• Insect;
  • Nicotinic acetylcholine receptor;
  • cDNA cloning;
  • Drosophila S2 cells;
  • Imidacloprid;
  • Ligand binding


  1. Top of page
  2. Abstract
  4. RT-PCR and rapid amplification of cDNA ends (RACE)
  6. Cloning and sequencing of full-length cDNAs of novel nAChR subunits
  8. Acknowledgements
  9. References

Abstract: The recent introduction of the chloronicotinyl insecticide imidacloprid, targeting insect nicotinic acetylcholine receptors (nAChRs), emphasises the importance of a detailed molecular characterisation of these receptors. We are investigating the molecular diversity of insect nAChR subunit genes in an important agricultural pest, the peach-potato aphid Myzus persicae. Two M. persicaeα-subunit cDNAs, Mpα1 and Mpα2, have been cloned previously. Here we report the isolation of three novel α-subunit genes (Mpα3-5) with overall amino acid sequence identities between 43 and 76% to characterised insect nAChR subunits. Alignment of their amino acid sequences with other invertebrate and vertebrate nAChR subunits suggests that the insect α subunits evolved in parallel to the vertebrate neuronal nAChRs and that the insect non-α subunits are clearly different from vertebrate neuronal β and muscle non-α subunits. The discovery of novel subtypes in M. persicae is a further indicator of the complexity of the insect nAChR gene family. Heterologous co-expression of M. persicae nAChR α-subunit cDNAs with the rat β2 in Drosophila S2 cells resulted in high-affinity binding of nicotinic radioligands. The affinity of recombinant nAChRs for [3H]imidacloprid was influenced strongly by the α subtype. This is the first demonstration that imidacloprid selectively acts on Mpα2 and Mpα3 subunits, but not Mpα1, in M. persicae.

Nicotinic acetylcholine receptors (nAChRs) play an important role in synaptic excitatory transmission at both neuronal and neuromuscular junctions of vertebrates. In insects, nAChRs are restricted to the nervous system (Sattelle, 1980; Breer and Sattelle, 1987) but are of particular interest because they are one of only a few validated target sites for insecticides (Benson, 1992; Soderlund, 1995). The recent introduction of imidacloprid, a chloronicotinyl insecticide targeting insect nAChRs (Leicht, 1996; Narahashi, 1996) and especially active against sucking pests (Elbert et al., 1996), emphasises the importance of investigating nAChR structure and function to provide a better understanding of the action of this class of insecticides and of potential changes that might confer target site resistance.

To date, 16 nAChR subunit genes have been identified and characterised in vertebrates (Lindstrom, 1995; McGehee and Role, 1995). Whereas muscle nAChRs form pentameric complexes of known subunit composition (α2βγδ or α2βεδ), the stoichiometries of neuronal nAChRs in vivo are still not well known, although various combinations drawn from eight α (α2—α9) and three β (β2—β4) isoforms can reconstitute functional receptors in vitro (Brammar, 1996). Molecular probes from various vertebrate nAChR isoforms have been used successfully to clone some insect nAChR genes. However, relatively few nAChR subtypes have been found in insects (Gundelfinger, 1992; Gundelfinger and Hess, 1992). Five nAChR genes have been cloned from Drosophila melanogaster, of which ALS (Bossy et al., 1988), SAD/Dα2 (Baumann et al., 1990; Sawruk et al., 1990a), and Dα3 (Schulz et al., 1998) are α-like and ARD (Hermans-Borgmeyer et al., 1986; Sawruk et al., 1988) and SBD (Sawruk et al., 1990b) are non-α-like. Two α-subunit cDNAs have been recently cloned from Myzus persicae, Mpα1 and Mpα2 (Sgard et al., 1998). Four α- (Locα1-4) and a non-α- (Locβ1) subunit cDNAs have been cloned from Locusta migratoria (Hermsen et al., 1998) and αL1 from Schistocerca gregaria (Marshall et al., 1990). In addition, two α subunits, MARA1 from Manduca sexta (Eastham et al., 1998) and Hvα1 from Heliothis virescens (AJ000399; unpublished), have also been identified. There have, however, been few reports of functional expression of cloned insect nAChRs. Homo-oligomeric insect nAChRs expressed in the Xenopus oocyte system typically generated only small wholecell currents (Marshall et al., 1990; Amar et al., 1995; Sgard et al., 1998). The three Drosophilaα subunits have been shown to form functional channels when co-expressed with a vertebrate β subunit in oocytes (Bertrand et al., 1994; Lansdell et al., 1997; Schulz et al., 1998). However, expression of Drosophila nAChR subunits in a variety of heterologous systems indicated that the folding and assembly of receptor subunits are temperature sensitive (Lansdell et al., 1997). Both evolutionary considerations (Le Novere and Changeux, 1995; Ortells and Lunt, 1995; Tsunoyama and Gojobori, 1998) and functional expression studies (Bertrand et al., 1994; Lansdell et al., 1997; Schulz et al., 1998) suggest that additional insect nAChR subunits remain to be cloned.

