, closed degree of modification in the F1519I mutant channel (Fig.
state inactivation, or voltage dependence of slow inactivation 4A, right), indicating the F1519I did not alter the channel (Fig. 2, D–F). However, although the wild-type sodium chan- sensitivity to Lqh␣IT. Furthermore, consistent with what nel was completely inactivated at the end of the depolarizing was observed in house fly neurons after application of pulse, the F1519I mutant channel exhibited a noticeable Lqh␣IT (Lee et al., 2000), Lqh␣IT shifted the voltage depen- sustained current (Fig. 2A), suggesting that the F1519I dence of activation by 10 mV in the depolarizing direction for change altered fast inactivation. In addition, the F1519I both wild-type and mutant channels (Fig. 4B). Lqh␣IT also channel activated more slowly than the wild-type channel increased the amplitudes of deltamethrin- and bioallethrin- (Fig. 2A). The F1519I mutation also shifted the voltage de- induced tail currents in the wild-type channel by 5-fold (Fig.
pendence of activation in the depolarizing direction by ca. 8 4, D and E). This enhancement likely results from an increas-

Molecular Determinants of the Pyrethroid Receptor Site
ing in the availability of open channels as the result of elim- Lqh␣IT-mediated enhancement, no tail current was detected inating channel inactivation and increasing the peak current in the F1519I channel. Thus, F1519I seems to be unique (Vais et al., 2000). However, even in the presence of the among all examined kdr mutations in that it alone is suffi- Fig. 2. The F1519I mutation affects sodium channel activation and inactivation. A, sodium current measured from a 20-ms test pulse at ⫺10 mV testing
potential from a holding potential of ⫺120 mV in BgNa 1-1 wild-type and F1519I mutant channels. B, voltage dependence of activation. The average of the
half-maximal voltage for activation (V ) and slope factor (k) for BgNa 1-1 wild-type and mutant channels are shown in Table 1. The normalized peak conductance was plotted against the potential of test pulses. C, recovery from fast inactivation for wild-type and F1519I mutant channels. The recovery ratefrom fast inactivation was measured using a two-pulse protocol, in which channels were fast inactivated by a 200-ms voltage pulse of ⫺10 mV, and then theywere allowed to recover at ⫺120 mV for an increasing time, and finally a 20 ms of ⫺10-mV test pulse was applied to test for the fraction of the channelsrecovered. Peak currents obtained during the test pulse were normalized to the peak current obtained during the inactivated pulse. The normalized peakcurrents were plotted versus recovery time. D, voltage dependence of steady-state inactivation for wild-type and F1519I mutant channels. The voltagedependence of fast inactivation was determined using 200-ms prepulse potentials ranging from ⫺120 to 40 mV in 5-mV increments and then a ⫺10-mV testpulse for 20 ms. The average of the half-maximal voltage for activation (V ) and slope factor (k) for BgNa 1⫺1 wild-type and mutant channels are shown in Table 1. The normalized peak current was plotted as a function of the prepulse potential. E, development of fast inactivation. The development of fastinactivation was determined by applying prepulse potentials ranging from ⫺80 to ⫺20 mV in 10-mV increments of varying duration from a holding potentialof ⫺120 mV and then applying a test pulse of ⫺10 mV for 20 ms to measure the fraction of sodium current inactivated during the prepulse. Data fordevelopment of inactivation (open symbols) and recovery from fast inactivation (filled symbols) were fitted to a single exponential function and plotted as afunction of the development-recovery pulse voltage. The average time constants from wild-type channel (squares, n ⫽ 5) and F1519I mutant channel(triangles, n ⫽ 4) are shown. F, voltage dependence of slow inactivation. Oocytes were held at prepulse potentials ranging from ⫺120 to ⫹10 mV in 10-mVincrements for 50 s. A 100-ms recovery pulse to ⫺120 mV and then a 20-ms test pulse to ⫺10 mV were given before returning to the holding potential of ⫺120 mV. Peak currents obtained during the test pulse were normalized with respect to the I and plotted as a function of the prepulse potential.
