- Top of page
- Materials and methods
Nicotinic acetylcholine (ACh) receptors (nAChRs) are the targets of several kinds of insecticides. Based on the mutagenesis studies of Torpedo californica nAChRs and solved structure of a molluscan, glial-derived soluble ACh-binding protein, a model of the agonist site was constructed with contributing amino acids from three distinct loops (A, B, and C) of the α subunits and another three loops (D, E, and F) of the non-α subunits. According to this model, most insect nAChR subunits can form the functional heteromeric or homomeric receptors. Actually, insect subunits themselves did not form any functional receptor at various combinations as yet, and only part of them can form the functional receptors with vertebrate non-α subunits. These findings suggested that the agonist binding for insect nAChRs was not only contributed by those key amino acids in six loops, but also some unidentified amino acids from other regions. In our previous studies on nAChRs for Nilaparvata lugens, a target-site mutation (Y151S) was found within two α subunits (Nlα1 and Nlα3). In Drosophila S2 cells and Xenopus oocytes, Nlα1 can form functional receptors with rat β2 subunit. However, the same thing was not observed in Nlα3. In the present paper, by exchanging the corresponding regions between Nlα1 and Nlα3 to generate different chimeras, amino acid residues or residue clusters in the regions outside the six loops were found to play essential roles in agonist binding, especially for the amino acid clusters between loop B and C. This result indicated that the residues in the six loops could be necessary, but not enough for the activity of agonist binding.
The nicotinic acetylcholine (ACh) receptors (nAChRs) are ligand-gated ion channels (LGICs) mediating fast cholinergic synaptic transmission in insect and vertebrate nervous systems (Matsuda et al. 2001). In mammals and other vertebrates, nAChRs are expressed at both the neuromuscular junction (the ‘muscle-type’ nAChR) and within the CNS and PNS (‘neuronal’ nAChRs). In insects, although nAChRs are not expressed at the neuromuscular junction (where synaptic transmission is glutamatergic), ACh is the major excitatory neurotransmitter in the insect brain (Breer and Sattelle 1987). The great abundance of nAChRs within the insect brain has led to the development of insecticides targeting these receptors. Neonicotinoid insecticides are insect-selective nAChR agonists that are used extensively in the areas of crop protection and animal health (Matsuda et al. 2001; Tomizawa and Casida 2005). Since the first commercial neonicotinoid insecticide, imidacloprid, was introduced in the early 1990s, the resistance to neonicotinoid insecticides had been observed in several pest species (Nauen and Denholm 2005). In brown planthopper (Nilaparvata lugens), a major rice pest in many parts of Asia, the significant resistance was reported both in the laboratory selected strain and field populations (Cheng and Zhu 2006; Liu and Han 2006).
The most extensively characterized nAChR is that expressed within the electric organ of fish such as the marine ray Torpedo (Unwin 1996). Affinity labeling, mutagenesis and structural studies have provided extensive evidence for a structure model of the agonist site with contributing amino acids from three distinct regions of the α-subunits (referred to as binding site segments A, B, and C) and from at least three regions of the non-α (β, γ, or δ) -subunits (segments D, E, and F, Fig. 1) (Prince and Sine 1998; Arias 2000; Corringer et al. 2000; Grutter and Changeux 2001). Most features of the model are present and confirmed in the binding site identified within the solved structure of a molluscan, glial-derived soluble ACh-binding protein (AChBP), a homopentameric structural, and functional homolog of the N-terminal ligand-binding domain of a nAChR α-subunit (Brejc et al. 2001; Smit et al. 2001).
Many insect nAChR subunits have been cloned, and genome sequencing of several insects also gives more information about insect nAChRs. In the fruit fly Drosophila melanogaster, an extensively studied model insect species, 10 nAChR subunits (α1-α7 and β1–3) have been identified by molecular cloning (Tomizawa and Casida 2001; Millar 2003). The proliferation of insect genome sequencing projects is now starting to reveal a similar level of nAChR subunit diversity in other species (Jones et al. 2005, 2006). The detailed pharmacological characterization of insect nAChRs has been considerably hindered by difficulties encountered in expressing recombinant insect nicotinic receptors (Millar and Denholm 2007), although they had all contributing amino acids in the agonist site structure model. Despite this, however, it has been possible to generate functional hybrid nicotinic receptors by the co-expression of some, but not all, insect α subunits with the vertebrate neuronal β subunits in the heterologous expression systems, such as Drosophila S2 cells and Xenopus oocytes (Bertrand et al. 1994; Lansdell et al., 1997; Lansdell and Millar 2000). Another way to do the pharmacological studies on insect nAChRs is to construct the artificial subunit chimeras, although it cannot reveal the complete features of its wildtype (Lansdell and Millar 2004; Shimomura et al. 2005).
To identify the key residues contributing to ligand binding outside the loops, experiments were carried out in this study to express and check different chimeras constructed by exchanging the corresponding regions between Nlα1 and Nlα3 subunits, which were previously identified from brown planthopper and proved that when co-expressed with the vertebrate neuronal β subunits, one produced active receptors and the other inactive in the heterologous expression systems.
