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RDL receptors Ian McGonigle and Sarah C.R. Lummis1 Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, U.K.
Abstract RDL receptors are invertebrate members of the Cys-loop family of ligand-gated ion channels. They are GABA (γ -aminobutyric acid)-activated chloride-selective receptors that are closely related to their vertebrate orthologues, the GABAA receptors, as well as other Cys-loop receptors such as the ionotropic glycine, nicotinic acetylcholine and 5-HT3 receptors. RDL receptors are widely expressed throughout the insect CNS (central nervous system) and are important in inhibitory neurotransmission. They are therefore a major insecticidal target site.
RDL receptors The RDL subunit was originally identified in mutant Drosophila strains showing resistance to the insecticide dieldrin, hence the name ‘RDL’ [1,2]. Dieldrin blocks the channel of RDL receptors and an alanine-to-serine mutation was identified in the pore-lining M2 region (Ala302 ), which conferred resistance. RDL receptors (made from RDL subunits) are the best studied insect GABA (γ -aminobutyric acid)-activated receptors. Heterologous expression in Xenopus oocytes has shown that they are related to their vertebrate counterparts, namely GABAA receptors, in that their activation opens a chloride channel [3]. They do, however, have significant differences in their pharmacological profile in that they cannot be inhibited by the classic GABAA receptor antagonist bicuculline, although they are inhibited by the channel blocker picrotoxin [4–6]. RDL receptor subunits in Drosophila melanogaster can occur as a variety of different splice variants, resulting in various agonist sensitivities [3,7]. The regions that are modified lie in exons 3 and 6 (Figure 1). These alternative transcripts are named a, b (exon 3), c and d (exon 6) and the RDLac variant is considered the canonical isoform. Other insect GABA receptor subunits from D. melanogaster that have been identified and successfully cloned include GRD (glycine-like receptor of Drosophila) [8] and LCCH3 (ligandgated chloride channel homologue 3) [9]. Heterologously expressed RDL receptors have similar characteristics to GABA receptors of cultured Drosophila neurons [4], and the subunits are widely distributed throughout the adult and embryonic Drosophila CNS (central nervous system) [6,10,11]; thus it is reasonable to assume that these receptors play a major role in inhibitory neurotransmission in the insect CNS. Indeed RDL receptors have been shown to be important in olfactory learning and in the associated conditioned stimulus pathway [12,13], linking RDL receptors
Key words: Cys-loop, Drosophila, γ -aminobutyric acid receptor (GABA receptor), RDL receptor. Abbreviations used: AChBP, acetylcholine-binding protein; CNS, central nervous system; GABA, γ -aminobutyric acid; GRD, glycine-like receptor of Drosophila, LCCH3, ligand-gated chloride channel homologue 3; nACh, nicotinic acetylcholine; TACA, (E)-4-aminobuten-2-oic acid. 1 To whom correspondence should be addressed (email
[email protected]).
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to normal neural function. As insect GABA receptors are a major insecticidal target [14,15], RDL receptors expressed in Xenopus oocytes therefore represent an effective drug screening platform.
RDL receptors are Cys-loop receptors RDL subunits are homologous with subunits from the Cys-loop family of LGICs (ligand-gated ion channels) whose vertebrate members include the GABAA , glycine, 5-HT3 (5-hydroxytryptamine 3) and nACh (nicotinic acetylcholine) receptors [16]. There are also a range of invertebrate Cys-loop receptors, many of which are activated by GABA, although, interestingly, GABA activation in several of these receptors (e.g. GRD, LCCH3 and EXP-1) results in the activation of a cation and not an anion selective channel [17,18]. Extrapolation from vertebrate receptor data suggests that all of these proteins are pentameric, consisting of five subunits arranged pseudo-symmetrically around a central ion pore [19,20]. The N-terminal extracellular domain of these receptors is largely β-sheet [as in the related AChBP (acetylcholine-binding protein)], whose structure ˚ (1 A ˚ =0.1 nm) [21]) and contains has been resolved to 2.2 A the signature disulfide Cys-loop. The four transmembrane regions (M1–M4) are α-helical in structure and the second region (M2) lines the integral ion channel [19].
