tant housefly phenotype. Our results indicate that the alleles analyzed from natural populations represent only a subset of mutations that can confer resistance.
Ó Springer-Verlag 1997
Mol Gen Genet (1997) 256: 602±610
ORIGINAL PAPER
B. Pittendrigh á R. Reenan á R. H. rench-Constant B. Ganetzky
Point mutations in the Drosophila sodium channel gene para associated with resistance to DDT and pyrethroid insecticides
Received: 9 May 1997 / Accepted: 21 July 1997
Abstract The gene para in Drosophila melanogaster encodes an a subunit of voltage-activated sodium channels, the presumed site of action of DDT and pyrethroid insecticides. We used an existing collection of Drosophila para mutants to examine the molecular basis of targetsite resistance to pyrethroids and DDT. Six out of thirteen mutants tested were associated with a largely dominant, 10- to 30-fold increase in DDT resistance. The amino acid lesions associated with these alleles de®ned four sites in the sodium channel polypeptide where a mutational change can cause resistance: within the intracellular loop between S4 and S5 in homology domains I and III, within the pore region of homology domain III, and within S6 in homology domain III. Some of these sites are analogous with those de®ned by knockdown resistance (kdr) and super-kdr resistance-associated mutations in house¯ies and other insects, but are located in dierent homologous units of the channel polypeptide. We ®nd a striking synergism in resistance levels with particular heterozygous combinations of para alleles that appears to mimic the super-kdr double mutant house¯y phenotype. Our results indicate that the alleles analyzed from natural populations represent only a subset of mutations that can confer resistance. The implications for the binding site of pyrethroids and mechanisms of target-site insensitivity are discussed. Key words para á Voltage-gated sodium channel á Insecticide resistance á DDT/pyrethroids á Drosophila melanogaster Communicated by M. Ashburner B. Pittendrigh á R. H. rench-Constant Department of Entomology and Center for Neuroscience, University of Wisconsin-Madison, 237 Russell Laboratories, 1630 Linden Drive, Madison, WI 53706, USA R. Reenan á B. Ganetzky (&) Department of Genetics, University of Wisconsin-Madison, Henry Mall, Madison, WI 53706, USA B. Pittendrigh and R. Reenan contributed equally to this work
Introduction Voltage-gated sodium channels play a key role in neural activity of both vertebrates and invertebrates by mediating the rapid in¯ux of sodium ions in the rising phase of action potentials. In vertebrates, the major functional component of the channel is the a subunit. This large (ca. 260-kDa) polypeptide may be associated with smaller b1 or b2 subunits in the mammalian brain (Catterall 1988). In insects the a subunit is coded for by the para locus, ®rst identi®ed in Drosophila mutants that showed a temperature-sensitive (ts) paralytic phenotype (Suzuki et al. 1971; Loughney et al. 1989). Although expression of para mRNA alone in Xenopus oocytes can produce functional sodium channels, expression is enhanced by co-injection of mRNA transcribed from the tip-E gene, which may encode a functional equivalent of the vertebrate b subunit (Feng et al. 1995; Warmke et al. 1997). Sodium channels are the primary sites of action of pyrethroid insecticides and DDT (eectively a type I pyrethroid) (Soderlund and Bloomquist 1989). Previous indirect evidence suggested that target site insensitivity to both pyrethroids and DDT resulted from genetic modi®cation of sodium channels. Recent studies of kdrtype resistance in house¯ies and other insects support this view (Miyazaki et al. 1996; Williamson et al. 1996; Dong 1997). Although possibly pre-selected by the use of DDT, the kdr phenotype was ®rst recognized in house¯ies as an ability to resist the rapid ``knockdown'' paralysis usually observed after pyrethroid exposure. Two mutant kdr alleles are found in house¯ies: kdr, which confers resistance to pyrethroids and also high levels of cross-resistance to type II pyrethroids, and super-kdr, which confers up to 500-fold resistance to type II pyrethroids such as deltamethrin (Sawicki 1978). Linkage studies in house¯ies, cockroaches, and the tobacco budworm, Heliothis virescens, have shown that RFLPs within the para orthologs of these species are tightly associated with the kdr phenotype (Dong and