TABLE 1Voltage dependence of activation and inactivation of wild-type and mutant sodium channels

Tan et al.
cient to completely block pyrethroid action on an insect so- tively (Fig. 5D), resulting in a reduction of 3- to 11-fold in dium channel.
their sensitivity to deltamethrin. However, the tail current An Aromatic Amino Acid Residue at Position 1519 Is
was not detectable for the F1519A channel at 1 ␮M delta- Required for the Action of Pyrethroids. To determine
methrin, and only a small tail current was detected at 100 how critical the amino acid side chain at 1519 position is for ␮M deltamethrin (Fig. 5C). These results demonstrated that the action of pyrethroids, we made four additional amino acid an aromatic residue at position 1519 in IIIS6 is required for substitutions at 1519: alanine (hydrophobic), arginine (posi- tively charged), tryptophan (aromatic), and tyrosine (aro- The F1519W Mutation Reduces the Binding of 1S-cis-
matic). The F1519R channel did not produce any detectable Permethrin to BgNa Channel. In previous studies by us
sodium current in oocytes, whereas the peak currents of and others, the effects of kdr mutations on pyrethroid bind- other three mutant channels were comparable with that of ing and action were assessed by quantifying the modification the wild-type BgNa 1-1 channel. Like the F1519I mutant of sodium channels by pyrethroids and fitting the data to the channel, these three mutant channels shifted the voltage Hill equation M M /{1 ⫹ (K /[P])nH}. Increases in K or dependence of activation in the depolarizing direction, with values for kdr mutant channels reflect reduced sensi- the largest shift of 13 mV for the F1519W channel (Fig. 5A; tivities of these mutant channels to pyrethroids. However, an Table1). None of the substitutions altered the voltage depen- increase in K or EC value does not necessarily represent a dence of inactivation (Fig. 3B; Table 1). Unlike the F1519I reduction in pyrethroid binding to the sodium channel. K is mutant channel, the other three mutant channels completely an apparent dissociation constant; a change in the K value inactivated at positive membrane potentials without gener- could be the result of 1) a direct alteration of the binding site ating a noninactivating current (data not shown).
and/or 2) a nonbinding-site alteration that allosterically un- We next examined the effect of the Ala, Trp, and Tyr amino couples pyrethroid binding from subsequent sodium channel acid substitutions on the pyrethroid-induced tail current.
modification. Therefore, whether the effect of a kdr mutation The aromatic residue substitutions Trp and Tyr only slightly on sodium channel sensitivity to pyrethroids results from a reduced the amplitude of tail current induced by 1 ␮M del- direct alteration of the pyrethroid binding site has not been tamethrin, compared with that induced in the wild-type determined in previous studies. In this study, we took advan- channel (Fig. 5C). The EC value was 0.2, 0.7, and 2.2 ␮M tage of the competitive binding of active and inactive pyre- for the wild-type, F1519Y, and F1519W channels, respec- throid isomers to the sodium channel to directly evaluate the Fig. 3. Effects of BTX on the functional properties of
wild-type (left) and F1519I mutant (right) channels. A,
sodium current traces elicited by a 20-ms test pulse to
⫺10 mV from a holding potential of ⫺120 mV before andafter the application of 100 nM BTX. With 100 nM BTXin recording chamber, a series of 3000 pulses to ⫺10 mVfor 10 ms was applied at 10 Hz, and then the sodiumcurrent was recorded (traces labeled BTX 100 nM). B,voltage dependence of activation curves. C, steady-stateinactivation curves generated before and after 100 nMBTX modification. Activation and inactivation curveswere determined as described in the legend of Fig. 2 (Band D).

Molecular Determinants of the Pyrethroid Receptor Site
pyrethroid binding affinity of the inactive isomer using the increasing concentrations of the inactive 1S-cis-isomer. The Schild analysis.
presence of the inactive 1S-cis-isomer shifted the dose-re- Consistent with the finding by Lund and Narahashi (1982), sponse curve for the active isomer to the right (Fig. 6, B–D), we found that the inactive 1S-cis-permethrin decreased the confirming that 1S-cis-isomer is an antagonist of the active amplitude of the tail current induced by the active 1R-cis- 1R-cis-isomer on the cockroach sodium channel.
permethrin, but it did not induce any tail current by itself The F1519I mutant channel is completely insensitive to (Fig. 6A). We then determined the percentage of channel pyrethroids and is thus not useful for binding affinity anal- modification by the active 1R-cis-isomer in the presence of ysis. We therefore conducted the Schild analysis using theF1519W channel that exhibits an intermediate sensitivity todeltamethrin. Schild analysis (Fig. 6, B–D) showed a linearregression with a slope of 0.84 and 0.62 for wild-type andF1519W channels, respectively. The x-intercept pA , which equals ⫺log K (the dissociation constant of the inactive 1S-cis-isomer), was 6.0 for the wild-type channel and 4.3 forthe F1519W channel (Fig. 6E), corresponding to a K value of 1.2 ␮M for the wild-type channel and a K value of 53 ␮M for the F1519W mutant channel. Therefore, the F1519W muta-tion caused a 45-fold reduction in the binding affinity of1S-cis-permethrin to the cockroach sodium channel, indicat-ing that Phe1519 is part of the pyrethroid binding site.
