InteractiveFly: GeneBrief

KaiR1D: Biological Overview | References


Gene name - CG3822

Synonyms - DKaiR1D, KaiR1D

Cytological map position - 93A2-93A2

Function - glutamate channel

Keywords - glutamate channel localized at presynaptic terminals of the neuromuscular junction, homeostatic potentiation of synaptic strength, eye, kainate family protein

Symbol - CG3822

FlyBase ID: FBgn0038837

Genetic map position - chr3R:20,860,187-20,866,231

Classification - Ligated ion channel L-glutamate- and glycine-binding site

Cellular location - transmembrane



NCBI links: EntrezGene
BIOLOGICAL OVERVIEW

Homeostatic signaling systems are thought to interface with other forms of plasticity to ensure flexible yet stable levels of neurotransmission. The role of neurotransmitter receptors in this process, beyond mediating neurotransmission itself, is not known. Through a forward genetic screen, the Drosophila kainate-type ionotropic glutamate receptor subunit DKaiR1D was identified as being required for the retrograde, homeostatic potentiation of synaptic strength. DKaiR1D is necessary in presynaptic motor neurons, localized near active zones, and confers robustness to the calcium sensitivity of baseline synaptic transmission. Acute pharmacological blockade of DKaiR1D disrupts homeostatic plasticity, indicating that this receptor is required for the expression of this process, distinct from developmental roles. Finally, this study demonstrates that calcium permeability through DKaiR1D is necessary for baseline synaptic transmission, but not for homeostatic signaling. It is proposed that DKaiR1D is a glutamate autoreceptor that promotes robustness to synaptic strength and plasticity with active zone specificity (Kiragasi, 2017).

The nervous system is endowed with potent and adaptive homeostatic signaling systems that maintain stable functionality despite the myriad changes that occur during neural development and maturation. The importance of homeostatic regulation in the nervous system is underscored by associations with a variety of neurological diseases, yet the genes and mechanisms involved remain enigmatic. A powerful model of presynaptic homeostatic plasticity has been established at the Drosophila neuromuscular junction (NMJ), a model glutamatergic synapse with molecular machinery that parallels central synapses in mammals. Here, genetic and pharmacological manipulations that reduce postsynaptic (muscle) glutamate receptor function trigger a trans-synaptic, retrograde feedback signal to the neuron that increases presynaptic release to precisely compensate for this perturbation. This process is referred to as 'presynaptic homeostatic potentiation' (PHP), because the expression mechanism requires a presynaptic increase in neurotransmitter release (Kiragasi, 2017).

In recent years, forward and candidate genetic approaches have revealed several new and unanticipated genes necessary for PHP expression. While perturbations to the glutamate receptors in muscle are crucial events in the induction of PHP, whether other ionotropic glutamate receptors (iGluRs) function in PHP or are even expressed at the Drosophila NMJ is unknown. Finally, although evidence has emerged that homeostatic modulation is synapse specific, no roles for neurotransmitter receptors or other factors have been found to enable the presynaptic tuning of release efficacy at individual synapses (Kiragasi, 2017).

This study has identified the kainate-type iGluR subunit DKaiR1D to be necessary for PHP expression at the Drosophila NMJ. DKaiR1D is necessary for the calcium sensitivity of baseline synaptic transmission, as well as for the acute and chronic expression of homeostatic potentiation. Recently, the functional reconstitution of DKaiR1D was achieved in heterologous cells, revealing that these receptors form homomeric calcium-permeable channels with atypical pharmacological properties compared to their vertebrate homologs (Li, 2016). This study has found that DKaiR1D is expressed in the nervous system and not the muscle, is present near presynaptic active zones, and is required specifically in motor neurons to enable the robustness of baseline neurotransmission and homeostatic plasticity. It is proposd that glutamate activates DKaiR1D at presynaptic release sites to translate autocrine activity into the robust stabilization of synaptic strength with active zone specificity (Kiragasi, 2017).

This unexpected role for iGluRs in sensing glutamate at presynaptic terminals indicates an autocrine mechanism that responds to glutamate release to adaptively modulate presynaptic activity at individual active zones (Kiragasi, 2017).

Glutamate receptors have diverse functions in modulating presynaptic excitability and short-term plasticity in addition to their established roles in postsynaptic excitation. Similar to what was observed with DKaiR1D at the Drosophila NMJ, rodent iGluRs also localize to presynaptic active zone, are activated by high concentrations of glutamate, and can modulate release during single action potentials (McGuinness, 2010; Pinheiro, 2007; Schmitz, 2000). This suggests conserved autocrine modulatory mechanisms shared between these systems (Kiragasi, 2017).

