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Point mutations in domain III of a
Drosophila
neuronal Na
channel confer resistance to allethrin
R.L. Martin
a, B. Pittendrigh
b, J. Liu
a, R. Reenan
c, R. ffrench-Constant
d, D.A. Hanck
a,*aDepartment of Medicine MC6094, University of Chicago, 5841 S. Maryland Avenue, Chicago, IL 60637, USA bDepartment of Entomology, Purdue University, West Lafayette, IN 47907, USA
cUniversity of Connecticut Health Center-MC3301, Department of Genetics and Developmental Biology, 263 Farmington Avenue, Farmington,
CT 06030, USA
dDepartment of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK
Received 3 December 1999; received in revised form 30 March 2000; accepted 30 March 2000
Abstract
Voltage-gated sodium channels are the presumed site of action of pyrethroid insecticides and DDT. We screened several mutant sodium channel Drosophila lines for resistance to type I pyrethroids. In insecticidal bioassays the para74and paraDN7 fly lines
showed greater than 4-fold resistance to allethrin relative to the allethrin sensitive Canton-S control line. The amino acid substitutions of both mutants are in domain III. The point mutation associated with para74lies within the S6 transmembrane region and the
amino acid substitution associated withparaDN7lies within the S4–S5 linker region. These sites are analogous to the mutations in
domain II underlyingknockdown resistance(kdr) andsuper-kdr, naturally occurring forms of pyrethroid resistance found in house-flies and other insects. Electrophysiological studies were performed on isolated Drosophila neurons from wild type and para74
embryos placed in primary culture for three days to two weeks. The mutantpara74sodium currents were kinetically similar to wild
type currents, in activation, inactivation and time to peak. The only observed difference betweenpara74and wild-type neurons was
in the affinity of the type I pyrethroid, allethrin. Application of 500 nM allethrin caused removal of inactivation and prolonged tail currents in wild type sodium channels but had little or no effect onpara74mutant sodium channels. 2000 Elsevier Science Ltd.
All rights reserved.
Keywords:Sodium channel; Allethrin;Drosophila; Paramutants; Electrophysiology
1. Introduction
The voltage-gated sodium channel is responsible for the rapid upstroke of the action potential in many excit-able tissues, including most neurons. In Drosophila, the
α-subunit is encoded by theparagene (Loughney et al., 1989) and it is highly homologous to sodium channels in other insects. Theα-subunit of the sodium channel is comprised of four domains, each with six transmem-brane spanning regions (Fig. 1). Several years ago pyr-ethroid and DDT resistant mutants in house fly were described (Williamson et al., 1996; Miyazaki et al., 1996), which were identified to result from point mutations in domain II of that voltage-gated sodium
* Corresponding author.
E-mail address:[email protected] (D.A. Hanck).
0965-1748/00/$ - see front matter2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 0 0 ) 0 0 0 8 0 - 1
channel (Smith et al., 1997; Lee et al., 1999b). The kdr
(knockdown resistant) mutation is located in S6 (Fig. 1). In a second pesticide resistant allele, the kdr mutation was found paired with another amino acid change in the intracellular linker between S4 and S5. The double mutant conferred much greater resistance to pyrethroids and was named super-kdr.
Pittendrigh et al. (1997) identified two mutations simi-lar to kdr and super-kdr in domain III of the voltage-gated Drosophila sodium channel, which confer resist-ance to pyrethroids and DDT. First, the para74 mutant
fly line has a mutation analogous to that of kdr, but in the third domain, with a Met to Ile (M1536I) change within ten amino acid positions of the analogous position thatkdroccupies in the second domain. Second, the par-aDN7 fly line has a Ala to Val (A1422V) change in the
anal-S2 S3 S4 S5
S1 S6
para74
paraDN7
S2 S3 S4 S5 S1
S6
S2 S3 S4 S5
S1 S6 S1 S2 S3 S4 S5 S6
super kdr kdr
A1422V
M1536I
Fig. 1. A schematic diagram of thepara voltage gated sodium channel indicating the location of the two amino acid replacements in DDT resistant parats mutants. A1422V inparaDN7and M1536I inpara74(numbering is according to GenBank accession number AAB59195). The relative location of thekdrandsuper-kdrreplacements documented in its homologs in the house fly and the German cockroach (Dong and Scott, 1994; Williamson et al., 1996; Miyazaki et al., 1996; Dong, 1997) are also shown.