The L993F kdr Mutation Also Reduces the Binding of
1S-cis-Permethrin to BgNa . A kdr mutation (correspond-
ing to L993F in the cockroach sodium channel) found in manyinsect species reduces the pyrethroid sensitivity of the cock-roach sodium channel by 5-fold (Tan et al., 2002a). We ex-amined the binding affinity of the inactive 1S-cis-permethrinisomer for the L993F channel using the Schild analysis. Ouranalysis showed that the x-intercept pA (i.e., ⫺logK ) was 4.8, corresponding to a K value of 16 ␮M for this mutant channel (Fig. 6, D and E). Therefore, L993F reduced thebinding affinity of the inactive isomer by 16-fold, comparedwith the wild-type channel. This result demonstrates thatLeu993 is also part of the pyrethroid receptor site.
As an important class of insecticides that target sodium channels, pyrethroids have attracted much attention in thestudy of the pharmacological and electrophysiological aspectsof sodium channels in the past several decades. However, thepyrethroid binding site on the sodium channel has eludedmolecular characterization despite serious efforts to under-stand it. Even though specific binding of pyrethroids to ratbrain membrane preparations has been reported (Trainer etal., 1997), many attempts to measure specific pyrethroidbinding in insect nerve tissue preparations using similarmethods were unsuccessful. The recent identification of aseries of kdr sodium channel mutations that confer pyre-throid resistance on insects brought us closer to the under-standing of this site, because some kdr mutations are ex-pected to affect pyrethroid binding. However, a directdemonstration of the effect of a kdr mutation on pyrethroidbinding is lacking. In this study, we show that 1) an F1519I Fig. 4. Effects of the scorpion toxin Lqh␣IT on the functional properties
mutation in IIIS6 abolished the sensitivity of the cockroach of wild-type (left) and F1519I mutant (right) channels. A, sodium currenttraces elicited by a 20-ms test pulse to ⫺10 mV from a holding potential sodium channel to diverse type I and type II pyrethroids; 2) of ⫺120 mV before and after the application of 10 nM Lqh␣IT. B and C, this mutation alters the channel gating properties, but it Lqh␣IT altered the voltage dependence of activation (B) and steady-state does not affect the action of site 2 (BTX) or site 3 (Lqh␣IT) inactivation (C) of the wild-type and F1519I mutant channels. Activationand inactivation curves were determined as described in the legend of sodium channel toxins; 3) an aromatic residue at position Fig. 2 (B and D). D and E, Lqh␣IT enhanced the amplitude of tail current 1519 is required for the action of pyrethroids; and 4) muta- induced by deltamethrin (D) and bioallethrin (E) in wild-type channels tions F1519W and L993F reduced the binding affinity of (left) but not in F1519I mutant channels (right). The tail current was pyrethroids to the cockroach sodium channel. Our work elicited by 100 pulses of a 67-Hz train of 5-ms depolarization from ⫺120to 0 mV.
therefore provides direct evidence for the involvement of

Tan et al.
Phe1519 in IIIS6 and Leu993 in IIS6 in forming the elusive studies using nerve preparations or sodium channels ex- pyrethroid binding site.
pressed in oocytes that pyrethroids interact with the sodium Our amino acid substitution experiments suggest that an channel at multiple sites (Lund and Narahashi, 1982; Vais et aromatic residue at position 1519 of the cockroach sodium al., 2002, 2003). For example, it was suggested that the channel is essential for pyrethroid binding. More than a super-kdr mutation M918T in the loop connecting IIS4-S5 of decade ago, Klaus Naumann proposed a "horseshoe model" the house fly sodium channel eliminates one of the pyre- for pyrethroid action, based on pyrethroid structure-activity throid action sites (Vais et al., 2000, 2003). We show here relations, stereochemical information, and toxicity data that the F1519I mutation abolishes sodium channel sensitiv- (Naumann, 1990). This model predicted that the binding site ity to structurally diverse pyrethroids. If the multiple bind- (pocket) for active pyrethroids, in which the pyrethroid ing site theory is correct, binding at Phe1519 must be a curves into a horseshoe shape, is possibly at an aromatic prerequisite for pyrethroid binding and action on other sites.
residue of the sodium channel backbone, and suggested a A lack of the initial binding of pyrethroids to Phe1519 may common active conformation for the structurally diverse py- prevent subsequent pyrethroid interactions with other resi- rethroids at the sodium channel. Our findings presented in dues, such as Leu993 and Met918. An initial binding at this report support this long-standing model and suggest Phe1519 could induce conformational changes necessary for that Phe1519 is probably the aromatic residue in Naumann's the formation of an optimal pyrethroid binding site. Both horseshoe model.
