21 S.H. Lee, D.M. Soderlund Insect Biochemistry and Molecular Biology 31 2001 19–29
2.2. Expression Oocytes were obtained surgically from female frogs
Nasco, Ft. Atkinson, WI and defolliculated by incu- bation with Type 1A collagenase Sigma, St. Louis, MO
followed by manual removal of remaining follicle cells. Stage IV–VI oocytes isolated by these methods were
incubated in ND-96 medium Goldin, 1992 sup- plemented with 1 sodium pyruvate, 1 vv of peni-
cillin 5000 unitsmlstreptomycin 5 mgml solution Sigma and 5 horse serum Sigma for 24 h at 18
° C
prior to injection. The cRNA used in expression experi- ments was synthesized from linearized plasmid Vssc1
or purified PCR fragment tipE templates using a com- mercial kit mMessage mMachine
, Ambion, Austin, TX. The integrity and approximate concentration of the
RNAs obtained by these methods were determined by electrophoresis in formaldehyde agarose gels. Oocytes
were injected with 25–50 nl of an aqueous solution of cRNA 1:1 molar ratio of Vssc1 and tipE cRNAs,
|1 ngnl and
incubated in
supplemented ND-96
medium at 18 °
C for up to 24 days prior to electrophysi- ological analysis of sodium currents.
2.3. Electrophysiology Electrophysiological recordings of sodium currents
were obtained from oocytes in ND-96 at 16–20 °
C. Rec- ordings were performed in Plexiglas recording chambers
200 µ
l bath volume with a glass coverslip bottom bonded with cyanoacrylate glue. Membrane currents of
oocytes were recorded using a two-microelectrode volt- age clamp with a virtual ground TEV-200, Dagan
Corp., Minneapolis, MN, filtered with a 2 kHz low pass four-pole Bessel filter, digitized at 10 kHz inactivation
experiments or 20 kHz all other experiments, and stored electronically MacLab, AD Instruments, Milford,
MA. Borosilicate glass recording electrodes 0.3– 2.0 M
V were filled with filtered 3 M KCl, coated with insulating resin, and shielded with grounded aluminum
foil. Compensation circuitry TEV-208, Dagan was used to remove leakage current. Net sodium currents,
with capacitive transients removed, were derived by sub- tracting traces from the same oocyte obtained in the pres-
ence of 1
µ M tetrodotoxin TTX; Sigma. Cismethrin
M. Elliott, Rothamsted Experimental Station, UK and BTX J. Daly, National Institute of Arthritis, Metab-
olism, and Digestive Disease, Bethesda, MD were pre- pared as stock solutions in dimethyl sulfoxide DMSO
and diluted with ND-96 immediately before bath appli- cation. Oocytes were incubated with cismethrin or BTX
in a non-perfused recording bath for 3 min prior to the collection of data. Each recording chamber was used
only once to prevent cross-contamination. The final DMSO concentration in the bath did not exceed 1, a
concentration that had no effect on sodium currents. Data analysis was performed in Axograph Axon Instru-
ments, Burlingame, CA. Midpoint potentials for acti- vation and steady-state inactivation e.g., test potentials
producing half-maximal
activation or
steady-state inactivation were determined by least-squares fits of
current–voltage data from individual experiments to the Boltzmann distribution r
.0.95. The unpaired Stud- ent’s t-test was used to compare midpoint potentials for
activation and steady-state inactivation obtained with different Vssc1 channel variants.
3. Results
In this study, we obtained the functional expression of house fly sodium channels in Xenopus oocytes by the
coinjection of cRNAs for wildtype or specifically mutated Vssc1 sodium channel
α subunits and the D.
melanogaster tipE protein, which is known to enhance the expression of Vssc1 sodium channels in this system
Smith et al., 1997. Expression of such channels Vssc1tipE channels containing the V410M mutation
resulted in sodium currents measured by two-electrode voltage clamp analysis under standard assay conditions
50 ms depolarizations from
2100 mV to 0 mV that were indistinguishable in mean amplitude and fast inacti-
vation kinetics from those measured for wildtype Vssc1tipE channels under the same assay conditions
data not shown.
