1053 R.L. Martin et al. Insect Biochemistry and Molecular Biology 30 2000 1051–1059
mM: CsF 100, CsCl 40, NaCl 10, HEPES 10, pH =
7.3. Allethrin 100
µ gml 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
µ M stock of allethrin was prepared in DMSO.
This stock was directly dissolved in the bath solution to give the desired concentration 50 nM–2
µ M 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 para
74
and para
DN7
were resistant to allethrin when compared with wild type Fig. 2A and B. The double mutant had an even
higher resistance to allethrin Fig. 2C. These data are summarized in Table 1. Because para
74
and para
DN7
had similar resistance to allethrin 5 fold, we chose one,
para
74
, for electrophysiological study. The double mutant para
74DN7
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 embryonic Drosophila. 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
µ m 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
. . . ..
. . . .
. .
.. .
. .
. ..
.. .
. ..
. .
. .. ...
.
Dose of Allethrin mgvial
99 90
70 50
30 10
1 1
10 100
99 90
70 50
30 10
1 1
10 100
99 90
70 50
30 10
1 1
10 100
A
B
C
Fig. 2. Lethality assays: Allethrin dose–response relationships for
para
ts
mutants right line for all figures compared to a standard sus- ceptible strain Canton-S left line for all figures. A Canton-S versus
para
74
. B Canton-S versus para
DN7
. C Canton-S versus para
74DN7
. 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.
Sodium channel density appeared to be independent of the number of processes extended. The presence of
detectable Na
+
current was a function of days in culture as well as the extension of processes. The percentage of
neurons from which Na
+
current could be elicited increased as a function of the number of days in culture,
with a plateau of 50 being reached in 5 to 7 days. After 7 days in the culture the number of neurons from
which Na
+
current could be elicited declined. No
1054 R.L. Martin et al. Insect Biochemistry and Molecular Biology 30 2000 1051–1059
Table 1 Resistance levels of para
ts
mutant lines of Drosophila melanogaster to allethrin. Mortality was recorded at the end of a 24 h treatment Line
n
a
LD
50
µ gvial
b
Slope
c
Resistance ratio
d
Allethrin Wild-type
920 9.61 7.89–11.45
1.55 ±
0.18 ;1.00
para
74
1050 50.5 34.9–88.6
1.55 ±
0.18 5.25
para
DN7
1099 48.6 38.9–63.0
2.10 ±
0.20 5.06
a
Total number of flies tested not including controls.
b
Values are shown as mean 95 confidence intervals.
c
Values 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 and para
74
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 near 240 mV. The half point of activation was 225
± 0.5
mV for wild type n =
11 sodium channels and 233.5
± 0.4 mV for para
74
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 para
74
vs 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 Cl
2
currents 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 both para
74
mutant 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 and para
74
. 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 from para
74
neurons with a representative family of current traces shown in the inset. V
h
= 2130 mV, V
t
= 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 at 210 mV. Fig. 5 shows
the fraction of available current for both wild type and para
74
mutant neurons. Typical current traces recorded at the test potential 210 mV are shown in the insets.
Channels began to inactivate at 270 mV and were fully inactivated by 220 mV for both wild type and para
74
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 para
74
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 para
74
neurons.
1055 R.L. Martin et al. Insect Biochemistry and Molecular Biology 30 2000 1051–1059
Fig. 4. Representative peak current–voltage relationship seen occasionally from either para
74
or 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
0.2 0.4
0.6 0.8
1.0
-120 -80
-40 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 and para
74
. 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 from para
74
neurons with a representative set of test potential current traces shown in the inset. V
h
= 2130 mV, V
c
= 2100
to 210 mV, V
t
= 210 mV.
Although there were no differences in the kinetics of Na
+
currents in wild type and para
74
Drosophila neu-
rons, the sensitivity to a type I pyrethroid differed. Allethrin was applied to the recording chamber and 20
ms depolarizing voltage steps to 230 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
and para
74
Na
+
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 para
74
Na
+
currents n =
3. Concentrations equal to or less than 100 nM had no effect on either wild
type or para
74
Na
+
current, and both the para
74
and wild type Na
+
currents were affected at concentrations of allethrin .1
µ M Fig. 7. Dose response data for
allethrin are summarized for both wild type and para
74
Na
+
currents in Fig. 8. The para
74
point mutation reduced the affinity of the Drosophila sodium channel
for allethrin approximately 2 fold without altering the kinetics or voltage dependence of the current.
1056 R.L. Martin et al. Insect Biochemistry and Molecular Biology 30 2000 1051–1059
-200 -400
-600 -800
-1000 20
40 60
80 100
120 -200
-400 -600
-800 -1000
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 and para
74
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. V
h
= 2130 mV,
V
t
= 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 para
74
ii neuronal sodium channels before and after addition of 500 nM allethrin. V
h
= 2130 mV, V
t
= 230 mV, test pulse
duration =
150 ms, tail pulse duration =
150 ms.
4. Discussion