The RDL receptor-binding site The high-resolution structure of AChBP and, more recently, of nACh receptor subunits and related prokaryotic receptors have allowed homology modelling of the ligand-binding pocket in Cys-loop receptors [19–21]. The binding site is located between two adjacent subunits in the extracellular domain, and aromatic residues from six discontinuous regions (loops A–F) have been shown to be important for receptor function [22–27]. A model of the RDL receptor is shown in Figure 2. Specific residues that are important for GABA binding in GABAA receptors have been identified, and include a loop A tyrosine residue that forms a cation–π interaction with the positively charged ammonium group of Biochem. Soc. Trans. (2009) 37, 1404–1406; doi:10.1042/BST0371404
Neuronal Glutamate and GABAA Receptor Function in Health and Disease
Figure 1 Regions of rdl (the gene for the RDL subunit) that are alternatively spliced Exons 3 and 6 undergo alternative splicing, resulting in altered agonist-sensitivity [7]. Exons 3 and 6 correspond to extracellular loops D and F respectively [7].
Figure 3 RDL agonist profile Top: concentration–response curves showing responses elicited by GABA analogues in RDL receptors expressed in Xenopus oocytes. Agonist potencies were muscimol>GABA>TACA>isoguvacine>β-alanine. Glycine did not elicit a response (>10 mM). Results are the means±S.E.M. for at least three oocytes. Bottom: structures of GABA analogues.
Figure 2 The RDL receptor binding site Top: RDL extracellular domain dimer. A three-dimensional homology model of the extracellular domain of an RDL GABA receptor was generated using MODELLER 6v2, based on the crystal structure of AChBP (PDB code 1I9B) at 2.7 Å resolution, and GABA was docked into the binding site (GOLD 3.0). Extracellular loops A–F contribute to the agonist-binding site. A GABA molecule is docked close to loops A, B and C. Bottom: aromatic residues that contribute to the binding pocket in the RDL receptor have mostly aromatic residues in the equivalent locations in other Cys-loop receptors.
GABA [23] and an arginine residue in loop C that interacts with the carboxylate end of the ligand [28]. Interestingly residue interaction with GABA is subtly different in GABAC receptors, a subclass of GABAA receptors: here there is a cation–π interaction with a loop B tyrosine residue [29], whereas the carboxylate interacts with a loop D arginine
residue [25]. The model of the RDL receptor-binding site indicates that GABA is located in a broadly similar orientation, with its ammonium group located between aromatic residues in loops B and C, and its carboxylate facing loop D. However, the specific interactions between GABA and residues in the RDL-binding site have not yet been clarified and are likely to differ from both of these as the pharmacological profile of RDL receptors is different from both of its vertebrate counterparts: the GABAC receptor agonist TACA [(E)-4-aminobuten-2-oic acid], a conformationally restricted GABA analogue, is reasonably potent, as is the GABAA receptor agonist isoguvacine, a pyridine GABA analogue (Figure 3). The GABAA agonists muscimol, isoguvacine and TACA [30,31] are agonists of RDL receptors with similar potencies to GABA. The GABAC and glycine receptor partial agonist, βalanine [32], is a poor agonist at RDL receptors; however, glycine does not activate RDL receptors (Figure 3). Other GABA analogues previously shown to activate RDL receptors with relatively low potencies include ZAPA {(Z)3-[(aminoiminomethyl)thio]prop-2-enoic acid}, isonipecotic acid, CACA (cis-aminocrotonic acid; the cis-isomer of TACA), 3-APS (3-aminopropanesulfonic acid) [31] and THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) [33]. C The
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Conclusion RDL receptors are an important class of insect GABA receptors. They are widely expressed in the Drosophila CNS, where they mediate inhibitory neurotransmission. RDL receptors are activated by a wide range of ligands and have a pharmacology that is distinct from their vertebrate orthologues. Resolving the molecular details of the RDL receptor would greatly facilitate the generation of RDLspecific antagonists, which could generate subunit-selective pesticides.
Funding This work was supported by a Wellcome Trust Senior Research Fellowship in Basic Biomedical Science to S.C.R.L. and a Medical Research Council graduate studentship to I.McG.
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