Postsynaptic Kainate Receptor Recycling and Surface Expression Are Regulated by Metabotropic Autoreceptor Signalling

Kainate receptors (KARs) play fundamentally important roles in controlling synaptic function and regulating neuronal excitability. Postsynaptic KARs contribute to excitatory neurotransmission but the molecular mechanisms underlying their activity‐dependent surface expression are not well understood. Strong activation of KARs in cultured hippocampal neurons leads to the downregulation of postsynaptic KARs via endocytosis and degradation. In contrast, low‐level activation augments postsynaptic KAR surface expression. Here, we show that this increase in KARs is due to enhanced recycling via the recruitment of Rab11‐dependent, transferrin‐positive endosomes into spines. Dominant‐negative Rab11 or the recycling inhibitor primaquine prevents the kainate‐evoked increase in surface KARs. Moreover, we show that the increase in surface expression is mediated via a metabotropic KAR signalling pathway, which is blocked by the protein kinase C inhibitor chelerythrine, the calcium chelator BAPTA and the G‐protein inhibitor pertussis toxin. Thus, we report a previously uncharacterized positive feedback system that increases postsynaptic KARs in response to low‐ or moderate‐level agonist activation and can provide additional flexibility to synaptic regulation.      

Differential trafficking of GluR7 kainate receptor subunit splice variants

Kainate receptors (KARs) are heteromeric ionotropic glutamate receptors that play a variety of roles in the regulation of synaptic network activity. The function of glutamate receptors (GluRs) is highly dependent on their surface density in specific neuronal domains. Alternative splicing is known to regulate surface expression of GluR5 and GluR6 subunits. The KAR subunit GluR7 exists under different splice variant isoforms in the C-terminal domain (GluR7a and GluR7b). Here we have studied the trafficking of GluR7 splice variants in cultured hippocampal neurons from wild-type and KAR mutant mice. We have found that alternative splicing regulates surface expression of GluR7-containing KARs. GluR7a and GluR7b differentially traffic from the ER to the plasma membrane. GluR7a is highly expressed at the plasma membrane, and its trafficking is dependent on a stretch of positively charged amino acids also found in GluR6a. In contrast, GluR7b is detected at the plasma membrane at a low level and retained mostly in the endoplasmic reticulum (ER). The RXR motif of GluR7b does not act as an ER retention motif, at variance with other receptors and ion channels, but might be involved during the assembly process. Like GluR6a, GluR7a promotes surface expression of ER-retained subunit splice variants when assembled in heteromeric KARs. However, our results also suggest that this positive regulation of KAR trafficking is limited by the ability of different combinations of subunits to form heteromeric receptor assemblies. These data further define the complex rules that govern membrane delivery and subcellular distribution of KARs.     

Assembly and cell surface expression of KA-2 subunit-containing kainate receptors

Kainate receptors (KARs) modulate synaptic transmission at both pre-synaptic and post-synaptic sites. The overlap in the distribution of KA-2 and GluR6/7 subunits in several brain regions suggests the co-assembly of these subunits in native KARs. The molecular mechanisms that control the assembly and surface expression of KARs are unknown. Unlike GluR5-7, the KA-2 subunit is unable to form functional homomeric KAR channels. We expressed the KA-2 subunit alone or in combination with other KAR subunits in HEK-293 cells. The cell surface expression of the KAR subunit homo- and heteromers were analysed using biotinylation and agonist-stimulated cobalt uptake. While GluR6 or GluR7 homomers were expressed on the cell surface, KA-2 alone was retained within the endoplasmic reticulum. We found that the cell surface expression of KA-2 was dramatically increased by co-expression with either of the low-affinity KAR subunits GluR5-7. However, co-expression with other related ionotropic glutamate receptor subunits (GluR1 and NR1) does not facilitate the cell surface expression of KA-2. The analysis of subcellular fractions of neocortex revealed that synaptic KARs have a relatively high KA-2 content compared to microsomal ones. Thus, KA-2 is likely to contain an endoplasmic reticulum retention signal that is shielded on assembly with other KAR subunits.     