ogous position in domain II of super-kdr. In this study we performed lethality assays on the domain III mutants and compared the electrophysiological properties of the wild type (Canton-S) voltage-gated sodium channel and the affinity of allethrin with thepara74mutant in
Droso-phila neurons. Some of these data have been presented in abstract form (Martin et al., 1998; Pittendrigh et al., 1998).
2. Methods
2.1. Drosophila melanogaster strains and assays of insecticide resistance
Drosophilacolonies were maintained at 20–25°C and were reared on Instant Drosophila Medium (Carolina Biological Supply, Burlington, NC). The parafly lines were EMS-mutagenized and originally scored for a para-lytic phenotype when held at 37°C. They were later scored for pesticide resistance. Bioassays were perfor-med as described by ffrench-Constant et al. (1990). Briefly, pesticides were placed evenly inside scintillation vials, and the flies were subsequently maintained in the vials, with a cotton plug containing 5% sucrose, for 24 h. Flies showing movement at 24 h from the beginning of the experiment were considered alive.
2.2. Isolation and culture of embryonic Drosophila neurons
For electrophysiological experimentsDrosophilaeggs (wild type and para74 mutant) were collected for
approximately 3 h post oviposition and placed at 25°C for 3.5 h to allow embryos to develop to the early gas-trula stage. Embryos were sterilized and dechorionated
by immersion for 10 min in 95% EtOH and 3% sodium hypochlorite (1:1 by volume) and rinsed in sterile water. Embryonic cells were dissociated by gentle disruption in a Dounce homogenizer containing modified Schneider’s media (GIBCO, Gaithersburg, MD) supplemented with 10% FBS; 200 ng/ml insulin; 50 µg/ml penicillin and 50µg/ml streptomycin. The homogenate was filtered and washed with culture media. Cells were gently resus-pended and plated on 35 mm dishes or on polylysine coated cover slips.
Isolated neuron cultures were grown at 25°C for 1–2 days in supplemented Schneider’s media containing 2
µg/ml cytochalasin B to inhibit mitosis and cytokinesis (Wu et al., 1990). After 48 h, cytochalasin B was removed from the media. One day after plating, most neuroblasts differentiated into single neurons with well extended processes exhibiting a variety of branching pat-terns. Distinct mono, bi- and multipolar cell types were observed. The diameter of the soma of a random sample of large treated cells reached 10.7±0.3µM (mean±SEM,
n=43) in five days. The cultures were maintained at 25°C for up to two weeks.
2.3. Electrophysiological recordings from embryonic neurons
Recordings were made 3 to 12 days after isolation on neurons adherent to 35 mm dishes or polylysine coated coverslips, which were rinsed with PBS (pH 7.4) for 30 min prior to beginning recording. Dishes and chambers were mounted on a Nikon inverted microscope, and cells were visualized at 600× magnification. Patch pipettes were constructed with alumina silicate glass capillary tubes with resistances of 0.8–2.0 MV. The bath solution contained (in mM): NaCl 140, KCl 2.5, MgCl21, CaCl2
mM): CsF 100, CsCl 40, NaCl 10, HEPES 10, pH=7.3. Allethrin (100 µg/ml in methylene chloride) was obtained from Chem Service (West Chester, PA). d-Allethrin was$95% (1R)-isomers and$75% trans iso-mers. Methylene chloride was removed (in vacuo), and a 328 µM stock of allethrin was prepared in DMSO. This stock was directly dissolved in the bath solution to give the desired concentration (50 nM–2 µM) immedi-ately before each experiment.