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Scott 1994; Knipple et al. 1994; Taylor et al. 1993; Williamson et al. 1993). Recently, para orthologs have been sequenced from both kdr and super-kdr house¯ies (Miyazaki et al. 1996; Williamson et al. 1996) and also from kdr-type resistant cockroaches (Miyazaki et al. 1996; Dong 1997). The kdr mutation in both house¯ies and cockroaches results from an L to F substitution in the S6 hydrophobic segment of homology domain II (Miyazaki et al. 1996; Williamson et al. 1996; Dong 1997). The super-kdr allele in house¯ies has the same L to F substitution in IIS6, but in addition has a second mutation resulting in an M to T change in the intracellular loop between IIS4 and IIS5. It has been suggested that these two mutations de®ne the binding site of pyrethroids at the intracellular mouth of the channel pore, in a region implicated as being important in channel inactivation (Williamson et al. 1996). Although the existence of independent mutant alleles of para orthologs with identical amino acid substitutions that confer pyrethroid resistance in several dierent insect species argues that this position is critical for binding of pyrethroids (Williamson et al. 1996), these mutations could represent a selected subset of all sodium channel mutations that alter the binding or eect of pyrethroids because other resistance mutations might also impair sodium channel function in other ways such that these mutations would not persist in natural populations. We have taken a dierent approach to examine this question in Drosophila. A large collection of viable para gene mutants has been identi®ed in Drosophila on the basis of their behavioral phenotypes without regard to insecticide resistance. These mutants are expected to represent amino acid substitutions distributed throughout the para polypeptide. We have now examined this collection of mutants to determine whether any of them also confer pyrethroid resistance. Our results suggest that pyrethroid resistance can be conferred by mutations at a larger number of residues in the sodium channel than have been identi®ed in natural populations. The implications for the binding site of pyrethroids and mechanisms of target-site insensitivity are discussed.
The para locus encompasses more than 85 kb of genomic DNA and contains an open reading frame (ORF) of about 6 kb (Hong and Ganetzky 1994; Loughney et al. 1989; Thackeray and Ganetzky 1994). Consequently, it was not feasible to sequence the entire coding region of each mutant allele. Instead, a modi®ed singlestrand conformation polymorphism (SSCP) technique was used to scan the entire ORF and pinpoint lesions caused by each mutation, and the relevant region was then sequenced. Full experimental details of this SSCP analysis of para mutants will be reported elsewhere (R. Reenan and B. Ganetzky, in preparation). Brie¯y, total RNA was isolated from each strain by LiCl/urea extraction and stored in 80% ethanol at )80° C prior to using this RNA as a template for the synthesis of cDNA by RT-PCR. Firststrand synthesis was performed using MMLV reverse transcriptase (Gibco-BRL) and a para-speci®c primer. cDNAs were then used as template for PCR using Taq polymerase and para-speci®c primers, which were designed with convenient restriction enzyme sites incorporated for later cloning steps. The para PCR ampli®cation products were analyzed for sequence polymorphisms by SSCP analysis on MDE acrylamide gels (A-T Biochem), following the manufacturer's instructions. When a polymorphism was detected, the PCR products were cloned into pBluescript and a minimum of three independent clones were sequenced. In every case, only one missense mutation was detected per mutant line in the para coding region. All other polymorphisms detected resulted in silent changes.