Phe1519 and Leu993 are located in the sixth transmem- Substitutions of the leucine residue in IIS6 (corresponding brane. However, other kdr or kdr-like mutations in different to Leu993 in BgNa ) have been found in diverse insect spe- arthropods are not confined in S6 segments. Several muta- cies and seem to be the most common type of naturally tions are found in the intracellular linkers or loops close to occurring kdr mutations in insects (Dong, 2002; Soderlund the transmembrane segments. In the folded sodium channel, and Knipple, 2003). We showed that the L993F mutation, many of these residues could be localized in proximity. There- like the F1519I mutation, reduced pyrethroid binding to the fore, conformational transitions induced by an initial pyre- cockroach sodium channel. Thus, it seems that natural selec- throid binding to Phe1519 may reorient some of these neigh- tion also favored residues in the pyrethroid binding site to boring residues to form optimal pyrethroid binding site(s) in reduce pyrethroid action.
the intracellular side of the channel. Interestingly, a similar It has been suggested from previous electrophysiological multistep binding model has been proposed for the interac- Fig. 5. Amino acid substitutions at Phe1519
alter sodium channel gating and sensitivity to
deltamethrin. A, voltage dependence of activa-
tion. B, voltage dependence of steady-state in-
activation. Activation and inactivation curves
were determined as described in the legend of
Fig. 2 (B and D). The average of the half-max-
imal voltage for activation (V
tor (k) for BgNa 1-1 wild-type and mutant channels are shown in Table 1. C, tail currentsinduced by deltamethrin. The tail current waselicited during a 100-pulse train of 5-ms depo-larization from ⫺120 to 0 mV with a 5-msinterpulse interval. The tail current decay wasbest fitted with two exponentials with the timeconstants ␶ ⫽ 1.5 ⫾ 0.3 s and ␶ ⫽ 0.3 ⫾ 0.03 s for the F1519Y channel and ␶ ⫽ 0.9 ⫾ 0.4 s and ␶ ⫽ 0.3 ⫾ 0.03 s for the F1519W channel.
However, the decay of the tail current in theF1519A channel was best fitted with one expo-nential with the time constant of ␶ ⫽ 103.9 ⫾47.7 ms. D, percentage of channel modificationby deltamethrin.

Molecular Determinants of the Pyrethroid Receptor Site
Fig. 6. L993F and F1519W mutations reduce the
binding of 1S-cis-permethrin to the cockroach so-
dium channel. A, inactive 1S-cis-permethrin antag-
onizes the action of 1R-cis-permethrin. 1S-cis-per-
methrin alone induced no tail current. Tail currents
induced by 1R-cis-permethrin were reduced with co-
application of 1S-cis-permethrin. Tail currents in-
duced by 1 ␮M 1R-cis-permethrin in the absence and
presence of the inactive 1 ␮M 1S-cis-permethrin are
shown. B to D, channel modification by 1R-cis-per-
methrin in the presence of a series of concentrations
of 1S-cis-permethrin in wild-type (B), F1519W (C),
and L993F (D) channels. The percentage of channels
modified by pyrethroids was calculated as described
in the legend of Fig. 5D. The modification curves
were linearized by a logarithm of modification per-
centage at the y-axis to estimate accurately the EC20
value of each line. E, Schild plots (i.e., pA plots)
showing the reduced binding affinity of 1S-cis-per-methrin to the L993F and F1519W channels com-pared with the wild-type channel. Each datum pointrepresents the average of four oocytes. K values were determined from the dose-response curves ofthe active 1R-cis-isomer on wild-type, F1519W, andL993F mutant sodium channels by measuring theamplitude tail current induced by 1R-cis-per-methrin and calculating percentage of channel mod-ification as described above. A series of K values (denoted as K ⬘) of the active 1R-cis-isomer were determined in the presence of increasing concentra-tions of the inactive 1S-cis-isomer. Schild analysiswas used to determine the affinity of the inactiveisomer, calculated from the equation log(dose ra-tio ⫺ 1) ⫽ logK ⫺ log[B]. The data were fitted with a linear regression, generating the Schild plot slopeand the x-intercept, pA , which equals ⫺logK .
tion between epinephrine and its receptor, ␤ adrenergic receptor (Kobilka 2004; Liapakis et al., 2004).
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