Pharmacological profiles of recombinant and native insect nicotinic acetylcholine receptors
Introduction
The nicotinic acetylcholine receptor (nAChR) is an agonist-gated ion channel complex for rapid excitatory neurotransmission. It is widely distributed in the insect central nervous system and constitutes a major target for insecticide action (Tomizawa and Casida, 2003). The functional architecture and diversity of insect nAChRs are superficially understood in contrast to their vertebrate counterparts. Genes encoding the ligand-binding α and structural β subunits have been cloned in several insect species. For instance in the fruit fly (Drosophila melanogaster), seven α (Dα1–7) and three β (Dβ1–3) subunit genes are cloned. Also in the peach–potato aphid (Myzus persicae), a major target pest for neonicotinoid insecticides, there are six identified genes for five α (Mpα1–5) and a β (Mpβ1) subunits (Gundelfinger and Schulz, 2000; Tomizawa and Casida, 2001; Grauso et al., 2002; Millar, 2003; Lansdell and Millar, 2004). The first four Drosophila α subunit genes (Dα1–4) and three β subunit genes (Dβ1–3), on expression in Xenopus oocytes, human embryonic kidney 293 cells or Drosophila S2 cells, singly or in various combinations, never produce any electrophysiological response or radioligand binding (Tomizawa and Casida, 2001; Millar, 2003). Dα6 and Dα7 genes can be expressed as chimera receptors containing the C-terminal domain of the serotonin-3A receptor to give [125I]α-bungarotoxin (α-BGT) and [3H]methyllycaconitine binding activities (Lansdell and Millar, 2004). Myzus Mpα1 and Mpα2 subunits form functional homomeric receptors expressed in Xenopus oocytes (Sgard et al., 1998) but no radioligand binding is detected with any of the four α subunits (Mpα1–4) alone or in combinations with Mpβ1 expressed in Drosophila S2 cells (Huang et al., 1999, Huang et al., 2000). However, functional ion channel property or radioligand binding activity is observed when any one of the four Drosophila α (Dα1–4) or three Myzus α (Mpα1–3) subunits is coexpressed in a hybrid receptor with a chick or rat β2 subunit (Bertrand et al., 1994; Schulz et al., 1998; Sgard et al., 1998; Huang et al., 1999; Lansdell and Millar, 2000a, Lansdell and Millar, 2000b).
Neonicotinoids (Fig. 1) are the only major new class of insecticides developed in the past three decades with worldwide annual sales currently accounting for 15% of the total insecticide market (Nauen et al., 2001; Matsuda et al., 2001; Kagabu, 2003; Tomizawa and Casida, 2003, Tomizawa and Casida, 2005). The excellent selective toxicity of neonicotinoids between insects and vertebrates is mainly conferred by the differential sensitivity of the insect versus vertebrate nAChRs (Tomizawa and Casida, 2003, Tomizawa and Casida, 2005). As an example, the neonicotinoid radioligand [3H]imidacloprid ([3H]IMI) serves as an excellent probe for insect but not vertebrate nAChRs (Liu and Casida, 1993; Tomizawa and Casida, 2003). [3H]Epibatidine ([3H]EPI) and [125I]- or [3H]α-BGT are important probes for characterizing the vertebrate α4β2 and α7 nAChR subtypes, respectively (Anand et al., 1993; Houghtling et al., 1995). In native insect nAChRs, the [3H]IMI binding site in Drosophila is distinct from that for [3H]α-BGT (Zhang et al., 2004). Specific [3H]EPI binding has been detected in some insects such as the American cockroach (Periplaneta americana) but not in others such as the housefly (Musca domestica) (Orr et al., 1997; Nauen et al., 2001).
Pharmacological profiles of the recombinant hybrid insect α/vertebrate β nAChRs are poorly defined and the binding sites are not established for identified subunits versus native receptors. Dα2 and Mpα2 are considered to be the main neonicotinoid-binding components based on protein biochemistry and immunology studies (Chamaon et al., 2002; Tomizawa and Casida, 1997; Tomizawa et al., 1996, Tomizawa et al., 2001a) and sensitivity to IMI for the hybrid receptors (Lansdell and Millar, 2000a; Huang et al., 1999; Ihara et al., 2003). In this report, we characterize the [3H]IMI and [3H]EPI binding sites in two recombinant hybrid nAChRs co-assembled by either Dα2 or Mpα2 with rat β2 subunits (Dα2/Rβ2 and Mpα2/Rβ2 receptors) and the pharmacological properties are compared with those of the native Drosophila and Myzus nAChRs and the vertebrate α4β2 subtype.
