20 S.H. Lee, D.M. Soderlund Insect Biochemistry and Molecular Biology 31 2001 19–29
residue of sodium channels from pyrethroid-resistant strains of several other insect species Miyazaki et al.,
1996; Dong, 1997; Guerrero et al., 1997; Martinez- Torres et al., 1998; Schuler et al., 1998; Lee et al.,
1999b, whereas some resistant populations of H. vires- cens have a histidine replacing leucine at this position
Park and Taylor, 1997. In the house fly, a second mutation M918T in the short cytoplasmic domain
between transmembrane segments IIS4 and IIS5 is found together with the L1014F mutation and is correlated with
enhanced pyrethroid resistance in strains carrying the super-kdr trait Williamson et al., 1996. The functional
significance of the L1014F and M918T mutations has been confirmed by inserting these mutations, singly or in
combination, into the wildtype house fly Vssc1 sodium channel sequence, expressing both wildtype and mutated
channels in Xenopus laevis oocytes, and documenting the reduced sensitivity of the mutated channels to pyr-
ethroids Smith et al., 1997; Lee et al., 1999c.
Although mutations at leucine residues aligning with Leu1014 of the house fly sodium channel are most com-
monly associated with knockdown resistance, other sodium channel point mutations are also associated with
kdr-like resistance in some species Williamson et al., 1996; Guerrero et al., 1997; Park et al., 1997; Pittendrigh
et al., 1997; Head et al., 1998; Schuler et al., 1998. Among these mutations, the V410M
2
mutation found in some H. virescens populations Park et al., 1997 is of
particular interest for three reasons. First, this mutation occurs in a position in transmembrane segment IS6 that
is similar to the location of the L1014F mutation in transmembrane segment IIS6. Second, neurons from
insects carrying this mutation have sodium channels with altered voltage dependence and pharmacological proper-
ties Lee et al., 1999a, but these altered properties have not been definitively correlated with the V410M
mutation. Finally, this mutation occurs in a region of the sodium channel protein where other mutations are
already known to alter both the pharmacology and func- tion of sodium channels. As shown in Fig. 1, Val410 of
the house fly Vssc1 protein aligns with Ile433 of the rat skeletal muscle sodium channel rSkM1 protein.
Mutagenesis studies with rSkM1 have shown that the I433K, N434K and L437K mutations each render the
rSkM1 channel completely insensitive to batrachotoxin BTX Wang and Wang, 1998, whereas the N434A
mutation confers partial resistance to BTX Wang and Wang, 1998, produces depolarizing shifts in the voltage
dependence of activation and steady-state inactivation and enhances slow inactivation Wang and Wang, 1997.
Photoaffinity labeling studies with a BTX derivative also
2
For clarity and consistency, all mutations in insect sodium channel genes are numbered throughout this report according to the amino-acid
sequence numbering of the house fly Vssc1 sodium channel GenBank accession number U38813.
Fig. 1. Alignment of the domain IS6 amino-acid sequences of the
house fly Vssc1 and rat skeletal muscle rSkM1 sodium channel α
subunits, with identical amino-acid residues indicated in bold face, illustrating the relationship between the resistance-associated mutation
in Vssc1 and mutations that alter the properties and BTX sensitivity of rSkM1.
implicate this region of domain IS6 as part of the BTX binding site Trainer et al., 1996.
In this paper, we describe the introduction of the V410M mutation into the wildtype house fly Vssc1
sodium channel by site-directed mutagenesis, the expression of functional wildtype and mutated channels
in Xenopus oocytes, and the biophysical properties and sensitivity to cismethrin and BTX of the expressed chan-
nels. Our results document the reduced sensitivity to cis- methrin but not BTX of channels containing the
V410M mutation.
2. Materials and methods
2.1. Site-directed mutagenesis and cDNA clone construction
The complete wildtype Vssc1 cDNA sequence, methods for preparing fly head first-strand cDNA, and
methods for amplification by the polymerase chain reac- tion PCR of segments of the Vssc1 cDNA from this
template are described elsewhere Ingles et al., 1996. The Vssc1 cDNA was amplified as two contiguous frag-
ments of 3.7 and 2.7 kb flanking a unique AatII restric- tion site as described previously Smith et al., 1997. For
the present study, the 3.7 kb fragment was mutated to introduce the resistance-associated V410M mutation
GTG to ATG by using oligonucleotide-mediated mutagenesis Altered Sites II in vitro mutagenesis sys-
tem, Promega, Madison, WI. A full-length cDNA clone containing the V410M mutation was assembled as
described previously Smith et al., 1997 and its integrity was confirmed by DNA sequencing prior to its use as
the template for cRNA synthesis. The D. melanogaster tipE cDNA, used as a template for in vitro transcription,
was obtained by PCR as described previously Smith et al., 1997.
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