Radioligand binding and electrophysiological studies have revealed that imidacloprid has selective toxicity on insects over mammals. [3H]Imidacloprid binds to head membranes of various insect species with a high affinity but with only low affinity to mammalian brain membranes (Liu and Casida, 1993; Chao and Casida, 1997; Chao et al., 1997). Electrophysiological studies using cockroach Periplaneta americana neurons showed that imidacloprid behaved largely as an agonist on at least two distinct populations of nAChRs that are sensitive or insensitive to α-bungarotoxin, respectively (Bai et al., 1991; Buckingham et al., 1997). In contrast, Nagata et al. (1998) reported that imidacloprid had both multiple agonist and antagonist effects on the neuronal nAChRs of clonal rat phaeochromocytoma (PC12) cells. More recently, Lind et al. (1998) have shown that there are two imidacloprid binding sites with high and low affinity in hemipteran insects including M. persicae, whereas only a single site exists in nonhemipteran insects such as Drosophila and houseflies. Thus, it is very likely that the nAChR is the molecular target of imidacloprid. However, its selectivity for nAChR subunits has not yet been investigated.

In this article, we report the cloning of a further three nAChR α subunit (Mpα3-5) cDNAs from M. persicae, an important plant-sucking pest. Co-expression of M. persicaeα subunits with the rat β2 subunit in Drosophila S2 cells combined with radioligand binding studies indicate their pharmacological diversity. For the first time, we demonstrate that imidacloprid selectively targets Mpα2 and Mpα3 subunits, but not Mpα1, in M. persicae. This will enable us to elucidate the molecular basis of any target site resistance to this compound that develops in response to selection pressure in the field.


  1. Top of page
  2. Abstract
  4. RT-PCR and rapid amplification of cDNA ends (RACE)
  6. Cloning and sequencing of full-length cDNAs of novel nAChR subunits
  8. Acknowledgements
  9. References


A clone (794J) of M. persicae, resistant to organophosphate insecticides but sensitive to imidacloprid, was used in this study.

Oligonucleotide primers

Degenerate primers, ACHR1 (ATT/C/A ATG ACI ACI AAT/C GTI TGG), ACHR2 (GCI GAT/C GGI AAC/T TAC/T GAG/A GT), ACHR3 (ATG AAG/A TTC/T GGI TCI TGG AC), and ACHR4 (ACI GTG/A TAG/A AAI AG/AI GTC/T TT), were designed from the conserved N-terminal regions of insect nAChR subunits. Gene-specific primers were based on the nucleotide sequences of individual genes.

RT-PCR and rapid amplification of cDNA ends (RACE)

  1. Top of page
  2. Abstract
  4. RT-PCR and rapid amplification of cDNA ends (RACE)
  6. Cloning and sequencing of full-length cDNAs of novel nAChR subunits
  8. Acknowledgements
  9. References

Total RNA was isolated from adult aphids by the modified guanidine-HCl method (Sambrook et al., 1989). Synthesis of first-strand cDNAs and RT-PCR were performed as described previously (Williamson et al., 1996). RACE was carried out using the 5′ and 3′ RACE Systems (GibcoBRL) according to the manufacturer’s instructions.

Cloning and sequencing

PCR fragments were recovered from agarose gels using the QIAquick gel extraction kit (Qiagen) and cloned into the pUAg vector (Ingenius), and plasmid DNAs were prepared using small- or medium-scale purification kits (Qiagen). Doublestrand plasmid DNAs (200-500 ng) and PCR fragments (30-90 ng) were taken for Taq Dye terminator cycle sequencing reactions (PE Biosystems) and analysed on a model 373A automatic DNA sequencer (PE Biosystems).

Northern hybridisation

Poly(A)+ RNA was prepared from adult aphids using the QuickPrep mRNA Purification Kit (Pharmacia), separated on an agarose gel containing formaldehyde, and transferred onto Hybond-N+ nylon membrane (Amersham). PCR fragments containing C-terminal cytoplasmic loops of Mpα3 (Leu258-end) and Mpα4 (Gly283-end) (Fig. 1) were 32P-labelled by nick translation (Amersham) and hybridised in a solution of 50% formamide, 5 × Denhardt’s, 6 × saline/sodium citrate (SSC), 0.5% sodium dodecyl sulfate (SDS), and 200 μg/ml salmon sperm DNA at 42°C overnight. The membranes were washed twice in 2 × SSC and 0.5% SDS for 20 min at 65°C and twice in 1 × SSC and 0.1% SDS for 20 min at 65°C.