Materials and methods
Results
Drosophila melanogaster strains
DDT and pyrethroid resistance of ¯ies bearing parats alleles
The various para alleles examined here were generated by mutagenesis with ethyl methansulfonate (EMS) and identi®ed on the basis of their temperature-sensitive paralytic or other behavioral phenotypes. The alleles parats1(Suzuki et al. 1971), parats2(Grigliatti et al. 1973), paraST76and paraST42(Siddiqi and Benzer 1976), para115(Ganetzky 1984), paraDTS2 (Loughney et al. 1989) and parasbl1(Lilly et al. 1994) have all been previously described. The mutations para63, para74, and para103 were identi®ed as suppressors of the Shaker mutation (Stern et al. 1990). paraMS2 was identi®ed by R. Kreber in the laboratory of B. Ganetzky among a collection of EMS-mutagenized X chromosomes. paraDN7and paraDN43 were isolated and provided by Dr. Satpal Singh (State Univ. of New York, Bualo). Although not all of the above mutants were derived from the same parental line, sequencing of several susceptible strains revealed no pre-existing polymorphisms. Two susceptible
strains, Canton-S and Harwich, were used as control lines in insecticide bioassays. Dp(peb) is an insertional translocation of a segment of the X chromosome containing para+ into the second chromosome. It is referred to by Lindsley and Zimm (1985) as Tp(1;2)r+75c. Assays of insecticide resistance The various para mutant strains were tested for insecticide resistance according to previously described protocols (rench-Constant et al. 1990). Brie¯y, 10±30 adults, 3±5 days old, were exposed to insecticide-coated glass scintillation vials for 24 h at 25° C. Control vials were treated with the solvent, acetone, alone. After 24 h, mortality was scored as inability to locomote. To test for metabolic resistance conferred by the activity of a mixed function oxidase, mutants were tested following pre-treatment in vials coated with 100 lg of piperonyl butoxide (PBO), an inhibitor of these enzymes. Dose-response curves were estimated from a minimum of ®ve dierent concentrations of insecticide with at least three replicates per dose. Probit analysis was performed using the computer program POLO (Robertson et al. 1980). Sequence analysis of mutant para alleles
We examined DDT resistance in a group of 12 independently isolated para mutants, including two alleles that cause dominant temperature-sensitive paralysis, nine that cause recessive temperature-sensitive paralysis, and one that causes a smell-blind phenotype (Fig. 1 and Table 1). Six of these mutants showed signi®cant (10±30 fold) resistance to DDT (Fig. 1 and Table 1). Resistance to DDT could result from mechanisms other than target site insensitivity. For example, increased mixed function oxidase activity is also known to confer resistance to a wide range of insecticides in some strains of Drosophila
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when they inherited their X chromosome from resistant females (compare Fig. 2a with 2c). Resistance in females was largely dominant; heterozygous females showed resistance irrespective of whether the resistant parent was a male or a female. In contrast, metabolic resistance associated with oxidase activity is known to have an autosomal pattern of inheritance (Dapkus 1992). Second, DDT resistance in the para mutants was not altered by treating the ¯ies with PBO, an inhibitor of mixed function oxidase activity. Similar treatment of 91-R, a positive control strain known to be associated with metabolic resistance (Waters and Nix 1988) resulted in a signi®cant decrease in resistance (Table 2). Finally, for two of the para alleles tested, parats1 and paraDTS2, a second independently isolated mutation exists (parats2 and paraDN43 respectively) with the same amino acid substitution. Since both pairs of mutant alleles exhibit similar levels of DDT resistance, the common lesion shared by these mutations is the most likely cause of resistance. Resistance-associated point mutations The amino acid substitution associated with each of the resistant para alleles is shown in the alignments with other sodium channel sequences in Fig. 3 and the relative positions of the mutations are shown in Fig. 4, which presents a schematic diagram of the proposed membrane topology of a sodium channel a subunit. It is of interest to note that two of the lesions (those in paraDN7 and parats1/parats2) reside at locations analogous to those of the super-kdr mutation in the house¯y ± in the intracellular loop between S4 and S5, but in homology domains I and III rather than II. Similarly, one of the para mutations (para74) occupies a site in S6 of homology domain III that is close to that of the kdr mutation in S6 of homology domain II. The remaining lesion in two of the para mutations (paraDTS2/paraDN43) identi®es a site in the pore region of homology domain III, a site not previously implicated in DDT or pyrethroid resistance. Fig. 1a±c DDT dose-response relationships for ®ve parats mutants compared to a standard susceptible strain Canton-S. a para74 compared to Canton-S. b paraDN43 and paraDTS2, which share the same point mutation. c parats1 and parats2, which share the same underlying point mutation