Small GTPase Rab17 Regulates the Surface Expression of Kainate Receptors but Not α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors in Hippocampal Neurons via Dendritic Trafficking of Syntaxin-4 Protein

Glutamate receptors are fundamental for control synaptic transmission, synaptic plasticity, and neuronal excitability. However, many of the molecular mechanisms underlying their trafficking remain elusive. We previously demonstrated that the small GTPase Rab17 regulates dendritic trafficking in hippocampal neurons. Here, we investigated the role(s) of Rab17 in AMPA receptor (AMPAR) and kainate receptor (KAR) trafficking. Although Rab17 knockdown did not affect surface expression of the AMPAR subunit GluA1 under basal or chemically induced long term potentiation conditions, it significantly reduced surface expression of the KAR subunit GluK2. Rab17 co-localizes with Syntaxin-4 in the soma, dendritic shaft, the tips of developing hippocampal neurons, and in spines. Rab17 knockdown caused Syntaxin-4 redistribution away from dendrites and into axons in developing hippocampal neurons. Syntaxin-4 knockdown reduced GluK2 but had no effect on GluA1 surface expression. Moreover, overexpression of constitutively active Rab17 promoted dendritic surface expression of GluK2 by enhancing Syntaxin-4 translocation to dendrites. These data suggest that Rab17 mediates the dendritic trafficking of Syntaxin-4 to selectively regulate dendritic surface insertion of GluK2-containing KARs in rat hippocampal neurons.     

CaMKII‐dependent phosphorylation of GluK5 mediates plasticity of kainate receptors

Calmodulin‐dependent kinase II (CaMKII) is key for long‐term potentiation of synaptic AMPA receptors. Whether CaMKII is involved in activity‐dependent plasticity of other ionotropic glutamate receptors is unknown. We show that repeated pairing of pre‐ and postsynaptic stimulation at hippocampal mossy fibre synapses induces long‐term depression of kainate receptor (KAR)‐mediated responses, which depends on Ca²⁺ influx, activation of CaMKII, and on the GluK5 subunit of KARs. CaMKII phosphorylation of three residues in the C‐terminal domain of GluK5 subunit markedly increases lateral mobility of KARs, possibly by decreasing the binding of GluK5 to PSD‐95. CaMKII activation also promotes surface expression of KARs at extrasynaptic sites, but concomitantly decreases its synaptic content. Using a molecular replacement strategy, we demonstrate that the direct phosphorylation of GluK5 by CaMKII is necessary for KAR‐LTD. We propose that CaMKII‐dependent phosphorylation of GluK5 is responsible for synaptic depression by untrapping of KARs from the PSD and increased diffusion away from synaptic sites.     

Subunit composition of kainate receptors in hippocampal interneurons

Kainate receptor activation affects GABAergic inhibition in the hippocampus by mechanisms that are thought to involve the GluR5 subunit. We report that disruption of the GluR5 subunit gene does not cause the loss of functional KARs in CA1 interneurons, nor does it prevent kainate-induced inhibition of evoked GABAergic synaptic transmission onto CA1 pyramidal cells. However, KAR function is abolished in mice lacking both GluR5 and GluR6 subunits, indicating that KARs in CA1 stratum radiatum interneurons are heteromeric receptors composed of both subunits. In addition, we show the presence of presynaptic KARs comprising the GluR6 but not the GluR5 subunit that modulate synaptic transmission between inhibitory interneurons. The existence of two separate populations of KARs in hippocampal interneurons adds to the complexity of KAR localization and function.     

Kainate receptors and RNA editing in cholinergic neurons

Parasympathetic ganglia are considered simple relay systems that have cholinergic input and output, with modulation occurring centrally. Greater complexity is suggested, however, by our showing here that avian ciliary ganglion (CG) neurons also express a different excitatory receptor type--ionotropic glutamate receptors of the kainate subtype (KARs). This is the first report of glutamate receptor expression in the CG and KAR expression in any cholinergic neuron. We show that KARs form functional channels on CG neurons. KARs localize to CG neuron axons and somata as well as axons and terminals of pre-synaptic inputs to the CG. Glutamate transporters are expressed on Schwann cells that surround synapses on neuronal somata, and may provide a local source of glutamate. CG neurons express multiple KAR subunit mRNAs (GluR5, GluR7, and KA1), and their relative levels change dramatically during axon outgrowth and synaptic differentiation. The developmental role for KARs may depend upon their calcium permeability, a property regulated by mRNA editing. We show GluR5 editing increases predominantly at the time CG axons contact peripheral targets. Our data suggest that glutamatergic signaling may function as a local circuit mechanism to modulate excitability and calcium signaling during synapse formation and maturation in the CG in vivo.     

