Health Articles
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 Lqh␣IT
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).
We thank Drs. Klaus Naumann and Ralf Nauen (Bayer Crop-
In summary, we show here that two residues defined by
Science) and John Daly (National Institutes of Health) for the gen-
kdr mutations in the sixth transmembrane segments of the
erous gift of permethrin isomers and other pyrethroids, and BTX,
cockroach sodium channel (Fig. 7) are part of the long-
respectively. We also thank Drs. Dalia Gordon and Noah Koller for
sought-after pyrethroid binding site. Further examination of
critical review of this manuscript.
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binding and action should lead to a better understanding of
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