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  • Novel functional properties of Drosophila CNS glutamate receptors


    Novel functional properties of Drosophila CNS glutamate receptors

    Phylogenetic analysis reveals AMPA, kainate, and NMDA receptor families in insect genomes, suggesting conserved functional properties corresponding to their vertebrate counterparts. However, heterologous expression of the Drosophila kainate receptor DKaiR1D and the AMPA receptor DGluR1A revealed novel ligand selectivity at odds with the classification used for vertebrate glutamate receptor ion channels (iGluRs). DKaiR1D forms a rapidly activating and desensitizing receptor that is inhibited by both NMDA and the NMDA receptor antagonist AP5; crystallization of the KaiR1D ligand-binding domain reveals that these ligands stabilize open cleft conformations, explaining their action as antagonists. Surprisingly, the AMPA receptor DGluR1A shows weak activation by its namesake agonist AMPA and also by quisqualate. Crystallization of the DGluR1A ligand-binding domain reveals amino acid exchanges that interfere with binding of these ligands. The unexpected ligand-binding profiles of insect iGluRs allows classical tools to be used in novel approaches for the study of synaptic regulation (Li, 2016). Video Abstract

    Glutamate is the major excitatory neurotransmitter in the vertebrate CNS; its actions are mediated largely via three classes of ionotropic glutamate receptors (iGluRs) named AMPA, kainate, and NMDA receptors. The classification of iGluRs into AMPA, kainate, and NMDA receptors was based on the efforts of medicinal chemists who identified subtype selective heterocyclic amino acids such as AMPA, kainate, and quisqualate and amino acid analogs such as NMDA and 2(R)-amino-5-phosphonopentanoic acid (D-AP5) that act as agonists and antagonists. This work was so successful that the selective action of NMDA and D-AP5 formed the corner stone on which the role of NMDA receptors in synaptic plasticity was established (Li, 2016).

    Subsequent cloning of insect iGluRs, which revealed sequence similarity with their vertebrate AMPA, kainate, and NMDA receptor counterparts, suggests that the same series of ligands can be used to investigate their role in CNS function. However, with the exception of the neuromuscular junction (NMJ) of larval Drosophila and the NMJ of adult locusts, the small size and inaccessibility of insect neurons has to date challenged characterization of the functional properties of native insect iGluRs. Sequence analysis of the Drosophila genome identified 14 iGluR genes that resemble vertebrate AMPA, kainate, and NMDA receptors. Transcript profiling revealed that nine of these iGluRs are expressed in the brain, with five expressed at the neuromuscular junction. Very little is known about the structure and functional properties of Drosophila iGluRs and only recently was a functional reconstitution achieved for recombinant Drosophila NMJ iGluRs (Han, 2015). As a result, iGluRs are understudied in model organisms like Drosophila for which powerful genetic techniques have otherwise yielded numerous insights into the molecular neurobiology of synapse development and function (Li, 2016).

    Four presumptive Drosophila kainate receptors (Clumsy, DKaiR1C, DKaiR1D, and CG11155) are functionally required for spectral preference behavior and are thought to mediate excitatory synaptic transmission from the second-order neuron Dm8 to the third-order neuron Tm5c (Karuppudurai, 2014). The eye-enriched kainate receptor (EKAR) is expressed in photoreceptors, receiving feedback glutamatergic signals from amacrine cells, but so far, in vitro reconstitution has not been achieved for any of these presumptive kainate receptors. Instead, functional analysis of their role in CNS glutamatergic circuits relies solely on chronic inactivation using genetic mutants and RNAi-mediated knockdown. This study combined electrophysiological, biochemical, and crystallographic analyses to determine receptor activity and ligand specificity of a Drosophila kainate receptor DKaiR1D and a Drosophila AMPA receptor DGluR1A. DKaiR1D was found to form functional homomeric channels in HEK cells and oocytes with pharmacological properties distinct from vertebrate and Drosophila NMJ iGluRs. Crystal structures of DKaiR1D ligand-binding dimer complexes with glutamate, NMDA, and AP5 revealed that only glutamate triggers domain closure and that NMDA and AP5 are antagonists. DGluR1A receptors respond weakly to AMPA and quisqualate; the crystal structure of DGluR1A revealed that the binding of these ligands is hindered by steric occlusion. Thus, despite structural and sequence similarity between insect and vertebrate iGluRs, insect iGluRs do not conform to the pharmacology-based classification of vertebrate iGluRs. However, the agonist/antagonist binding properties of insect iGluRs we report here provide a new approach for acute inactivation/activation in vivo and for dissecting their functions in complex neural circuits (Li, 2016).