Figure 1. Amino acid sequence alignments of M. persicae nAChR α subunits using the PILEUP program of the Genetics Computer Group package. Predicted signal peptide (SP) and transmembrane domains (TM1, TM2, TM3, and TM4) are underlined. Two conserved N-glycosylation sites (CHO) are shown. Key residues thought to be involved in ligand binding (#) (Hucho et al., 1996; Arias, 1997) in the N termini and to form “rings” (*) (Arias, 1997) around TM2 are indicated. Four important cysteines corresponding to positions 128, 142, 192, and 193 in the Torpedoα subunit and unusual residue substitutions mentioned in the text are in bold and larger font.

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Sequence analysis and construction of phylogenetic tree

Sequences were edited and analysed using a Genetics Computer Group software package (version 8.0, University of Wisconsin) (Devereux et al., 1984). Homology comparisons were made using the PILEUP and GAP programs and phylogeny relationships calculated with the DISTANCES (Kimura protein distance algorithm) program based on multiple alignment of amino acid sequences from PILEUP. The phylogenetic tree was made with the GROWTREE (UPGMA algorithm) program.

Preparation of expression plasmid constructs

The expression vector for Drosophila S2 cells pRmHa3, a modified form of pRmHa1 (Bunch et al., 1988), was provided by Dr. T. Bunch (University of Arizona, Tucson, AZ, U.S.A.). The hygromycin selection plasmid pCOHygro (van der Straten et al., 1989) was provided by Dr. M. Rosenberg (SmithKline Beecham Pharmaceuticals, U.S.A.). M. persicae Mpα1 and Mpα2 cDNA clones have been described previously (Sgard et al., 1998). The coding sequences of Mpα1, Mpα2, Mpα3, and Mpα4 were PCR amplified using modified gene-specific primers that introduced an optimal sequence for translational initiation (GCCACCATG (Kozak, 1987, 1990) upstream of the start codon and then cloned into pRmHa3. The rat α4 and β2 cDNA clones, provided by Dr. J. Patrick (Baylor College of Medicine, Waco, TX, U.S.A.), were subcloned into pRmHa3 as previously described (Lansdell et al., 1997).

Heterologous expression of M. persicae nAChRs

Drosophila S2 cells were maintained in Shields and Sang M3 medium (Sigma) containing 12.5% heat-inactivated fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (GibcoBRL) at 25°C. Cells were transfected with 20 μg of plasmid DNAs by a modified calcium phosphate method (Chen and Okayama, 1987) as previously described (Millar et al., 1994). For stable expression, cells were co-transfected with the selection plasmid pCOHygro and expression plasmid DNAs in the ratio of 2:1. Stable transfectants were selected and maintained in M3 medium containing 300 μg/ml hygromycin B. Transfected S2 cells were induced by addition of CuSO4 (0.6 mM) for 24 h prior to protein assays.

Radioligand binding assays

Cells were resuspended in 10 mM potassium phosphate buffer (pH 7.2) containing the protease inhibitors leupeptin (2 μg/ml) and pepstatin (1 μg/ml). The suspension was taken up five times through a 23-gauge needle to obtain membrane preparations. Aliquots were stored at —80°C until use. Proteins were quantified using the BCA protein assay reagents (Pierce). Radioligand binding assays using [3H]epibatidine (56 Ci/mmol; DuPont—NEN) and [3H]imidacloprid (30 Ci/mmol) (Lind et al., 1998) were performed as previously described (Lansdell et al., 1997) except that nonspecific binding was determined in the presence of 1 mM carbachol and 1 mM nicotine. For saturation binding, the concentration ranges of 0.01-10 nM [3H]epibatidine and 0.1-100 nM [3H]imidacloprid were examined.

Binding data analysis

Curves for saturation binding were fitted with GraFit Version 3.0, Erithacus Software (Leatherbarrow, 1992), using the equation y = (Bmax·x)/(KD + x), where y is the amount of radioligand bound, x is the free radioligand concentration, Bmax is the maximal binding, and KD is the dissociation constant of ligand.