All recordings were made using pCLAMP6 in con-junction with an Axopatch 200 or EPC7 amplifier. Low pass filter was set to 5 kHz. In order to guarantee full channel availability, the membrane potential was set at
2130 mV. Na+current responses were elicited by depol-arizing voltage steps between 290 mV and +30 mV in 5 mV increments. Recordings were made at room tem-perature (22–24°C) or elevated temperature (36–40°C) using a thermocouple driven Peltier feedback system on the microscope stage (Sensortek TS4, Bailey, NJ). Data were analyzed using MATLAB (Mathworks, Natick, MA). All data are reported as means±S.E.M.
3. Results
In lethality assays the mutants para74 and paraDN7
were resistant to allethrin when compared with wild type (Fig. 2(A) and (B)). The double mutant had an even higher resistance to allethrin (Fig. 2(C)). These data are summarized in Table 1. Becausepara74andparaDN7had
similar resistance to allethrin (5 fold), we chose one,
para74, for electrophysiological study. The double
mutant (para74/DN7) was not readily available for further
study because of the sex linked nature of the gene. Only the females could be heterozygous for the para alleles and there is no easy way to determine the sex of the eggs.
In order to study the mutant electrophysiologically, neurons were isolated from embryonicDrosophila. The isolation procedure favors survival of neurons over other cell types. Cytochalasin B was added to the culture media to inhibit mitosis and cytokinesis (Wu et al., 1990) for one to two days. This allowed the cell soma to increase in size up to 10 µm in diameter. Na+ currents could be recorded from many of the cells with extended processes. Neurons would typically extend one or more processes after 24 h in primary culture. Sixty-four per-cent of the cells extended a single process and 28% extended two processes while only 8% of cells extended 3 or more processes. It is presumed that most of the sodium channels are located on the processes rather than on the cell body because when cells were trypsinized and most processes were lost, we never were able to observe Na+ current (n.10). Because of this, neurons were left attached to the coverslip or bottom of the cul-ture dish during recording. This cell configuration made
. . .
..
Fig. 2. Lethality assays: Allethrin dose–response relationships for
paratsmutants (right line for all figures) compared to a standard sus-ceptible strain (Canton-S) (left line for all figures). (A) Canton-S versus
para74. (B) Canton-S versusparaDN7. (C) Canton-S versuspara74/DN7. Mortality was recorded at the end of a 24 h treatment with the pestic-ide. The non-parallel shift in the dose–response data most likely results from the mixture of allethrin isoforms present in the commercially available agent.
voltage clamping the Na+ current technically challeng-ing.
Table 1
Resistance levels ofparatsmutant lines ofDrosophila melanogasterto allethrin. Mortality was recorded at the end of a 24 h treatment
Line na LD
50(µg/vial)b Slopec Resistance ratiod
Allethrin
Wild-type 920 9.61 (7.89–11.45) 1.55±0.18 ;1.00
para74 1050 50.5 (34.9–88.6) 1.55±0.18 5.25
paraDN7 1099 48.6 (38.9–63.0) 2.10±0.20 5.06
aTotal number of flies tested (not including controls). b Values are shown as mean (95% confidence intervals). cValues are shown as mean±the standard error of the estimate. d With respect to wild-type.
attempts were made to record from cells that had been in culture for only 1 or 2 days.
Na+currents were recorded from wild type andpara74
neurons. Mean current voltage relationships and an example of families of current traces from both groups of neurons are illustrated in Fig. 3. The current activated near240 mV. The half point of activation was225±0.5 mV for wild type (n=11) sodium channels and
233.5±0.4 mV for para74 (n=9) sodium channels. The
more negative value for the mutant channels is probably due to poorer voltage control in these cells (dx=4.4±0.3 mV for para74vs dx=6.8±0.3 mV for wild type). These
currents were blocked by 100 nM tetrodotoxin (n=5). The outward currents were more than likely Cl2currents
since the intracellular Cs+solutions used would not sup-port currents through potassium channels. These varied in magnitude from neuron to neuron as can be appreci-ated in the raw data shown in the insets.