(Waters and Nix 1988; Sundseth et al. 1990) and DDT dehydrochlorinase may also be responsible for DDT metabolism. Several observations indicate that it is the para mutation that confers DDT resistance in our strains, rather than genetic polymorphisms in the background associated with increased metabolic activity. First, the DDT resistance in each para mutant line co-segregates with the X chromosome bearing the para allele in reciprocal crosses between sensitive and resistant ¯ies (Fig. 2). Male ospring were resistant only
Synergistic levels of resistance in heteroallelic combinations The coincidence of the lesions in the resistant para alleles with analogous mutations in kdr and super-kdr strains in house¯ies and other insects led us to examine the eects of combining dierent para alleles in heterozygous combination. The high levels of resistance in super-kdr house¯y strains are associated with a sodium channel gene mutated at two sites in homology domain II. The para74 and paraDN7 alleles mutate analogous sites in homology domain III. We therefore asked whether a heterozygous combination of these alleles could mimic the enhanced resistance conferred by the doubly mutant super-kdr house¯y sodium channel. Although para74 and
605
para DN7 alone confer only modest levels of resistance to the type II pyrethroid, deltamethrin (2.58 and 9.76-fold, respectively), the para74/paraDN7 heterozygote showed strikingly increased levels of resistance to deltamethrin (105-fold, Table 3). Similar to super-kdr mutants in house¯ies, the para74/paraDN7 heterozygote also had increased resistance to DDT but to a lesser extent than to deltamethrin (data not shown). Furthermore, this
Table 1 Resistance levels of parats mutant lines of D. melanogaster to dichloro-diphenyltrichloro ethane (DDT)
Fig. 2a±d Sex-linkage of para-associated DDT resistance. Doseresponse curves for male and female progeny from reciprocal crosses of either resistant para females (a, b) or resistant para males (c, d) to the susceptible Canton-S strain. Note that both male and female progeny of resistant para females are resistant (a, b), whereas only the female progeny of resistant para males are themselves resistant (compare d with c)
Line
na
Susceptible Canton-S MS2 para paraST76 parats115 paraST42 para63 para103 parasb11
860 1020 990 1320 1220 1320 1380 1320
2.86 2.77 3.36 3.37 3.41 5.88 6.07 6.58
Resistant para74 parats1 paraDN7 paraDTS-2
2350 2860 1380 855
29.04 40.69 51.03 58.34
a b c
LC50b (95% CL)
Slope (S.E.)
Resistance ratioc
(2.22±3.40) (1.99±3.43) (2.87±3.79) (2.93±3.76) (2.97±3.80) (4.24±7.21) (5.30±3.96) (4.31±7.85)
2.41 3.33 3.05 3.40 3.89 2.80 2.60 3.26
(0.22) (0.22) (0.21) (0.21) (0.26) (0.23) (0.15) (0.50)
1.00 0.97 1.17 1.18 1.19 2.06 2.12 2.30
(24.61±33.56) (32.93±48.67) (25.98±74.66) (52.40±64.95)
2.03 1.57 1.28 4.26
(0.09) (0.08) (0.19) (0.36)
10.15 14.23 17.84 20.40
Total number of ¯ies tested (not including controls) Expressed as lg per vial Resistance ratio with reference to Canton-S
606 Table 2 Eects of piperonyl butoxide on resistance levels of lines of D. melanogaster resistant to dichloro-diphenyl-trichloro ethane (DDT)
Line
Treatment
na
LC50b (95% CL)
Slope (S.E.)
Resistance ratioc
Canton-S
No PBO PBO No PBO PBO No PBO PBO No PBO PBO No PBO PBO No PBO PBO
482 510 505 468 286 320 383 375 468 400 150 390
2.62 2.96 28.22 20.76 26.34 24.49 39.79 22.32 57.79 63.37 400 107.86
2.82 3.98 2.42 1.83 1.67 1.55 2.00 1.58 4.36 3.56 ± 3.13
1.00 1.13 10.77 7.92 10.05 9.35 15.19 8.52 22.06 24.19 152 41.17
para74 parats1 paraDN7 paraDTS-2 91-R
(2.05±3.07) (2.38±3.42) (21.62±34.68) (9.99±29.31) (13.01±35.53) (15.16±31.38) (29.93±51.35) (9.53±31.09) (51.67±64.75) (53.01±77.48) (92.28±132.28)
(0.33) (0.45) (0.26) (0.25) (0.35) (0.24) (0.31) (0.29) (0.44) (0.33) (0.36)
a
Total number of ¯ies tested (not including controls) Expressed as lg per vial c Resistance ratio with reference to untreated Canton-S b
Table 3 Resistance levels of parats mutant lines of D. melanogaster to deltamethrin
Genotype
na
LC50b (95% CL)
Slope (S.E.)