  1. Top of page
  2. Abstract
  4. RT-PCR and rapid amplification of cDNA ends (RACE)
  6. Cloning and sequencing of full-length cDNAs of novel nAChR subunits
  8. Acknowledgements
  9. References

Identification of novel members of M. persicae nAChR gene family

Degenerate primers were used to amplify nAChR subunit cDNA fragments from M. persicae by RT-PCR, and the resulting fragments (∼240, ∼400, and ∼540 bp) were cloned into the vector pUAg. Several different clones were identified in this pool of cDNA inserts (data not shown). Selected cDNA clones were sequenced and in most cases were found to encode open reading frames with significant amino acid sequence identity to known insect nAChR subunits. Previously, two α subunits, Mpα1 and Mpα2, have been identified in M. persicae (Sgard et al., 1998). We found that Mpα2 was the most common sequence and represented 70% of clones in the pool. The second most common sequence (20% of total) was a novel non-α subunit similar to Drosophila ARD (Y. Huang et al., in preparation). Of the remaining 10% of clones, four novel genes encoding putative α subunits were identified and designated Mpα3, Mpα4, Mpα5, and Mpα6. The previously identified Mpα1 sequence was not detected in the pool. Within the amplified region, the predicted amino acid sequences of Mpα3, Mpα4, Mpα5, and Mpα6 show 68, 66, 67, and 79% identity to Mpα1 and 69, 69, 70, and 73% to Mpα2, respectively.

Cloning and sequencing of full-length cDNAs of novel nAChR subunits

  1. Top of page
  2. Abstract
  4. RT-PCR and rapid amplification of cDNA ends (RACE)
  6. Cloning and sequencing of full-length cDNAs of novel nAChR subunits
  8. Acknowledgements
  9. References

Screening of an M. persicae cDNA library (Sgard et al., 1998) for clones containing the novel nAChR sequences (Mpα3, Mpα4, Mpα5, and Mpα6) was unsuccessful, and therefore a RACE strategy was used to amplify the remaining coding sequences. This resulted in the isolation of full-length sequences for Mpα3 and Mpα4 and an almost complete sequence for Mpα5 (lacking the N-terminal 50 amino acids and signal peptide). Attempts to obtain a full-length Mpα6 sequence by RACE were not successful possibly because of its unusually high GC content (data not shown). The EMBL/GenBank accession nos. for these sequences are AJ236786 (Mpα3), AJ236787 (Mpα4), and AJ236788 (Mpα5). The predicted nAChR subunits encoded by Mpα3 and Mpα4 are 515 and 505 amino acids, respectively, with molecular masses of 60 and 59 kDa. This excludes signal peptide sequences of 22 and 27 amino acids, as based on N-terminal alignments with other insect nAChR subunit amino acid sequences (Fig. 1). The partial Mpα5 sequence encodes a polypeptide of 482 amino acids but lacks part of the 5′ coding region.

Northern blot analysis

Northern hybridisations were performed to confirm whether the two fully sequenced, novel M. persicae nAChR subunits (Mpα3 and Mpα4) are expressed in vivo. PCR fragments spanning the weakly conserved cytoplasmic loop between transmembrane domains TM3 and TM4 were used as hybridisation probes under conditions of high stringency to exclude any cross-hybridisation. The results revealed that the major transcripts of Mpα3 and Mpα4 were ∼2 and 9 kb, respectively (Fig. 2). Clearly, the Mpα4 transcript is much larger than that required for the 1,596-bp open reading frame, a feature that has also been reported for both Drosophila ALS and Dα3 transcripts (Bossy et al., 1988; Schulz et al., 1998).


Figure 2. Northern blot analysis of M. persicae Mpα3 and Mpα4 transcripts. Each lane contains ∼10 μg of poly(A)+ RNA. The sizes of major transcripts are given in kilobases on the right.

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Characterisation of cloned M. persicae nAChR subunits

The proteins encoded by Mpα3, Mpα4, and Mpα5 genes have features typical of members of the nAChR family. First, the deduced protein sequences of these genes show substantial sequence homology to some previously characterised insect nAChR subunits, with overall amino acid identities between 43 and 76% (Table 1). Second, they contain the secondary sequence elements and features characteristic of the nAChR gene family (Karlin, 1993): that is, a signal peptide, a conserved disulfide-linked loop (15 amino acids) formed by two cysteines at positions 128 and 142 (Torpedoα-subunit numbering), and the four hydrophobic putative transmembrane domains TM1-4 (Fig. 1). Third, they share two potential N-glycosylation sites present at positions 24 and 212 of the mature proteins. The former (Asn24) is conserved in all insect nAChR subunits reported to date and most of the vertebrate neuronal receptors (Marshall et al., 1990; Gundelfinger and Hess, 1992; Lindstrom, 1995), and the latter (Asn212) seems to be unique to the insect α subunits (Marshall et al., 1990).