The culture procedure did not give a homogenous population of cells. Fig. 4 shows a representative peak current–voltage relationship that was seen occasionally (,5% of recordings) from bothpara74mutant and wild
type neurons. Unlike typical neuronal Na+currents, these currents were much slower in both their onset and rate of decay, although their voltage dependence of activation
Fig. 3. Comparison of Na+currents from wild type andpara74. (A) Mean (n=11) peak current–voltage relationship from Canton-S (wild type) neurons with a representative family of current traces shown in the inset. (B) Mean (n=9) peak current–voltage relationship frompara74neurons with a representative family of current traces shown in the inset.Vh=2130 mV,Vt=290 to+30 mV.
was the same. Allethrin studies were not done using neu-rons with sodium channels that had this type of kinetics. Steady state availability of the Na+ current was also investigated. Neurons were conditioned at a variety of potentials (290 to +30 mV) for 1 s from a holding potential of 2130 mV. Na+ current inactivation was assessed with a 10 ms test step at210 mV. Fig. 5 shows the fraction of available current for both wild type and
para74 mutant neurons. Typical current traces recorded
at the test potential (210 mV) are shown in the insets. Channels began to inactivate at270 mV and were fully inactivated by 220 mV for both wild type and para74
sodium channels. The half point of availability was also similar, 242.7±0.6 mV for wild type (n=13) and
247.0±2.2 mV for para74 ( n=8).
Because the mutants were originally selected for tem-perature sensitivity, we investigated the effect of elev-ated temperature on the sodium channel. In a subset of neurons, Na+current was elicited at 36–40°C as well as at room temperature. As would be expected at the higher temperature current kinetics were speeded and current amplitude was increased. Voltage dependence of the cur-rent was unaffected and, interestingly, there were no dif-ferences in these assays between wild type and para74
Fig. 4. Representative peak current–voltage relationship seen occasionally from eitherpara74or wild type neurons in the absence of pesticide. Peak currents are plotted as open circles and current remaining at 20 ms are plotted as asterisks. Allethrin studies were not carried out using neurons with this type of sodium channel.
-120 -80 -40 0
0.0 0.2 0.4 0.6 0.8 1.0
-120 -80 -40 0
0.0 0.2 0.4 0.6 0.8 1.0
2 ms
2 ms
A.
B.
Voltage (mV)
Voltage (mV)
Fig. 5. Comparison of Na+current steady state availability from wild type andpara74. (A) Mean (n=13) sodium channel steady state availability from Canton-S (wild type) neurons with a representative set of test potential current traces shown in the inset. (B) Mean (n=8) sodium channel steady state availability frompara74neurons with a representative set of test potential current traces shown in the inset.V
h=2130 mV,Vc=2100
to210 mV,Vt=210 mV.
Although there were no differences in the kinetics of Na+ currents in wild type and para74 Drosophila
neu-rons, the sensitivity to a type I pyrethroid differed. Allethrin was applied to the recording chamber and 20 ms depolarizing voltage steps to230 mV from a holding potential of 2130 mV, applied at 1 Hz, were used to assess the insecticide effect on the Na+ current. Fig. 6 shows the effect of 500 nM allethrin on both wild type andpara74Na+ currents. This concentration of allethrin
removed inactivation from wild type Na+currents (n=6). The upper panels of the figure show the effect of wash-ing in allethrin on the current amplitude at the end of 20 ms depolarizing pulses. Also, with the removal of
inactivation, a prominent tail current developed (see lower panels). This concentration of allethrin had no effect on para74 Na+ currents (n=3). Concentrations
equal to or less than 100 nM had no effect on either wild type orpara74Na+current, and both thepara74and wild
type Na+ currents were affected at concentrations of allethrin .1 µM (Fig. 7). Dose response data for allethrin are summarized for both wild type and para74
Na+ currents in Fig. 8. The para74 point mutation
0
-200
-400
-600
-800
-1000
0 20 40 60 80 100 120 0
-200
-400
-600
-800
-1000
0 100 200 300 400 500
Time (s) Time (s)
500 nM allethrin 500 nM allethrin
i) ii)
A.
i) ii)
B.