- Top of page
- Materials and methods
The superfamily of pentameric LGICs, including nAChR, 5-HT3, GABAA and GABAC, and glycine receptors, mediates chemical synaptic transmission. All of these LGICs form homo- or heteropentamers of related subunits, implying a common evolutionary origin (Ortells and Lunt 1995). In nAChRs, the ligand-binding site is located at the interface between two subunits (Arias 2000; Corringer et al. 2000). Numerous biochemical studies showed that the principal part is always formed by the α-subunit residues – contributing to the so-called ‘loops’ A, B and C – whereas the neighboring subunit residues – contributing to the ‘loops’ D, E and F – form the complementary part of the binding pocket (Brejc et al. 2001; Grutter and Changeux 2001). Most studies on the agonist site structure in nAChRs are based on the most extensively characterized nAChRs in Torpedo and a molluscan, glial-derived soluble AChBP (Grutter and Changeux 2001). Figure 1 shows the important residues contributing to six loops based on the studies of Torpedo nAChRs and studies on AChBP confirmed these findings (Brejc et al. 2001; Grutter and Changeux 2001).
The agonist site structure model of nAChRs was derived from few species up to the present and it remains unknown whether the structure is suitable for all animals because of the diversity in nAChRs. A total of 17 nAChR subunits (α1–α10, β1–β4, γ, δ, and ε) have been identified in vertebrate species, which can co-assemble to form multiple functional homopentamers (α7, α8, and α9) or heteropentamers (Corringer et al. 2000). The genome sequencing projects of insects had revealed 10, 10, 11, 12, and 12 subunits in D. melanogaster (Adams et al. 2000), Anopheles gambiae (Jones et al. 2005), Apis mellifera (Jones et al. 2006), Bombyx mori (Shao et al. 2007) and Tribolium castaneum (Jones and Sattelle 2007), respectively. The combinatorial assembly of these subunits produces a wide structural diversity of receptor oligomers, targeted to different subcellular compartments, which exhibit variable electrical properties (conductance, ion selectivity, and rectification), pharmacologic characteristics (affinities for agonists, competitive antagonists and allosteric effectors, and potency orders), and kinetics of activation and desensitization (Le Novere et al. 2002).
The abundant nAChRs identified in different animal species can generate a wide variety of nAChRs with much different pharmacological properties (Millar 2003). The agonist site structure model derived from Torpedo nAChRs and molluscan AChBP might not suitable for all nAChRs from different animal species, although most nAChR subunits possess the key residues included in the agonist site structure model. According to the model, most vertebrate α and non-α subunits can generate the functional heteropentamers and some α subunits can generate the functional homopentamers, which is far away from the fact that vertebrate nAChRs have the fixed subunits combinations (Millar 2003). Caenorhabditis elegans is the most abundant resource of nAChRs, but most subunits have not so far been functionally characterized and it is yet to be determined which neurotransmitters/ligands act on them because of the difficulties in the heterologous expression of C. elegans nAChR subunits (Jones et al. 2007). The same difficulties were encountered in the assembly and pharmacological studies on insect nAChRs (Tomizawa and Casida 2001; Millar and Denholm 2007). No functional nAChR pentamers were identified even when insect nAChR subunits were heterologously expressed together with different insect subunit combinations or all subunits from one insect species (Lansdell and Millar 2000; Liu et al. 2005, 2006). The fact that the key residues in the agonist structure model were included in insect nAChRs and no functional pentamers were identified in the expression of recombinant nAChRs from insect species in heterologous expression systems, gives some indications that the model is not generally suitable for some species, or other important residues in the regions outside these six loops also play essential roles in nAChRs function.
Co-expression of the insect α-type subunit with vertebrate β-type subunit constitutes the best available model at present, although these hybrid receptors may not faithfully reflect the insect nAChRs (Tomizawa and Casida 2001). However, even in this co-expression system, not all insect α subunits could generate functional pentamers with vertebrate β subunits (Lansdell and Millar 2004). In our previous studies, it was found that N. lugensα1 subunit (Nlα1) could be functionally co-expressed with rat β2 both in Drosophila S2 cells or Xenopus oocytes, but N. lugensα3 subunit (Nlα3) could not (Liu et al. 2005, 2006). By exchanging the corresponding regions between Nlα1 and Nlα3 to generate different chimeras and checking the binding activity of the S2 cells co-expressed these chimeras with rat β2, present work demonstrated that amino acid residues in the regions outside the six loops really play essential roles in nAChRs function. Actually, exchange of any region of the extracellular N-coding fragment could affect the co-expressed binding activity, and a few of them did it significantly. Especially, exchange of the region CH9 in the loop B–C interval could totally delete the agonist binding ability of Nlα1 and activate Nlα3. The loop B–C interval was previously report to be important in the selective interactions with imidacloprid in insects (Shimomura et al. 2004), which also implied its importance for agonist binding. Our discovery obviously means that the residues in the six loops could be necessary, but not enough for agonist binding.
And the accumulative effects were found in the influence on the ligand binding when two or more regions (subregions) were exchanged. CH1, CH4, and CH8 regions all showed the additional effects on ligand binding activities when CH9 region was exchanged together with these regions. Subregions II, IV, and V, co-exchanged with subregions I and III, showed the accumulative effects too. It seems that the amino acid residues out and in the loops do not behavior alike. The residues in the loops interact with agonists directly and the substitution in these residues always produces crucial influence on agonist binding. For the residues out of the loops, the substitutions might only show the quantitative effects. However, the decisive effects of the substitutions in the residues out of the loops could not be excluded, such as the results of our present study. At present, it could be concluded that not only the residues in the six loops as these residues identified from Torpedo nAChRs and molluscan AChBP, but also some other residues or residue clusters out of the loops in the extracellular N-domain contribute to the activity of agonist binding in insect nAChRs.