Resistance ratioc
Canton-S parats115 para74 parats2 paraDN43 paraDN7 para74/paraDN7; Dp(peb) cn /+ para74/paraDN7; SM6/+
900 670 760 740 664 630 800 360
1.08 0.63 2.61 6.35 7.63 9.86 126.42 114.36
1.76 1.31 1.82 1.43 3.60 2.88 2.43 1.83
1.00 0.62 2.58 6.29 7.55 9.76 117.06 105.89
a b c
(0.81±1.55) (0.42±0.87) (1.54±3.59) (4.44±13.86) (5.78±9.11) (7.82±12.14) (98.40±163.06) (89.65±114.75)
(0.18) (0.16) (0.19) (0.20) (0.32) (0.29) (0.15) (0.20)
Total number of ¯ies tested (not including controls) Expressed as lg per vial Resistance ratio with reference to Canton-S
synergistic eect on resistance was dominant because it was not altered by the introduction of an extra copy of para+ carried by Dp(peb) (Table 3). Thus, a large increase in resistance to deltamethrin is seen not only in super-kdr double house¯y mutants, but also when comparable mutations are expressed on dierent sodium channel polypeptides in heterozygotes.
Discussion A range of point mutations in the para gene confer insecticide resistance The demonstration that a variety of mutant para alleles, originally identi®ed on the basis of their temperaturesensitive paralytic phenotypes, confer DDT resistance supports the conclusion emerging from studies in other insects that resistance to pyrethroids and related insecticides results from mutations in the gene encoding the primary structural component of sodium channels. Although both our analysis and investigations of naturally occurring pyrethroid resistance in other insect species support this conclusion, there are some important dierences in the results for these dierent studies that are worth noting. A striking outcome from the analysis of kdr mutations in both house¯ies and cockroaches is that both
share a leucine to phenylalanine substitution in the IIS6 membrane-spanning region (Miyazaki et al. 1996; Williamson et al. 1996; Dong 1997). The only other naturally occurring mutation is an additional methionine to threonine substitution in the intracellular loop between IIS4 and IIS5 in super-kdr house¯y strains, which are double mutants (Williamson et al. 1996). This limited allelic diversity has contributed to the conclusion that the sites identi®ed by these mutations are directly involved in pyrethroid binding (Williamson et al. 1996). However, there may be other constraints that also contribute to the restricted number of sodium channel mutations in natural populations that confer pyrethroid resistance. Because all aspects of electrical signaling in the nervous system are critically dependent on sodium channel function, mutations that even slightly impair the normal activity of these channels are likely to have deleterious eects and are therefore unlikely to be maintained in natural populations. Consequently, it may be inferred that the kdr alleles that have been found are those that confer pyrethroid resistance without interfering signi®cantly with normal sodium channel function. Perhaps there are only a limited number of ways in which a sodium channel polypeptide can be mutated while still satisfying both criteria. Our approach ± examining previously identi®ed mutations in a sodium channel structural gene for insecticide resistance ± is not subject to the same constraints. In
607
an experimental organism such as Drosophila, sodium channel mutations can be maintained in laboratory conditions even when these mutations are associated with severe behavioral and electrophysiological defects. Indeed, we have found a larger array of sodium channel mutations that confer resistance to DDT (four additional point mutations), indicating that the sites identi®ed in the kdr and super-kdr house¯y strains are not the only amino acid positions that aect the binding or action of pyrethroids. Nonetheless, it is noteworthy that several of the para mutations that confer DDT resistance occupy positions analogous to those of the kdr and super-kdr mutations but in dierent homology domains. For example, para74 lies within 10 amino acids of the residue altered in kdr strains, but in homology domain III rather than II. Similarly, the residues aected by parats1 in homology domain I and by paraDN7 in homology domain III lie within one amino acid of the corresponding position in homology domain II aected by the super-kdr house¯y mutation. Surprisingly, none of the mutations we identi®ed map to the second homology domain and represent the exact counterparts of kdr mutations in Drosophila. Indeed, none of over a dozen para alleles that we have characterized molecularly map to the second homology domain (R. Reenan and B. Ganetzky, unpublished). One possible explanation for this outcome depends on the complementary