500 nM allethrin
500 nM allethrin
control control
50 ms 50 ms
Fig. 6. Comparison of the effect of allethrin on wild type (i) andpara74(ii) neuronal sodium channels. (A) Na+current was elicited with a train
of 20 ms depolarizing pulses at 1 Hz. 500 nM allethrin (presence represented by the solid bar) was initially applied at pulse 20.Vh=2130 mV,
Vt=230 mV. Measurements of Na+current amplitude (depicted as filled circles) were made at the end of each 20 ms depolarizing pulse and are
plotted as a function of time from allethrin application. (B) Representative Na+current traces elicited with a 300 ms voltage clamp protocol from
wild type (i) and para74 (ii) neuronal sodium channels before and after addition of 500 nM allethrin.Vh=2130 mV, Vt=230 mV, test pulse duration=150 ms, tail pulse duration=150 ms.
4. Discussion
MutantDrosophila(para74) were isolated on the basis
ofts-paralysis (Stern et al., 1990), and the survivors were found to be resistant to DDT, type I and type II pyrethro-ids. Similar tokdr, the amino acid substitution (M1536I) associated withpara74lies within the S6 transmembrane
region, but unlike kdr, the mutation is found in domain III instead of domain II. The paraDN7 line has a Ala to
Val change (A1422V) in the intracellular loop between S4 and S5 of the III domain, a single amino acid position away from the analogous position in domain II of super-kdr. Unlikekdrandsuper-kdrthese amino acid changes in the Drosophila para gene exist on separate alleles, allowing for (1) comparisons of the effects of each mutation on pesticide resistance and (2) the potential for the study of these mutations together, in trans, using het-erozygotes (Pittendrigh et al., 1997).
In the lethality assay the mutants para74and
paraDN7
caused a 5-fold decreased sensitivity to allethrin (see
Fig. 2 and Table 1) and the combined mutation (para74/DN7) caused a 12-fold decrease in allethrin
sensi-tivity relative to Canton-S (wild type). The correspond-ing mutations in domain II,kdrandsuper-kdr, caused a greater than 688-fold decrease in permethrin (a type I pyrethroid) sensitivity in horn files,Haematobia irritans
(Guerrero et al., 1997). Our electrophysiological deter-mination of pyrethroid sensitivity indicated only a 2-fold reduction in sensitivity to allethrin. Differences between lethality assays and electrophysiological data are not sur-prising. Toxicity in whole animals can be multi-factorial, while the electrophysiological data speak more directly to interactions of the toxin and the channel.
B.
A.
C.
D.
50 ms
50 ms
50 ms 50 ms
100 nM allethrin
1000 nM allethrin
Fig. 7. Comparison of the effect of allethrin on wild type andpara74neuronal sodium channels. Representative Na+current traces elicited with a 300 ms voltage clamp protocol from wild type (A) and para74(C) neuronal sodium channels before and after addition of 100 nM allethrin. Representative Na+current traces elicited with a 300 ms voltage clamp protocol from wild type (C) andpara74(D) neuronal sodium channels before and after addition of 1000 nM allethrin.Vh=2130 mV,Vt=230 mV, test pulse duration=150 ms, tail pulse duration=150 ms for all current traces.
10
100
1000
0.0
0.5
1.0
1.5
2.0
Allethrin [nM]
Fig. 8. Allethrin dose response curve for wild type (j) andpara74 (G) neuronal sodium channels. Tail currents were measured as the increase in tail current amplitude (Vt=2130 mV) at 50 ms due to the presence of allethrin divided by the peak Na+current at210 mV. Data
are plotted as mean±S.E.M. (n=3 for each point).