We assessed the voltage dependence of activation of Vssc1tipE channels carrying the V410M mutation
V410M channels by measuring the amplitude of the peak transient sodium currents obtained upon 50 ms
depolarizations from a holding potential of 2120 mV to
test potentials ranging from 290 to 90 mV in 5–10 mV
increments. Fig. 2 shows a family of sodium current traces obtained under these conditions from a representa-
tive oocyte expressing the V410M channel [Fig. 2a], plots of the current–voltage relationship for mean nor-
malized peak transient currents obtained from multiple data sets [Fig. 2b], and a fit of mean conductance
values derived from these data to the Boltzmann distri- bution [Fig. 2c]. The conductance values in Fig. 2c
were calculated for each data set using the value for the sodium reversal potential determined in each experi-
ment. Table 1 compares the midpoint potential for volt- age-dependent activation of the V410M channel, calcu-
lated from individual fits of multiple data sets, with previously published values for the wildtype and L1014F
variants of the Vssc1tipE sodium channel Lee et al., 1999c. The |9 mV depolarizing shift in the midpoint
potential for the activation of the V410M channel com- pared with wildtype channel was statistically significant
P
,0.0001, df =
15. However, the midpoint potentials for the V410M and L1014F channel variants were not
significantly different P =
0.0618, df =
14.
22 S.H. Lee, D.M. Soderlund Insect Biochemistry and Molecular Biology 31 2001 19–29
Fig. 2. Voltage dependence of activation of the V410M variant of the Vssc1tipE sodium channel. a Family of sodium currents obtained from
a single oocyte by 50 ms depolarizations from 2120 mV to test potentials of 290 to 90 mV. b Current–voltage relationship derived from multiple
data set such as that shown in a. c Conductance transformation of the current–voltage data shown in b. Table 1
Comparison of the voltage dependence of activation and steady-state inactivation of wildtype and specifically mutated house fly Vssc1 volt-
age-sensitive sodium channels expressed in Xenopus oocytes
Vssc1 variant Midpoint potential mV
± standard
deviation n Activation
Steady-state inactivation
Wildtype 219.7
± 1.3 8
a
234.5 ±
3.8 6
a
V410M 210.6
± 2.9 9
b
229.5 ±
1.6 8
c
L1014F 213.8
± 3.6 7
a
231.1 ±
2.0 7
a a
Previously published data Lee et al., 1999c used in statistical comparisons.
b
Calculated from data from multiple experiments such as that shown in Fig. 2a.
c
Calculated from data from multiple experiments such as that shown in Fig. 3a.
We also examined the effects of the V410M mutation on the voltage dependence of steady-state inactivation
of Vssc1tipE sodium channels. The two-pulse protocol employed in these experiments began with a step from
a holding potential of 2100 mV to a conditioning poten-
tial ranging from 290 mV to 30 mV in 5–10 mV
increments for 160 ms, which was followed by a second pulse to
210 mV for 50 ms after a brief step 1 ms to the holding potential. The short step to the holding
potential between pulses was inserted to facilitate the subtraction of capacitive transients and did not affect
inactivation. Fig. 3a shows a family of current traces obtained using this pulse protocol from a single oocyte
expressing the V410M channel, and Fig. 3b shows a plot of mean normalized peak current against condition-
ing prepulse potential derived from the multiple data sets such as that shown in Fig. 3a. Table 1 compares the
midpoint potential for steady-state activation of the V410M channel, calculated from individual fits of mul-
tiple data sets, with previously published values for the wildtype and L1014F variants of the Vssc1tipE sodium
channel Lee et al., 1999c. The |5 mV depolarizing shift in the midpoint potential for steady-state inacti-
vation of the V410M channel compared with wildtype channel was statistically significant P
= 0.0054, df
= 12
but the midpoint potentials for the V410M and L1014F channel
variants were
not significantly
different P
= 0.1097, df
= 13.
