Insect Biochemistry and Molecular Biology 31 2001 19–29 www.elsevier.comlocateibmb
The V410M mutation associated with pyrethroid resistance in Heliothis virescens reduces the pyrethroid sensitivity of house fly
sodium channels expressed in Xenopus oocytes
Si Hyeock Lee
1
, David M. Soderlund
Department of Entomology, New York State Agricultural Experiment Station, Cornell University, Geneva, NY 14456, USA Received 22 November 1999; received in revised form 18 April 2000; accepted 19 April 2000
Abstract
Some strains of Heliothis virescens carry a novel sodium channel mutation, corresponding to the replacement of Val410 by Met designated V410M in the house fly Vssc1 sodium channel, that is genetically and physiologically associated with pyrethroid
resistance. To test the functional significance of this mutation, we created a house fly Vssc1 sodium channel containing the V410M mutation by site-directed mutagenesis, expressed wildtype and specifically mutated sodium channels in Xenopus laevis oocytes, and
evaluated the effects of the V410M mutation on the functional and pharmacological properties of the expressed channels by two- electrode voltage clamp. The V410M mutation caused depolarizing shifts of |9 mV and |5 mV in the voltage dependence of
activation and steady-state inactivation, respectively, of Vssc1 sodium channels. The V410M mutation also reduced the sensitivity of Vssc1 sodium channels to the pyrethroid cismethrin at least 10-fold and accelerated the decay of cismethrin-induced sodium tail
currents. The degree of resistance conferred by the V410M mutation in the present study is sufficient to account for the degree of pyrethroid resistance in H. virescens that is associated with this mutation. Although Val410 is located in a sodium channel segment
identified as part of the binding site for batrachotoxin, the V410M mutation did not alter the sensitivity of house fly sodium channels to batrachotoxin. The effects of the V410M mutation on the voltage dependence and cismethrin sensitivity of Vssc1 sodium channels
were indistinguishable from those caused by another sodium channel point mutation, replacement of Leu1014 by Phe L1014F, that is the cause of knockdown resistance to pyrethroids in the house fly. The positions of the V410M and L1014F mutations in
models of the tertiary structure of sodium channels suggest that the pyrethroid binding site on the sodium channel
α subunit is
located at the interface between sodium channel domains I and II.
2001 Elsevier Science Ltd. All rights reserved.
Keywords: Insecticide; Resistance; Pyrethroid; Batrachotoxin; Voltage-sensitive sodium channel; Musca domestica
1. Introduction
In the house fly Musca domestica, the knockdown resistance kdr trait confers resistance to pyrethroid and
diphenylethane [e.g.,
dichlorodiphenyltrichloroethane DDT] insecticides by a reduction in the sensitivity of
the nervous system to these compounds, and resistance traits similar to kdr have also been identified in numer-
ous other insect species Soderlund and Bloomquist, 1990; Soderlund, 1997. In the house fly Williamson et
Corresponding author. Tel.: +
1-315-787-2364; fax: +
1-315-787- 2326.
E-mail address: dms6cornell.edu D.M. Soderlund.
1
Present address: Department of Entomology, Fernald Hall, Uni- versity of Massachusetts, Amherst, MA 01003, USA.
0965-174801 - see front matter
2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 0 0 0 0 0 8 9 - 8
al., 1993; Knipple et al., 1994, Heliothis virescens Taylor et al., 1993, Blattella germanica Dong and
Scott, 1994 and Leptinotarsa decemlineata Lee et al., 1999b, these resistance traits are genetically linked to
sodium channel
α subunit genes that are orthologous to
the para sodium channel gene of Drosophila melanogas- ter Loughney et al., 1989.
Comparison of sodium channel gene sequences from susceptible and knockdown-resistant insects has ident-
ified point mutations in sodium channel gene sequences that are associated with knockdown resistance traits.
Replacement of Leu1014 by Phe designated L1014F in transmembrane segment IIS6 of the house fly sodium
channel is associated with the kdr trait of the house fly Miyazaki et al., 1996; Williamson et al., 1996. The
same mutation is also found at the corresponding leucine
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