Rodent autoreceptors are known to modulate presynaptic activity on rapid timescales. In these cases, most of the impact on release is likely to derive from a calcium store-dependent mechanism, or from modulation of the action potential during the repolarization phase, when most of the calcium influx that drives vesicle release occurs (Schneggenburger, 2015). In a similar fashion, activation of presynaptic DKaiR1D during a single action potential could lead to a rapid additional source of presynaptic calcium influx from DKaiR1D itself and/or through modulation of presynaptic membrane potential to drive increased vesicle release. Voltage imaging at the Drosophila NMJ has found the half width of the action potential waveform to be ~5 ms (Ford, 2014), which is sufficient time to be modulated through such a mechanism. Therefore, dynamic changes in voltage or calcium influx at or near active zones could, in principle, drive additional vesicle release during a single action potential. This modulation may be restricted to nearby active zones and compartments relative to the site of glutamate release. Indeed, presynaptic kainate autoreceptors have the capacity to confer short-range, synapse-specific modulation to synaptic transmission (Scott, 2008), while presynaptic ligand-gated ion channels in C. elegans can also rapidly modulate synaptic transmission (Takayanagi-Kiya, 2016). Local activation of DKaiR1D could, therefore, subserve a powerful and flexible means of tuning presynaptic efficacy at or near individual release sites (Kiragasi, 2017).

How does DKaiR1D promote the expression of presynaptic homeostatic plasticity? In contrast to the role of DKaiR1D in baseline release, the data indicate that the DKaiR1D-dependent mechanism that drives PHP is calcium independent. This implies two changes to DKaiR1D functionality that are unique to homeostatic adaptation compared to baseline transmission. First, because presynaptic release is acutely potentiated following application of PhTx, the activity, levels, and/or localization of DKaiR1D receptors must change to acquire a novel influence on neurotransmitter release following PHP induction. The activity of synaptic glutamate receptors can change through associations with additional subunits and auxiliary factors such as Neto (Kim, 2012; Straub, 2011). Furthermore, various forms of plasticity are expressed through dynamic changes in the levels and localization of glutamate receptors trafficking between active zones and endosome pools or extra-synaptic membrane (Anggono, 2012; Kneussel, 2016; Yan, 2013). Indeed, when DKaiR1D is overexpressed in motor neurons, it rescues baseline transmission and PHP expression while localizing to heterogeneous puncta of varying distances relative to active zones. Notably, there is evidence that DKaiR1D interacts with other glutamate receptor subunits in vivo (Karuppudurai, 2014), which may contribute to the pharmacological differences observed in this study compared with the in vitro characterization (Li, 2016) and may also be targets of modulation during PHP (Kiragasi, 2017).

Second, calcium signaling through DKaiR1D differentially drives baseline release and homeostatic plasticity. Therefore, mechanisms distinct from calcium permeability of the channel must contribute to PHP expression. One possibility is that DKaiR1D signals through an undefined metabotropic mechanism during PHP, which might contribute to the ability of the calcium impermeable DKaiR1DR transgene, with reduced conductance (Li, 2016), to rescue PHP expression. Alternatively, an attractive possibility is that following PHP induction, glutamate released from nearby active zones may dynamically modulate the presynaptic membrane potential and/or action potential waveform to promote additional synaptic vesicle release. Indeed, small, sub-threshold depolarizations of the presynaptic resting potential, as small as 5 mV, are sufficient to induce a 2-fold increase in presynaptic release. The timescale of this activity could occur within a few milliseconds as discussed above, and studies at the Drosophila NMJ have revealed that glutamate is released from single synaptic vesicles over timescales of milliseconds. Interestingly, epithelial sodium channels (ENaCs) have been proposed to enable PHP expression through changes in the presynaptic membrane potential, and such a mechanism could be shared by DKaiR1D but gated by glutamate release at individual active zones. Thus, DKaiR1D may serve to homeostatically modulate presynaptic release through modulation of presynaptic voltage and, intriguingly, with active zone specificity (Kiragasi, 2017).

This characterization of DKaiR1D has revealed a role for presynaptic glutamate signaling in homeostatic plasticity. In the mammalian CNS, glutamate signaling drives the adaptive regulation of postsynaptic AMPA receptor insertion and removal, known as homeostatic scaling (Turrigiano, 2008). Further, kainate receptors were recently demonstrated to regulate postsynaptic homeostatic scaling (Yan, 2013). Together with the present study, these results demonstrate that glutamatergic signaling through kainate receptors orchestrate the potent and adaptive homeostatic control of synaptic strength on both sides of the synapse. Future studies will reveal the integration between synaptic glutamate signaling and other forces that modulate synaptic strength to enable robust, flexible, and stable neurotransmission(Kiragasi, 2017).