Table 1. Percent identity of mature protein among members of insect nAChR gene family, calculated with GAP program of Genetics Computer Group package
  Mpα1 Mpα2 Mpα3 Mpα4 Mpα5
  1. The EMBL/GenBank accession nos. are as follows: ALS, X07194; SAD, X53583; Dα3, Y15593; ARD, X04016; SBD, X55676 and Y14678; αL1, X55439; MARA1, Y09795; Mpα1, X81887; Mpα2, X81888; Mpα3, AJ236786; Mpα4, AJ236787; Mpα5, AJ236788.

  2. aAmino acid sequence identity of ≥65%.

ALS57 72a595856
SAD 75a59545350
Dα3 5656 76a60 65a
SBD556160 67a59
αL1 76a57545553
MARA15561 76a58 69a

Mpα3, Mpα4, and Mpα5 have been designated as three α subunits because they contain not only two adjacent cysteines (Cys192 and Cys193) but also several conserved tryptophan and tyrosine residues, such as Trp86, Tyr93, Trp149, Tyr151, Tyr190, and Tyr198 (Torpedoα-subunit numbering), which are involved in ligand binding (Hucho et al., 1996; Arias, 1997). The only exception is the conservative substitution of tyrosine by phenylalanine at position 93 in Mpα4 (Fig. 1). A single mutation of Tyr93[RIGHTWARDS ARROW]Phe in the mouse muscle α subunit has been reported to cause 50-fold reduction of acetylcholine binding when receptors were expressed in HEK-293 cells (Sine et al., 1994). Interestingly, although Mpα4 is designated as an α subunit, like Locα2 (Hermsen et al., 1998), it shows greater sequence similarity to the Drosophila non-α subunit SBD (Table 1). This situation is similar to that of the vertebrate neuronal α5 and β3 subunits, which also show considerable sequence homology (Boulter et al., 1990; Hernandez et al., 1995). Mpα3 and Mpα5 are most similar to Drosophila Dα3 with 76 and 65% identity, respectively, although the sizes of the cytoplasmic loops between TM3 and TM4 of Mpα3 (156 amino acids) and Mpα5 (183 amino acids) are much shorter than that of Dα3 (415 amino acids) (Schulz et al., 1998). In addition, unusual residue changes in different subunit isoforms, such as Gly248[RIGHTWARDS ARROW]Lys (Mpα1), Gly248[RIGHTWARDS ARROW]Asp (Mpα3), Ser256[RIGHTWARDS ARROW]Asp (Mpα4), Leu259[RIGHTWARDS ARROW]Val (Mpα4), and Val263[RIGHTWARDS ARROW]Met (Mpα1), around TM2, which is thought to line the ion channel (Karlin, 1993), have been found here (Fig. 1) and previously (Sgard et al., 1998).

Phylogenetic analysis of insect nAChR subunits

A phylogenetic tree was generated using the present sequence data together with other previously reported insect subunits, a number of sequences representing various vertebrate muscle and neuronal nAChR subtypes, and some nematode sequences (Fig. 3). This tree shows that the insect nAChRs are divided into two major groups: α and non-α. The α group appears to have evolved in parallel to vertebrate neuronal nAChRs, and the non-α group (ARD-like) is distinct from vertebrate neuronal β and muscle non-α subunits. Surprisingly, the Drosophila non-α subunit SBD falls into the insect α group rather than the non-α group. The insect α group is most similar to two nematode α subunits (unc38 and tar1) and then to the vertebrate neuronal α and β group (including muscle α1). Clearly, the insect α group comprises at least four subtypes, as defined by Mpα1, Mpα2, Mpα3, and Mpα4. The fifth subtype, including Mpα5 and Locα1 (Hermsen et al., 1998), which share 74% amino acid identity, may be considered to have split from the Mpα3 subtype. The tree also suggests that the group of vertebrate α7-9 and nematode ce21 (Ballivet et al., 1996), which can function as homo-oligomeric receptors when expressed in vitro, can be regarded as a more ancestral form of nAChR subunit, which is in agreement with previous results (Le Novere and Changeux, 1995; Ortells and Lunt, 1995; Tsunoyama and Gojobori, 1998).


Figure 3. Evolutionary tree of the nAChR family. Symbols used for vertebrate nAChR subunits are the following: r, rat; c, chick. Database accession nos. for vertebrate muscle subunits are as follows: rα1 (X74832), rβ1 (X74833), rδ (X74835), rγ (X74834), rε (X13252); for vertebrate neuronal subunits: rα2 (L10077), rα3 (L31621), rα4 (L31620), rα5 (J05231), rα6 (L08227), cα7 (X68586), cα8 (X52296), rα9 (U12336), rβ2 (L31622), rβ3 (J04636), rβ4 (U42976); for nematode subunits: ce21 (X83887), unc38 (X98600), tar1 (U56903), acr2 (X86403), acr3 (Y08637), ce13 (X83888), lev1 (X98601); and for insect subunits: ALS (X07194), SAD (X53583), Dα3 (Y15593), ARD (X04016), SBD (X55676 and Y14678), αL1 (X55439), Locα1 (AJ000390), Locα2 (AJ000391), Locα3 (AJ000392), Locβ1 (AJ000393), MARA1 (Y09795), Hvα1 (AJ000399), Mpα1 (X81887), Mpα2 (X81888), Mpα3 (AJ236786), Mpα4 (AJ236787), Mpα5 (AJ236788).

