1052 R.L. Martin et al. Insect Biochemistry and Molecular Biology 30 2000 1051–1059
S2 S3 S4 S5 S1
S6
para74
paraDN7
S2 S3 S4 S5 S1
S6 S2 S3 S4 S5
S1 S6
S2 S3 S4 S5 S1
S6
super kdr kdr
A1422V M1536I
Fig. 1. A schematic diagram of the para voltage gated sodium channel indicating the location of the two amino acid replacements in DDT
resistant para
ts
mutants. A1422V in para
DN7
and M1536I in para
74
numbering is according to GenBank accession number AAB59195. The relative location of the kdr and super-kdr replacements 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 the para
74
mutant 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
Drosophila colonies were maintained at 20–25
° C and
were reared on Instant Drosophila Medium Carolina Biological Supply, Burlington, NC. The para fly 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 experiments Drosophila eggs wild type and para
74
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 ngml insulin; 50
µ gml penicillin and
50 µ
gml 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
µ gml 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
± 0.3
µ M 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, MgCl
2
1, CaCl
2
1, HEPES 10, pH =
7.4. The pipette solution contained in
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