Channels normally open for milliseconds, but in the presence of pyrethroids they can remain open as long as several seconds. Increased toxicity of pyrethroids in insects over mammals is thought to be due mainly to the increased sensitivity of the insect sodium channel (Warmke et al., 1997; Narahashi et al., 1998).
The mechanism of resistance to pyrethroids conferred by kdr and super-kdr mutations remains somewhat unclear. Recently, Vais et al. (1997) introduced a kdr
mutation into a rat sodium channel to determine the functional changes in the channel that would allow for pyrethroid resistance. They found that as compared to wild-type channels the steady state activation curve of the mutant was shifted 14 mV in the depolarizing direc-tion. They proposed that resistance to permethrin, in insects, may be due to a shift in the steady-state acti-vation curve. In contrast, Williamson et al. (1996) sug-gest that the mutations simply alter a binding site for pyrethroids at the intracellular mouth of the sodium channel pore. In a pyrethroid resistant tobacco budworm,
inacti-vation, but they also attributed the|21-fold lower sensi-tivity to permethrin of the mutant sodium channels to structural changes in the channel that created a reduced sensitivity of the pyrethroid target site.
Pittendrigh et al. (1997) suggested that the pyrethroid binding site is likely composed of residues from all four sodium channel domains and that single mutations in any of the domains will alter binding. The budworm mutant (Lee et al., 1999a) also provides supportive evidence for mutations in other sodium channel domains contributing to pyrethroid sensitivity. The mutant they studied had a single amino acid substitution in domain I S6.
Our data suggest that the resistance to allethrin found in thepara74mutant sodium channel is not due to a shift
in activation as activation was not shifted relative to con-trol sodium channels.Para74mutants produced Na+
cur-rents with a normal kinetic phenotype (see Fig. 3 and Fig. 5). These data are similar to those previously obtained by O’Dowd and Aldrich (1988). In recordings from wild type embryonicDrosophilaneurons with 140 mM Na+as the charge carrier, they found the half point of activation for Na+ current to be |222 mV and the
half point of availability to be|248 mV, with channels
beginning to inactivate at270 mV and fully inactivated by 220 mV. The Na+ currents from the mutants they studied were expressed at a lower density relative to wild type but the current kinetics were similar. O’Dowd et al. (1989) also examined the temperature sensitivity of wild type and parats mutant embryonicDrosophila neuronal
Na+ current. They found decreased current density for all mutants and mutant and wild-type currents behaved similarly at elevated temperature, exhibiting a 5–7 mV depolarizing shift for activation. We also found that volt-age dependence of the current was unaffected, and there were no differences between wild type and para74
neu-rons at elevated temperature.
Furthermore, these values are similar to those obtained forparasodium channels expressed inXenopusoocytes; activation V1/2=212 to 217 mV and inactivation
V1/2=240 to 250 mV (Warmke et al., 1997). Smith et
al. (1997) found no differences between para/TipE and
para/TipE L1014F (kdr) expressed inXenopus oocytes, with the V1/2 for activation=216.9 mV vs 215.1 mV
and theV1/2for inactivation=234 mV vs231 mV. Also,
Vais et al. (2000) found no differences between
para/TipE, para/TipE L1014F (kdr) and para/TipE L10114F+M918T (super-kdr) expressed in Xenopus
oocytes; activation V1/2=213 to 217 mV and
inacti-vationV1/2=238 to244 mV. These data support the idea
that the mutations do not alter the basic kinetic proper-ties of the sodium channel, but target the interaction site of pyrethroids with the channel.
Our data do not provide support for the concept that pyrethroid resistance-associated mutations must alter the kinetics of the mutant sodium channel and highlight the need for further comparative electrophysiological
analy-sis of the range of different mutations found in differ-ent insects.
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