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Expression of M. persicae nAChR subunits in Drosophila S2 cells

The original Mpα1 (similar to Drosophila SAD) cDNA lacks part of the N-terminal signal sequence (Fig. 1). To facilitate heterologous expression, the SAD signal sequence (41 amino acids) was attached to the first-codon Asn (ACC) of the Mpα1 mature protein (532 amino acids), as previously described (Sgard et al., 1998). Mpα2, Mpα3, and Mpα4 contain putative signal sequences of 25, 22, and 27 amino acids, respectively. To facilitate translation in the heterologous expression system, a consensus initiation sequence was introduced immediately upstream of the start codon of each M. persicae nAChR subunit cDNA (see MATERIALS AND METHODS).

The four M. persicae nAChR α-subunit coding sequences were subcloned into the expression vector pRmHa3 and transiently transfected into Drosophila S2 cells either alone or in combination with the rat β2 structural subunit. Expression was then evaluated by measuring the specific binding of [3H]epibatidine, a high-affinity nicotinic ligand (Briggs et al., 1995; Gerzanich et al., 1995). Preliminary results showed no specific binding was detected with any of M. persicae nAChR α subunits expressed singly. In contrast, specific binding of [3H]epibatidine was detected when M. persicae Mpα1, Mpα2, or Mpα3 was co-transfected with the vertebrate (rat) β2 subunit, a strategy that has been successful for cloned Drosophilaα subunits in Xenopus oocytes and Drosophila S2 cells (Bertrand et al., 1994; Lansdell et al., 1997; Schulz et al., 1998). Similarly, a high level of specific binding was observed in S2 cells transfected with the rat α4β2 subunit combination, as reported previously (Lansdell et al., 1997).

Pharmacological properties of M. persicae nAChR subunits expressed in S2 cells

Stably transfected S2 cell lines were established for each of the four M. persicaeα subunits together with the rat β2 to investigate their pharmacological properties in more detail. Saturation binding studies confirmed specific binding of [3H]epibatidine to the Mpα1/rβ2, Mpα2/rβ2, and Mpα3/rβ2 subunit combinations (Table 2), as had been observed after transient transfection. An accurate KD value was not determined due to the very high affinity of epibatidine for all three nAChR subtypes, which led to ligand depletion at low concentrations, but for all subunit combinations, it was <0.1 nM. In the context of the present work to study imidacloprid binding, it was considered sufficient to demonstrate the high affinity of this ligand for hybrid (M. persicae/rat) recombinant receptors. Ligand depletion did not prevent the measurement of Bmax values, which were 1.0 ± 0.1, 2.2 ± 0.1, and 0.14 ± 0.01 pmol/mg for Mpα1/rβ2, Mpα2/rβ2, and Mpα3/rβ2 receptors, respectively. The Bmax value of [3H]epibatidine for the rat α4β2 (6.5 ± 2.3 pmol/mg) has been reported previously (Lansdell et al., 1997).

Table 2. Stable expression of nAChR subunits in Drosophila S2 cells assayed by saturation binding of two cholinergic radioligands
  [3H]Epibatidine [3H]Imidacloprid
Subunit combinationBmax (pmol/mg)Bmax (pmol/mg)KD (nM)
  1. rα4 and rβ2 represent rat neuronal nAChR α4 and β2 subunits, respectively. (—) represents the absence of specific binding with up to 30 nM [3H]epibatidine or 100 nM [3H]imidacloprid. Bmax and KD values are means ± SD, from at least three separate determinations. ND, data not determined in this study.