Section snippets
Chemicals
Sources for the compounds were: [3H]EPI (53 Ci/mmol) and [3H]α-BGT (37 Ci/mmol) from Amersham Biosciences (Piscataway, NJ, USA); [3H]IMI (32 Ci/mmol) from Syngenta Crop Protection (Basel, Switzerland); (−)-nicotine hydrogen tartrate (NIC) and α-BGT from Sigma (St. Louis, MO, USA); (±)-EPI hydrochloride from TOCRIS (St. Louis, MO, USA). The neonicotinoids and nicotinoids were available from previous studies in the Berkeley laboratory (Tomizawa et al., 2000).
Cell culture
Schneider's Drosophila S2 cells stably
Radioligand binding profiles of Dα2/Rβ2 hybrid, Mpα2/Rβ2 hybrid, Drosophila native, and vertebrate α4β2 nAChRs (Table 1)
The four receptor preparations were compared with [3H]IMI, [3H]EPI, and [3H]α-BGT. Specific binding of [3H]IMI was detected to hybrid Dα2/Rβ2 nAChRs (50 fmol/mg protein and 68%) but, surprisingly, the level was lower than that detected with [3H]EPI. In contrast, no significant binding of [3H]α-BGT was observed in Dα2/Rβ2. With hybrid Mpα2/Rβ 2 nAChRs, [3H]IMI and [3H]EPI gave relatively high levels of specific binding (1000–1500 fmol/mg protein and 98–99%) and [3H]α-BGT had low levels of specific
Unique pharmacological profiles of insect α/vertebrate β hybrid nAChRs
Due to difficulties which have been encountered in the heterologous expression of insect nAChR subunits, the pharmacological properties of recombinant nAChRs can only be defined for now by hybrid receptors consisting of insect α subunits and a vertebrate β subunit. By adopting this strategy, previous studies have revealed that [3H]IMI binds with high affinity to hybrid receptors in which Dα1, Dα2, Dα3, Mpα2, or Mpα3 was coassembled with rat β2 (Huang et al., 1999; Lansdell and Millar, 2000a).
Acknowledgements
The project described was supported by Grant number ES08424 (to Berkeley) from the National Institute of Environmental Health Sciences (NIEHS), NIH. The contents of this paper are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS, NIH. Valuable advice was received from our Berkeley laboratory colleague Gary Quistad. NSM acknowledges support from the Biotechnology and Biological Sciences Research Council for the United Kingdom.
References (52)
- et al.
Desnitroimidacloprid and nicotine binding site in rat recombinant α4β2 neuronal nicotinic acetylcholine receptor
Pestic. Biochem. Physiol.
(1999) - et al.
Cloning, heterologous expression and co-assembly of Mpβ1, a nicotinic acetylcholine receptor subunit from the aphid Myzus persicae
Neurosci. Lett.
(2000) - et al.
Diverse actions of neonicotinoids on chicken α7, α4β2 and Drosophila-chicken SADβ2 and ALSβ2 hybrid nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes
Neuropharmacology
(2003) - et al.
The influence of nicotinic receptor subunit composition upon agonist, α-bungarotoxin and insecticide (imidacloprid) binding affinity
Neuropharmacology
(2000) - et al.
Cloning and heterologous expression of Dα4, a Drosophila neuronal nicotinic acetylcholine receptor subunit: identification of an alternative exon influencing the efficiency of subunit assembly
Neuropharmacology
(2000) - et al.
[3H]Imidacloprid labels high- and low-affinity nicotinic acetylcholine receptor-like binding sites in the aphid Myzus persicae (Hemiptera: Aphididae)
Pestic. Biochem. Physiol.
(1998) - et al.
Characterisation of multiple α-bungarotoxin binding sites in the aphid Myzus persicae (Hemiptera: Aphididae)
Insect Biochem. Mol. Biol.
(1999) - et al.
High affinity binding of [3H]imidacloprid in the insect acetylcholine receptor
Pestic. Biochem. Physiol.
(1993) - et al.
Neonicotinoids: insecticides acting on insect nicotinic acetylcholine receptors
Trends Pharmacol. Sci.
(2001) - et al.
Pharmacological characterization of an epibatidine binding site in the nerve cord of Periplaneta americana
Pestic. Biochem. Physiol.
(1997)