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- Materials and methods
Insect nicotinic acetylcholine receptors (nAChRs) play a central role in mediating neuronal synaptic transmission and are the target sites for the increasingly important group of neonicotinoid insecticides. Six nicotinic acetylcholine receptor (nAChR) subunits (four α-type and two β-type) have been cloned previously from the model insect species Drosophila melanogaster. Despite extensive efforts, it has not been possible to generate functional recombinant nAChRs by heterologous expression of any combination of these six subunits. It has, however, been possible to express functional hybrid receptors when Drosophilaα subunits are co-expressed with vertebrate β subunits. This has led to the assumption that successful heterologous expression might require an, as yet, uncloned β-type insect subunit. Examination of the recently completed Drosophila genomic sequence data has identified a novel putative nAChR β-type subunit. Here we report the molecular cloning, heterologous expression and characterization of this putative Drosophila nAChR subunit (Dβ3). Phylogenetic comparisons with other ligand-gated ion channel subunit sequences support its classification as a nAChR subunit but show it to be a distantly related member of this neurotransmitter receptor subunit family. Evidence that the Dβ3 subunit is able to coassemble with other Drosophila nAChR subunits and contribute to recombinant nAChRs has been obtained by both radioligand binding and coimmunoprecipitation studies in transfected Drosophila S2 cells.
Nicotinic acetylcholine receptors (nAChRs) play a central role in mediating fast synaptic transmission in the insect brain (reviewed by Gundelfinger and Schulz 2000). They are also the target site for the economically important class of neonicotinoid insecticides (Narahashi 1996; Tomizawa et al. 1999). These factors have prompted an extensive investigation of the molecular and functional properties of insect nAChRs. Recent studies of both native nAChRs and cloned nAChR subunits have been aimed at identifying the subunit composition of insect nAChRs. Substantial progress has been made in identifying the subunit composition of insect nAChRs using immunoprecipitation studies with native nAChRs from Drosophila (Schloss et al. 1991, 1992; Chamaon et al. 2000; Schulz et al. 2000). In contrast, a detailed study of cloned nAChR subunits has been hindered by difficulties which have been encountered in generating functional insect nAChRs in heterologous expression systems (Millar 1999; Sivilotti et al. 2000).
Six nAChR subunits have been identified and cloned from the fruit fly Drosophila melanogaster (Hermans-Borgmeyer et al. 1986; Bossy et al. 1988; Baumann et al. 1990; Sawruk et al. 1990a,b; Schulz et al. 1998; Lansdell and Millar 2000a). Four have been classified as α subunits (ALS, Dα2/SAD, Dα3 and Dα4) and two as non-α- or β subunits (ARD and SBD). To date, no combination of these Drosophila nAChR subunits has enabled the successful expression of recombinant nAChRs. Despite these problems, however, it has been possible to detect functional nAChRs and/or high-affinity binding of nicotinic radioligands when any of the four Drosophilaα subunits are co-expressed with vertebrate non-α subunits (Bertrand et al. 1994; Lansdell et al. 1997; Schulz et al. 1998; Lansdell and Millar 2000a,b). Similar results have been reported with nAChR α subunits cloned from other insect species (Huang et al. 1999, 2000). Such studies have led to the assumption that problems encountered in heterologous expression of insect nAChRs may be due to a requirement for an, as yet unidentified, insect β-type subunit (Millar 1999). The recently completed sequencing of the Drosophila genome (Adams et al. 2000) has provided an opportunity to identify additional genes encoding potential nAChR subunits. Recent analysis of the Drosophila genome sequence data identified a single novel putative nAChR β subunit gene which encodes a protein which is distantly related to other nAChR subunits (Littleton and Ganetzky 2000). In this study we have characterized this putative nAChR β subunit gene by isolation of a full-length cDNA clone and its heterologous expression in a Drosophila cell line. Direct evidence that the Dβ3 subunit is able to coassemble with other Drosophila nAChR subunits and contribute to recombinant nAChRs has been obtained by both radioligand binding and coimmunoprecipitation studies.
- Top of page
- Materials and methods
A novel putative nAChR subunit, Dβ3, has been cloned from Drosophila and shown, by heterologous expression studies to coassemble with previously cloned nAChR subunits and contribute to the formation of recombinant nAChRs. Although the Dβ3 subunit displays features which are characteristic of nAChR subunits (as is described in Results), we consider it appropriate to describe Dβ3 as an atypical nAChR subunit. This is illustrated by the fact that, whereas previously cloned nAChRs subunits typically display 45–65% amino acid sequence identity in pair-wise comparisons (Gundelfinger and Hess 1992), Dβ3 shows only ≈ 20% identity with other nAChR subunits (even when comparisons ignore the highly divergent M3−M4 loop region). The length of the predicted M3−M4 intracellular loop in Dβ3 is shorter than that of other previously cloned nAChR subunits and, consequently, the size of the predicted mature Dβ3 subunit (402 amino acids) is significantly smaller than that of other insect or mammalian nAChR subunits, the size of which range, typically, from ≈ 440 to 600 amino acids (Claudio 1989). The recently characterized vertebrate α10 nAChR subunit (Elgoyhen et al. 2001) also has a relatively small ORF (predicted mature protein of 423 amino acids; mature unglycosylated molecular mass of 47 kDa). Despite this, the α10 subunit, like other vertebrate nAChR subunits, contains a predicted M3−M4 intracellular loop region which is considerably longer than that of Dβ3. The Dβ3 coding sequence ends at a position immediately after the predicted M4 domain, in contrast to other Drosophila nAChR subunits which extend for a further 20–40 amino acids (Fig. 2). There are, however, several examples of nAChR subunits which terminate at a position similar to that of Dβ3. These include the mammalian α9 and α10 subunits (Elgoyhen et al. 1994, 2001) and several nAChR subunits identified in C. elegans (Mongan et al. 1998). Although Dβ3, contains potential N-linked glycosylation sites within its predicted extracellular domain, as do other nAChR subunits, these do not occur at positions which are conserved in other subunits. Although nAChRs can differ widely in their exon−intron structure (Le Novére and Changeux 1995), the structure of the Dβ3 gene is somewhat unusual, containing only three introns, none of which are larger than ≈ 250 bp. As it contains very little intronic DNA, the Dβ3 gene occupies < 1800 bp of genomic sequence. This is in stark contrast to, for example, the recently cloned Drosophila nAChR Dα4 subunit which is encoded by 11 exons spanning ≈73 000 bp of genomic sequence (Lansdell and Millar 2000a).