    This study found that DGluR1A and DKaiR1D, similar to vertebrate GluA1-4 AMPA and GluK1-3 kainate receptor subunits, form homomeric calcium-permeable channels. Based on sequence alignments and the lack of RNA-editing of Drosophila iGluRs mRNA at their Q/R sites, it is likely that most insect iGluRs are calcium permeable and that they are inhibited by endogenous cytoplasmic polyamines and by spider venom polyamine toxins. It is noted that homomeric DKaiR1D has a very fast desensitization rate, while for DGluR1A, fit has not yet been possible to achieve sufficient expression to allow recording from outside-out patches with rapid perfusion. Structural analyses revealed that DKaiR1D LBD dimers contain conserved Na+ ion binding sites characteristic of vertebrate kainate receptors, but these appear to not strongly modulate the activation or desensitization of KaiR1D, perhaps because the Cl- binding site found in vertebrate kainate receptors is absent in insect kainate receptors. Sequence analysis revealed that this separation of Na+ and Cl- binding sites in KaiR1D subunits occurs in all insect species examined. Structure-aided sequence analysis also reveals that in the other three groups of fly kainate receptors, different combinations of amino acid substitutions destroy or significantly weaken both the Na+ and Cl− binding sites. Thus, the allosteric modulation by both anions and cations that is characteristic of vertebrate kainate receptors is uncoupled in insect kainate receptors and for the majority of cases both ion binding sites are eliminated (Li, 2016).

    Previous phylogenetic studies suggest that most bilateria, including insects, worms, and vertebrates, have three major classes of cation-selective iGluRs, corresponding to vertebrate AMPA, kainate, and NMDA receptors. The current analysis reveals that in insects, the kainate receptor family is expanded into four groups, while a prior phylogenetic analysis revealed that in Mollusca the AMPA receptor family is expanded. At the neuromuscular junction of Drosophila and the locust Schistocerca gregaria, iGluRs have been extensively studied, in part serving as a surrogate model for CNS iGluRs. Interestingly, this study found that late in evolution, in higher Diptera, the five Drosophila NMJ iGluR subunits, GluRIIA-E, were derived from two separate kainate receptor subtypes, KaiR1C and Clumsy. Thus, despite their unique obligate heterotetrameric subunit stoichiometry and insensitivity to kainate), fly NMJ iGluRs evolved from ancestral kainate-sensitive iGluRs, and it is likely that in other insect species iGluRs related to KaiR1C and Clumsy may function in both the CNS and NMJ (Li, 2016).

    Although phylogenetic analysis supports classification of Drosophila and other insect iGluRs into the familiar AMPA, kainate, and NMDA receptor families, the current results reveal unexpected differences in their ligand-binding properties. The most dramatic change was the conversion of NMDA from an agonist for vertebrate NMDA receptors to an antagonist for Drosophila KaiR1D, a kainate receptor that is also inhibited by both isomers of AP5, while D-AP5, but not L-AP5 acts as a potent vertebrate NMDA receptor antagonist. The crystal structures solved in this study for the DKaiR1D LBD establish that NMDA and AP5 inhibit activation of DKaiR1D by stabilizing an open cleft conformation, similar to the action of competitive antagonists for vertebrate iGluRs from each of the three major families. In addition, NMDA triggered separation of the upper lobes of the DKaiR1D LBD dimer assembly, a conformational change that occurs during desensitization of vertebrate AMPA and kainate receptors, may in addition contribute to the inhibitory action of NMDA on DKaiR1D (Li, 2016).

    AMPA receptors were initially identified by and named in response to their activation by quisqualic acid, a glutamate bioisostere that is nonselective and which activates all of the major vertebrate iGluR subtypes, in addition to acting as a potent agonist for G protein-coupled glutamate receptors. Prior to the cloning of GluA1–4 subunits, the so-called quisqualate receptors were renamed AMPA receptors, following the synthesis of AMPA and the discovery that it was a highly selective agonist, without activity at kainate, NMDA, or G protein-coupled glutamate receptors. These serendipitous events in the history of the development of selective ligands for iGluR subtypes were strongly reinforced when a large family of vertebrate iGluR subunits were cloned, and it was discovered that these encoded discrete families of iGluR subtypes, each with high sequence identity, the ligand-binding properties of which corresponded to the familiar AMPA, kainate, and NMDA receptor subtypes. The current experiments reveal an unexpected breakdown of the classification scheme for Drosophila and most likely other insect species iGluRs (Li, 2016).

    With the plethora of genetic tools and advanced connectome analyses, Drosophila has emerged as a key model organism for studying the circuit basis of behavior. It is now evident that like vertebrates, glutamatergic synapses are abundantly utilized in fly CNS circuits. Functional and structural analyses revealed that Drosophila iGluRs have agonist and antagonist selectivity very different from those of vertebrates, indicating that sequence and structural homology does not confer conserved pharmacological properties. However, the unique pharmacology of Drosophila iGluRs reported in this study has proven of use to reveal the role of KaiR1D in presynaptic homeostasis. It is envisioned that appropriate use of pharmacological tools in combination with powerful fly genetics will greatly aid studies of complex neural circuits in Drosophila (Li, 2016).



    REFERENCES

    Li, Y., Dharkar, P., Han, T. H., Serpe, M., Lee, C. H. and Mayer, M. L. (2016). Novel functional properties of Drosophila CNS glutamate receptors. Neuron 92(5): 1036-1048. PubMed ID: 27889096; Video Abstract



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