Mpα1/rβ2 1.0 ± 0.1
Mpα2/rβ2 2.2 ± 0.11.3 ± 0.13.0 ± 0.5
Mpα3/rβ2 0.14 ± 0.010.08 ± 0.012.8 ± 0.1
rα4/rβ2 6.5 ± 2.3ND>100

Previous studies have shown that imidacloprid exhibits high potency against native insect nAChRs (Liu and Casida, 1993; Zwart et al., 1994; Chao et al., 1997; Lind et al., 1998), but the molecular basis of the insecticidal action is not understood. Our aim in cloning M. persicae nAChR subunits was to investigate their roles as potential target sites for this insecticide. We therefore examined the influence of different M. persicae nAChR α subunits on the affinity of [3H]imidacloprid by saturation binding. Both Mpα2/rβ2 and Mpα3/rβ2 receptors showed high-affinity binding of imidacloprid with KD values of 3.0 ± 0.5 and 2.8 ± 0.1 nM and Bmax values of 1.3 ± 0.1 and 0.08 ± 0.01 pmol/mg, respectively (Table 2; Fig. 4A and B). Despite showing very high affinity to [3H]epibatidine, the Mpα1/rβ2 receptor was insensitive to imidacloprid up to 100 nM (Table 2). Likewise, no specific binding of either [3H]imidacloprid (100 nM) or [3H]epibatidine (30 nM) could be detected for the Mpα4/rβ2 receptor. The rat α4β2, the predominant receptor in the vertebrate brain, showed very low affinity for [3H]imidacloprid, but the binding did not appear to be saturated even at a high concentration (100 nM), which prevented an accurate estimate of its KD and Bmax values (Fig. 4C).


Figure 4. Saturation binding of [3H]imidacloprid to the nAChRs stably expressed in Drosophila S2 cells. Specific binding was transformed as Scatchard plots (insets in A and B) showing the linear relationship. A: Mpα2/rβ2; B: Mpα3/rβ2; C: rα4/rβ2; D: chemical structures of epibatidine and imidacloprid.

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  1. Top of page
  2. Abstract
  4. RT-PCR and rapid amplification of cDNA ends (RACE)
  6. Cloning and sequencing of full-length cDNAs of novel nAChR subunits
  8. Acknowledgements
  9. References

We have cloned three novel nAChR genes (Mpα3-5) using RT-PCR and RACE strategies. Thus, together with Mpα1 and Mpα2 (Sgard et al., 1998), at least five α subunits exist in M. persicae. M. persicaeα subunits have been successfully co-expressed in Drosophila S2 cells with the rat β2 subunit. Saturation binding has demonstrated that the affinity of the insecticide imidacloprid for recombinant nAChRs is influenced strongly by the nature of α subunits. The viability of heterologous expression of M. persicae nAChRs opens up the way to investigate biochemical, pharmacological, and physiological profiles of single-receptor subtypes.

Heterologous expression of M. persicae nAChRs

It was previously reported that both Mpα1 and Mpα2 could form homo-oligomeric receptors in Xenopus oocytes, with the latter displaying only small agonist-induced currents (Sgard et al., 1998). Here we show that expression of Mpα1, Mpα2, and Mpα3 gives rise to specific radioligand binding only when they are coexpressed with a vertebrate β2 subunit in Drosophila S2 cells, as found previously with Drosophila SAD, ALS, and Dα3 (Bertrand et al., 1994; Lansdell et al., 1997; Schulz et al., 1998).

Mpα4 shares many features with the vertebrate neuronal α5 subunit. First, Mpα4 (505 amino acids) is the smallest of all full-length insect α subunits isolated so far, and α5 is the smallest of the eight vertebrate neuronal α isoforms (α2-9) (Brammar, 1996). Second, Mpα4 together with its homologue Locα2 (Hermsen et al., 1998) are most similar to the Drosophila non-α subunit SBD, and the α5 subunit falls into a subgroup with β3 (Fig. 3). Third, two unusual substitutions of Tyr93[RIGHTWARDS ARROW]Phe and Leu259[RIGHTWARDS ARROW]Val in Mpα4 (Fig. 1) are also present in α5, and the latter is shared by SBD and β3. Finally, Mpα4 does not form homo-oligomeric or hetero-oligomeric nAChRs with rat β2; neither does α5 with any of the β subunits (McGehee and Role, 1995). However, as α5 has been shown to generate functional nAChR channels when co-expressed with both another α and β subunit in oocytes, for example, α5α4β2 (Ramirez-Latorre et al., 1996), it is possible that successful heterologous expression of Mpα4 may also require the presence of two additional subunits.

How complex are insect nAChRs?

It has been suggested that the nAChR family of insects may exhibit less subunit diversity than that of vertebrates because of their confinement to the nervous system (Sattelle, 1980; Breer and Sattelle, 1987) and the fact that fewer receptor subtypes had been cloned (Gundelfinger, 1992). Until recently, knowledge of recombinant insect nAChRs has largely been limited to Drosophila (Gundelfinger and Hess, 1992). The present study and previous data (Sgard et al., 1998) indicate that at least five α (Mpα1-5) subunits exist in M. persicae. These results, together with the recent data from L. migratoria (Hermsen et al., 1998), suggest that insect nAChRs may indeed be as complex as their vertebrate counterparts. Phylogenetic comparisons suggest that insect α subunits evolved in parallel to the vertebrate neuronal nAChRs (this study; Le Novere and Changeux, 1995; Ortells and Lunt, 1995; Tsunoyama and Gojobori, 1998). Heterologous expression of insect α subunits also indicates considerable diversity of pharmacological and physiological properties.