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 (Alberstein, 2015). 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 (Han, 2015), 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

Search PubMed for articles about Drosophila DKaiR1D

Alberstein, R., Grey, R., Zimmet, A., Simmons, D. K. and Mayer, M. L. (2015). Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes. Proc Natl Acad Sci U S A 112(44): E6048-6057. PubMed ID: 26460032

Anggono, V. and Huganir, R. L. (2012). Regulation of AMPA receptor trafficking and synaptic plasticity. Curr Opin Neurobiol 22(3): 461-469. PubMed ID: 22217700

Ford, K. J. and Davis, G. W. (2014). Archaerhodopsin voltage imaging: synaptic calcium and BK channels stabilize action potential repolarization at the Drosophila neuromuscular junction. J Neurosci 34(44): 14517-14525. PubMed ID: 25355206

Han, T. H., Dharkar, P., Mayer, M. L. and Serpe, M. (2015). Functional reconstitution of Drosophila melanogaster NMJ glutamate receptors. Proc Natl Acad Sci U S A 112(19): 6182-6187. PubMed ID: 25918369

Karuppudurai, T., Lin, T. Y., Ting, C. Y., Pursley, R., Melnattur, K. V., Diao, F., White, B. H., Macpherson, L. J., Gallio, M., Pohida, T. and Lee, C. H. (2014). A hard-wired glutamatergic circuit pools and relays UV signals to mediate spectral preference in Drosophila. Neuron 81(3): 603-615. PubMed ID: 24507194

Kim, Y.J., Bao, H., Bonanno, L., Zhang, B., and Serpe, M. (2012). Drosophila Neto is essential for clustering glutamate receptors at the neuromuscular junction. Genes Dev. 26: 974-987. PubMed ID: 22499592

Kiragasi, B., Wondolowski, J., Li, Y. and Dickman, D. K. (2017). A presynaptic glutamate receptor subunit confers robustness to neurotransmission and homeostatic potentiation. Cell Rep 19(13): 2694-2706. PubMed ID: 28658618

Kneussel, M. and Hausrat, T. J. (2016). Postsynaptic neurotransmitter receptor reserve pools for synaptic potentiation. Trends Neurosci 39(3): 170-182. PubMed ID: 26833258

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

McGuinness, L., Taylor, C., Taylor, R. D., Yau, C., Langenhan, T., Hart, M. L., Christian, H., Tynan, P. W., Donnelly, P. and Emptage, N. J. (2010). Presynaptic NMDARs in the hippocampus facilitate transmitter release at theta frequency. Neuron 68(6): 1109-1127. PubMed ID: 21172613

Pinheiro, P. S., Perrais, D., Coussen, F., Barhanin, J., Bettler, B., Mann, J. R., Malva, J. O., Heinemann, S. F. and Mulle, C. (2007). GluR7 is an essential subunit of presynaptic kainate autoreceptors at hippocampal mossy fiber synapses. Proc Natl Acad Sci U S A 104(29): 12181-12186. PubMed ID: 17620617

Schmitz, D., Frerking, M. and Nicoll, R. A. (2000). Synaptic activation of presynaptic kainate receptors on hippocampal mossy fiber synapses. Neuron 27(2): 327-338. PubMed ID: 10985352

Schneggenburger, R. and Rosenmund, C. (2015). Molecular mechanisms governing Ca(2+) regulation of evoked and spontaneous release. Nat Neurosci 18(7): 935-941. PubMed ID: 26108721

Scott, R., Lalic, T., Kullmann, D. M., Capogna, M. and Rusakov, D. A. (2008). Target-cell specificity of kainate autoreceptor and Ca2+-store-dependent short-term plasticity at hippocampal mossy fiber synapses. J Neurosci 28(49): 13139-13149. PubMed ID: 19052205

Straub, C., Hunt, D. L., Yamasaki, M., Kim, K. S., Watanabe, M., Castillo, P. E. and Tomita, S. (2011). Distinct functions of kainate receptors in the brain are determined by the auxiliary subunit Neto1. Nat Neurosci 14(7): 866-873. PubMed ID: 21623363

Takayanagi-Kiya, S., Zhou, K. and Jin, Y. (2016). Release-dependent feedback inhibition by a presynaptically localized ligand-gated anion channel. Elife 5. PubMed ID: 27782882

Turrigiano, G. G. (2008). The self-tuning neuron: synaptic scaling of excitatory synapses. Cell 135(3): 422-435. PubMed ID: 18984155

Yan, D., Yamasaki, M., Straub, C., Watanabe, M. and Tomita, S. (2013). Homeostatic control of synaptic transmission by distinct glutamate receptors. Neuron 78(4): 687-699. PubMed ID: 23719165


Biological Overview

date revised: 8 July 2017

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