Fig. 3a±d The amino acid replacements associated with the resistant para alleles analyzed in this study. In each case, the wild-type para sequence (para1) is shown on the top line. Shown above this line is the single amino acid replacement found in the indicated mutants. For comparison, the sequence in these regions is aligned with the sodium channel sequences from a group of vertebrate and invertebrates species. Abbreviations are: para1, para; lopa1, Lopa opalescens (squid); eSKM1, equine skeletal muscle; hSKM1, human skeletal muscle (SCN4A), FUGU, Fugu rubripes (®sh); rNaCH6, rat brain neuronal/glial sodium channel; rBRAIN2, rat brain II; rBRAIN3, rat brain III; rBRAIN1, rat brain I; hNE, human neuroendocrine cell sodium channel; hCARDIAC, human cardiac sodium channel gene (SCN5A); eelNaCH, Electrophorus electricus (eel); hATYP, human atypical sodium channel from heart and uterus; TUNA1, Halocynthia roretzi (ascidian); Lbleek, Loligo bleekeri (squid); jellyNaCH, Cyanea capillata (jelly®sh)
limitation of our approach compared with that associated with the analysis of naturally occurring kdr mutations: the only para alleles that we have identi®ed are those associated with striking behavioral defects. If for some reason mutations in homology domain II are less severe in their eects than the analogous mutation in one of the other homology domains, we would be less likely to identify such mutations. Thus, our failure to identify temperature-sensitive/resistant mutations in homology domain II, and the failure to identify naturally occurring DDT/pyrethroid resistance-associated mutations in homology domains other than II, may be opposite aspects
608 Fig. 4 A schematic diagram of the para voltage-gated sodium channel, indicating the locations of the four amino acid replacements in DDT-resistant parats mutants, alongside the relative location of the kdr and super-kdr replacements documented in homologs from the house ¯y and the German cockroach
of the same problem. This interpretation makes several predictions that can be tested by site-directed mutagenesis and functional expression of insect sodium channel in heterologous systems. Implications for the mechanism of resistance Regardless of the above interpretation, the collection of DDT-resistant para alleles identi®ed here sheds additional light on the mechanism of resistance. The lesion associated with both paraDN43 and paraDTS2 resides in the pore segment of homology domain III and represents a site not previously implicated in insecticide resistance. In addition, the resistance to both DDT (Table 1) and deltamethrin (Table 3) associated with parats1and paraDN7, suggests that mutations in the intracellular loop between the S4 and S5 segments in homology domains I and III, respectively, are sucient by themselves to confer resistance. The analogous mutation in homology domain II found in super-kdr house¯y strains has not been examined for resistance by itself because it is found only in association with the kdr mutation in IIS6. Our results suggest that a house¯y strain in which only the amino acid change caused by the super-kdr alteration was present would also show signi®cant pyrethroid resistance. Moreover, we had the unique opportunity in our experiments to examine the resistance phenotype of individuals that were heterozygous for two para mutations (paraDN7and para74) analogous to those found in doubly mutant super-kdr house¯y strains. Surprisingly, the heterozygous combination of these alleles conferred much greater resistance to deltamethrin than expected from the sum of their individual eects (approximately 10-fold resistance in the single mutants but 100-fold in the double mutants; Table 3). It has been suggested that the kdr and super-kdr mutations de®ne the binding site of pyrethroid insecticides. Our results suggest the possibility of a dierent interpretation. The identi®cation of DDT-resistant mutations at nearly equivalent sites to those altered in kdr and super-kdr, but in other homology domains, suggests that if these mutations do indeed de®ne a binding site, this site is likely to be composed of residues contributed