The nomenclature for genes encoding Drosophila nAChR subunits, as proposed by FlyBase (http://flybase.bio.indiana.edu), includes information derived from the gene's chromosomal localization. The predicted gene CG11822 has been cytogenetically mapped to chromosomal location 21C5-21C6 (see http://flybase.bio.indiana.edu/.bin/fbidq.html?FBgn0031261). Consequently, although we have suggested the trivial name Dβ3 for this putative Drosophila nAChR subunit, in accordance with the suggested FlyBase nomenclature, we propose that the formal name for this gene should be nAcRβ-21C.
To date, reconstitution of functional nAChRs by heterologous expression of nAChR subunits cloned from Drosophila or from other insect species has been largely unsuccessful (Hermsen et al. 1998; Millar 1999; Gundelfinger and Schulz 2000). It has, however, been possible to detect functional nAChRs and/or high-affinity binding of nicotinic radioligands when any of the four Drosophilaα subunits is co-expressed with any one of several vertebrate non-α subunits (Bertrand et al. 1994; Lansdell et al. 1997; Schulz et al. 1998; Lansdell and Millar 2000a,b). Such findings have led to the assumption that problems encountered in heterologous expression of insect nAChRs may be due to a requirement for an, as yet unidentified, insect β-type subunit (Millar 1999). Vertebrate subunits which have been used successfully in co-expression studies with Drosophilaα subunits include the neuronal nAChR β2 and β4 subunits and the vertebrate muscle nAChR γ- and δ subunits (Bertrand et al. 1994; Lansdell and Millar 2000b). The recently completed Drosophila genome sequence has enabled a detailed search for putative novel genes but has not identified any novel putative Drosophila nAChR subunits which show particularly close sequence similarity to vertebrate subunits such as β2, β4, γ or δ subunits (see, for example, Littleton and Ganetzky 2000). Three further putative nAChR α-type subunits have been identified, all of which appear to be most closely related to the vertebrate neuronal nAChR α7 subunit (Littleton and Ganetzky 2000). The only putative novel Drosophila nAChR β subunit (here called Dβ3) which has emerged from analysis of the Drosophila genome shows closest sequence similarity to a C. elegans nAChR β subunit ‘Ce13/UNC-29’ (Ballivet et al. 1996) a nAChR subunit␣which has been shown by heterologous expression to be capable of generating functional nAChRs (Fleming et al.␣1997).
Although the cloning and heterologous expression of Dβ3 has not enabled the generation of functional recombinant nAChRs in the absence of a co-expressed vertebrate β subunit, we have obtained evidence to indicate that Dβ3 can coassemble with other Drosophila nAChR subunits and can contribute to␣the formation of hybrid (insect/vertebrate) recombinant nAChRs. This has been demonstrated by an increase in radioligand binding seen when Dβ3 is co-expressed as a third subunit with combinations of Drosophilaα subunit and␣vertebrate β subunits (e.g. Dα3 + rat β2). Interestingly, we also observe a dominant-negative effect when Dβ3 is co-expressed with the vertebrate nAChR α4 + β2 subunit combination.
Because no difference in the affinity of radioligand binding is observed when Dβ3 is co-expressed with subunit combinations such as Dα3 + β2, we can conclude that the increase in the level of specific radioligand binding represents an increase in the steady-state level of assembled Dα3-containing nAChRs (Bmax). This may be a consequence of Dβ3 increasing the efficiency of coassembly of Dα3 with the β2 subunit. The decrease in total radioligand binding detected when Dβ3 is co-expressed with rat α4 and β2 subunits suggests that coassembly of Dβ3 reduces the efficiency with which α4 and β2 subunits can coassemble with one another. This may be a consequence of α4 and Dβ3 coassembling into ‘dead-end’ subunit complexes which preclude the subsequent association of α4 with β2.
As discussed previously (Lansdell et al. 1997), one explanation for difficulties encountered in heterologous expression of Drosophila and other insect nAChRs is the unsuitability of commonly used heterologous expression systems. However, considering the success that has been achieved in heterologous expression studies when insect α subunits have been co-expressed with vertebrate β subunits, a more plausible explanation is that additional (presently uncloned) insect nAChR subunits are required. We have obtained evidence to suggest that Dβ3 is able to coassemble with other Drosophila nAChR subunits and contribute to the formation of recombinant nAChRs. However, as these receptors still require the co-expression of a nAChR β subunit the search must continue (either for the elusive ‘missing’ subunit or for a more optimal expression system).