Molecular action of imidacloprid on insect nAChRs

The action of imidacloprid in selectively targeting insect nAChRs with little mammalian toxicity is well documented (Bai et al., 1991; Liu and Casida, 1993; Buckingham et al., 1997; Chao and Casida, 1997; Chao et al., 1997; Lind et al., 1998; Nagata et al., 1998). The different profiles of imidacloprid sensitivity characterised in these studies may be due to the influence of different nAChR populations. Binding of [3H]imidacloprid to native M. persicae nAChRs (Lind et al., 1998) led to two possibilities either that imidacloprid may bind to more than one receptor population, each with a different affinity, or that it may bind differently to two separate binding sites within a single receptor, perhaps with allosteric interaction between them. These authors recognised that it would be very difficult to investigate the pharmacological profile of the low-affinity binding site using native receptors without a selective blocker to inhibit the high-affinity binding. Our binding results from single-receptor subtypes expressed in S2 cells clearly indicate that Mpα2- and Mpα3-containing nAChRs (with rat β2) are highly sensitive to imidacloprid (KD∼3 nM), whereas Mpα1-containing nAChR (also with rat β2) is insensitive to this insecticide. Although the present data do not suffice to assign these cloned subunits to native M. persicae nAChR populations, it is likely that Mpα1, Mpα2, and Mpα3 contribute to at least two distinct receptor populations in vivo that are imidacloprid-sensitive and -insensitive. The Bmax values determined with [3H]imidacloprid are approximately half of those determined with [3H]epibatidine (Table 2). It is possible, therefore, that the number of binding sites is different for these two cholinergic ligands on these hybrid (M. persicae/rat) nAChRs.

Our data also confirm that imidacloprid has very low-affinity binding to vertebrate neuronal α4β2 receptors (KD > 100 nM;Fig. 4C), consistent with previous electrophysiological results (Matsuda et al., 1998). These authors also reported that, unlike the recombinant chick α4β2 receptor, the hybrid Drosophila SAD/chick β2 nAChR expressed in Xenopus oocytes is sensitive to imidacloprid. Our study indicated that Mpα1/rat β2 nAChR expressed in S2 cells is insensitive to imidacloprid. Surprisingly, the Mpα1 and SAD subunits share 89% amino acid identity and 94% similarity in the N-terminal half of the protein (from the first amino acid of the mature protein to the end of TM3) within which the binding site is located. The different sensitivities of Mpα1, Mpα2, and Mpα3 to epibatidine and imidacloprid can be explained if these two ligands use different agonist binding sites (probably very close) in the N-terminal regions of these subunits and the 2-nitroiminoimidazolidine moiety of imidacloprid (Fig. 4D) is recognised by Mpα2 and Mpα3 but not by Mpα1. It has been proposed that the nitrogen atom of the imidazolidine ring (at 1-position) and of the pyridine ring interact with the electron-rich and hydrogen-donating sites on the nAChR, respectively (Yamamoto et al., 1995; Okazawa et al., 1998). Given the current value of these insecticides, it will be of particular interest to elucidate which functional domains or key residues within these subunits determine this selectivity.

Furthermore, studies over the years indicate that multiple resistance mechanisms have evolved in M. persicae (Devonshire, 1989; Elbert et al., 1996). It is notable that imidacloprid is playing an increasingly important role in controlling this pest and some resistance has already developed (Devine et al., 1996; Nauen et al., 1996). Based on the finding that imidacloprid targets only some nAChR α subunits from M. persicae and by means of molecular cloning and heterologous expression of nAChRs, we will aim to investigate whether changes in this target are involved in the development of insecticide resistance.


  1. Top of page
  2. Abstract
  4. RT-PCR and rapid amplification of cDNA ends (RACE)
  6. Cloning and sequencing of full-length cDNAs of novel nAChR subunits
  8. Acknowledgements
  9. References

We thank Drs. Thomas Bunch, Jim Patrick, Martin Rosenberg, and Frederic Sgard for generously providing plasmid vectors and cDNA clones used in this study. Y.H. is the recipient of a Rothamsted International—Zeneca Studentship, N.S.M. acknowledges support from the Wellcome Trust, and IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.


  1. Top of page
  2. Abstract
  4. RT-PCR and rapid amplification of cDNA ends (RACE)
  6. Cloning and sequencing of full-length cDNAs of novel nAChR subunits
  8. Acknowledgements
  9. References
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