We evaluated the effect of the V410M mutation on the sensitivity of Vssc1tipE sodium channels to pyr-
ethroids in assays comparing the effects of cismethrin on the wildtype and V410M channel variants. Fig. 4a
shows sodium current traces obtained from an oocyte expressing the wildtype channel in the presence or
absence of cismethrin 0.3–600
µ M. As documented in
previous reports Smith et al. 1997, 1998; Lee et al., 1999c, cismethrin produced both a prolonged sodium
current “late current” during a 50 ms depolarization and a slowly decaying biphasic tail current following
repolarization. The amplitudes of both the late current and the tail current were concentration-dependent. The
decay of the falling phase of the tail current in the pres- ence of 30
µ M cismethrin was fitted to a single-
exponential curve with a time constant of 465 ±
262 ms n
= 5. Fig. 4b shows traces obtained from an oocyte
expressing the V410M channel in the presence or absence of the same concentrations of cismethrin. In
contrast to wildtype channel, the amplitudes of the late and tail currents were markedly reduced relative to the
amplitude of the peak current obtained prior to cis- methrin treatment. In addition, the tail current observed
in assays with the V410M variant decayed |10 times more rapidly time constant
= 47.2
± 38 ms; n
= 4 than that
obtained with wildtype channels. We quantified the extent of modification of the wild-
type and V410M sodium channel variants by different
23 S.H. Lee, D.M. Soderlund Insect Biochemistry and Molecular Biology 31 2001 19–29
Fig. 3. Voltage dependence of steady-state inactivation of the V410M variant of the Vssc1tipE sodium channel. a Family of sodium currents
obtained from a single oocyte by 50 ms depolarizations from 2100 mV to 210 mV following 160 ms conditioning prepulses from 2100 mV to
test potentials of 290 to 30 mV. b Steady-state inactivation curve derived from multiple data set such as that shown in a.
Fig. 4. Effects of the V410M mutation on the response of the Vssc1tipE sodium channel to cismethrin. Sodium currents were measured from
oocytes expressing wildtype a or V410M b channels using the indicated pulse protocols before and after bath application of cismethrin a the concentrations shown. c Comparison of the normalized amplitudes of cismethrin-induced late currents from the wildtype and channels n
= 2-4
independent experiments per data point. The late currents were measured at the end of a 50 ms depolarization.
cismethrin concentrations by determining the normalized amplitude
of the
cismethrin-induced late
current measured at the end of a 50 ms depolarization. Plots
of normalized late current amplitudes against cismethrin concentration [Fig. 4c] identified a threshold for detect-
able cismethrin modification of the wildtype channel of 0.3
µ M, whereas the threshold for detectable modifi-
cation of the V410M channel was 10 times higher 3
µ M. In these experiments the responses to cismethrin
were not saturated at the highest concentration attain- able, thus preventing the calculation of cismethrin con-
centrations causing half-maximal modification. As an alternative, we determined the concentrations of cis-
methrin that produced the same normalized late current amplitude in both channels. As shown in Fig. 4c,
600
µ M cismethrin was required to produce late currents
from V410M that were of the same amplitude as currents produced by 30
µ M cismethrin from wildtype channels.
By this criterion, the V410M mutation reduces the sensi- tivity of Vssc1tipE sodium channels to cismethrin by
approximately 20-fold. We also compared the sensitivities of wildtype and
24 S.H. Lee, D.M. Soderlund Insect Biochemistry and Molecular Biology 31 2001 19–29
V410M sodium channels to BTX. Fig. 5 shows typical sodium current traces obtained from oocytes expressing
wildtype Vssc1tipE sodium channels upon 50 ms depol- arizations from
2100 mV to 25 mV in the presence of 2
µ M BTX after the indicated number of 30 ms depolar-
izing prepulses from 2100 mV to 25 mV at a pulse fre-
quence of 25 Hz. The extent of sodium channel modifi- cation by BTX was strongly use-dependent. There was
little or no detectable modified current after 10 prepul- ses, but further stimulation produced a sustained late cur-
rent without inducing a tail current following repolariz- ation. To compare the sensitivities of the wildtype and
V410M chanel variants to BTX, we determined the con- centration-dependent modification of sodium currents
measured after 1000 depolarizing 30 ms prepulses deliv- ered at a frequency of 25 Hz. Fig. 6a and b shows
typical sodium currents obtained from oocytes express- ing either the wildtype or V410M channel, respectively,
following exposure to BTX concentrations ranging from 0.08
µ M to 4.2
µ M. The responses of both channel vari-
ants to BTX in these assays was very similar. Compari- son of the normalized amplitudes of the BTX-induced
late currents obtained in such assays [Fig. 6c] showed that the BTX sensitivity of the V410M channel appeared
to be slightly less than that of the wildtype channel, but the mean amplitudes of the normalized late currents
obtained at each BTX concentration did not differ sig- nificantly between the two channel variants.
4. Discussion