by each of the four homology domains. This possibility seems reasonable given the essentially tetrameric structure of the sodium channel a subunit. Moreover, it is also possible that the sodium channel mutations characterized here, and in previous studies, confer resistance to pyrethroids and DDT by a mechanism other than direct alteration of the binding site. The pharmacological eect of these insecticides is to cause persistent activation of sodium channels by delaying the normal voltage-dependent mechanism of inactivation (Soderlund and Bloomquist 1989). Rather than directly affecting the binding of pyrethroids and DDT, the mutations may confer resistance by causing functional changes in sodium channel properties that compensate for, or alleviate the consequences of exposure to the insecticide. For example, if the mutations altered the voltage dependence or kinetics of activation or inactivation, the neurotoxic eects of the insecticide could be reduced or overridden. This interpretation is particularly attractive in the case of the resistance mutations that map to the S4-S5 loop in the dierent homology domains, because evidence from various systems suggests the importance of this region in channel function. Mutations in this loop are known to reduce fast inactivation of potassium channels (Isaco et al. 1991). Furthermore, mutations in mammalian sodium channels at or near the site de®ned by the paraDN7 mutation are associated with a variety of abnormalities. One form of long-QT syndrome, an inherited cardiac arhythmia, is caused by an amino acid substitution in the SCN5A channel at a site adjacent to the residue aected by paraDN7 (Wang et al. 1995). At the identical residue as paraDN7, an alanine to threonine replacement in the human SCN4A channel causes paramyotonia congenita, a disorder associated with decreased kinetics of sodium channel inactivation (McClatchey et al. 1992; Yang et al. 1994). The same replacement in a mouse neuronal sodium channel produces the jolting phenotype (Kohrman et al. 1996). Functional studies indicate that this mutation signi®cantly shifts the voltage dependence of activation in the depolarizing direction. The parats1 mutation resides at the identical location in homology domain I and may cause functional perturbations analogous to those caused by mutations in the vicinity of paraDN7. The kdr/ para74 mutations in S6 transmembrane segments might
609
also alter the gating properties of the sodium channel in some distinctive manner. Our ®nding of synergistic resistance to pyrethroids in para74/paraDN7 heterozygotes is of particular interest. The lesions in these two mutations in homology domain III are in analogous locations to the two mutant sites in homology domain II in super-kdr strains of house¯ies, and the elevated levels of resistance in the heterozygote approximate to the enhanced resistance in kdr vs. superkdr strains. But the two circumstances are very dierent. In super-kdr mutants the two lesions reside in the same polypeptide, in para74/paraDN7 heterozygotes the lesions reside in dierent polypeptides. The increase in resistance in super-kdr house¯y strains is presumed to re¯ect a combined de®cit in pyrethroid binding by the doubly mutant polypeptide. This explanation seems inadequate to account for the phenotype of para74/paraDN7 heterozygotes. However, this phenotype can be reconciled with the alternative view that resistance is mediated via functional alterations of the encoded sodium channel polypeptides. The particular functional defect associated with one mutation could enhance the perturbation caused by the other. For example, one mutation could alter the inactivation mechanism, leading to a slight depolarization of membrane potential, and the other could alter the voltage dependence of inactivation. The combined eect in double heterozygotes would be very dierent from the eect of either mutation alone or when heterozygous with a wild-type allele. This interpretation is also consistent with the observed phenotypic dominance of the double heterozygote in the presence of an extra copy of the para locus encoding wild-type sodium channel subunits. This situation is analogous to the dominant eect of mutations causing hyperkalemic periodic paralysis (Ptacek et al. 1991; Rojas et al. 1991; McClatchey et al. 1992) in humans, where the malfunction of a small percentage of mutant sodium channels at the resting membrane potential shifts all the rest of the channels (including wild-type channels) into an inactivated state (Cannon et al. 1991). Direct electrophysiological studies of the properties of normal and resistant sodium channels will be needed to resolve these various possibilities. In any case, it is clear that the array of sodium channel mutations available in Drosophila will provide an important additional tool in dissecting the mechanism of target-site resistance to pyrethroids and related insecticides. Acknowledgements We thank R. Kreber for technical assistance. Supported by PHS Grants to GM43100 to B.G and NS29623 to R. -C. This is paper 3493 from the Laboratory of Genetics, University of Wisconsin-Madison.
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