Assembly and Trafficking of Homomeric and Heteromeric Kainate Receptors with Impaired Ligand Binding Sites

Kainate receptors (KARs) are a subfamily of ionotropic glutamate receptors (iGluRs) mediating excitatory synaptic transmission. Cell surface expressed KARs modulate the excitability of neuronal networks. The transfer of iGluRs from the endoplasmic reticulum (ER) to the cell surface requires occupation of the agonist binding sites. Here we used molecular modelling to produce a range of ligand binding domain (LBD) point mutants of GluK1–3 KAR subunits with and without altered agonist efficacy to further investigate the role of glutamate binding in surface trafficking and activation of homomeric and heteromeric KARs using endoglycosidase digestion, cell surface biotinylation and imaging of changes in intracellular Ca²⁺ concentration [Ca²⁺]_i. Mutations of conserved amino acid residues in the LBD that disrupt agonist binding to GluK1–3 (GluK1-T675V, GluK2-A487L, GluK2-T659V and GluK3-T661V) reduced both the total expression levels and cell surface delivery of all of these mutant subunits compared to the corresponding wild type in transiently transfected human embryonic kidney 293 (HEK293) cells. In contrast, the exchange of non-conserved residues in the LBD that convert antagonist selectivity of GluK1–3 (GluK1-T503A, GluK2-A487T, GluK3-T489A, GluK1-N705S/S706N, GluK2-S689N/N690S, GluK3-N691S) did not alter the biosynthesis and trafficking of subunit proteins. Co-assembly of mutant GluK2 with an impaired LBD and wild type GluK5 subunits enables the cell surface expression of both subunits. However, [Ca²⁺]_i imaging indicates that the occupancy of both GluK2 and GluK5 LBDs is required for the full activation of GluK2/GluK5 heteromeric KAR channels.

     

PKC-dependent autoregulation of membrane kainate receptors

Agonists of kainate receptors (KARs) cause both the opening of the associated ion channels and the activation of signalling pathways driven by G-proteins and PKC. Here we report the existence of an unknown mechanism of KAR autoregulation, involving the interplay of this two signalling mechanisms. Repetitive activation of native KARs evoked the rundown of the ionotropic responses in a manner that was dependent on the activation of PKC. Experiments on recombinant GluR5 expressed in neuroblastoma cells indicated that KARs trigger the activation of PKC and induce the internalization of membrane receptors. This phenomenon depends on the PKC-mediated phosphorylation of serines 879 and 885 of the GluR5-2b subunits, since mutation of these two residues abolished internalization. These results reveal that the non-canonical signalling of KARs is associated with a sensitive mechanism that detects afferent activity. Such a mechanism represents an active way to limit overactivation of the KAR system, by regulating the number of KARs in the cell membrane.     

Profilin II regulates the exocytosis of kainate glutamate receptors

The trafficking of ionotropic glutamate receptors to and from synaptic sites is regulated by proteins that interact with their cytoplasmic C-terminal domain. Profilin IIa (PfnIIa), an actin-binding protein expressed in the brain and recruited to synapses in an activity-dependent manner, was shown previously to interact with the C-terminal domain of the GluK2b subunit splice variant of kainate receptors (KARs). Here, we characterize this interaction and examine the role of PfnIIa in the regulation of KAR trafficking. PfnIIa directly and specifically binds to the C-terminal domain of GluK2b through a diproline motif. Expression of PfnIIa in transfected COS-7 cells and in cultured hippocampal neurons from PfnII-deficient mice decreases the level of extracellular of homomeric GluK2b as well as heteromeric GluK2a/GluK2b KARs. Our data suggest a novel mechanism by which PfnIIa exerts a dual role on the trafficking of KARs, by a generic inhibition of clathrin-mediated endocytosis through its interaction with dynamin-1, and by controlling KARs exocytosis through a direct and specific interaction with GluK2b.     

Metabotropic-mediated kainate receptor regulation of IsAHP and excitability in pyramidal cells

Kainate receptors (KARs) on CA1 pyramidal cells make no detectable contribution to EPSCs. We report that these receptors have a metabotropic function, as shown previously for CA1 interneurons. Brief kainate exposure caused long-lasting inhibition of a postspike potassium current (I(sAHP)) in CA1 pyramidal cells. The pharmacological profile was independent of AMPA receptors or the GluR5 subunit, indicating a possible role for the GluR6 subunit. KAR inhibition of I(sAHP) did not require ionotropic action or network activity, but was blocked by the inhibitor of pertussis toxin-sensitive G proteins, N-ethylmaleimide (NEM), or the PKC inhibitor calphostin C. These data suggest how KARs, putatively containing GluR6, directly increase excitability of CA1 pyramidal cells and help explain the propensity for seizure activity following KAR activation.     

Expression of functional kainate and AMPA receptors in developing lateral superior olive neurons of the rat

A functional analysis of AMPA and kainate receptors (AMPARs and KARs) in the lateral superior olive (LSO), a major nucleus in the auditory brainstem, has not been performed so far, to our knowledge. Here we investigated the presence and characteristics of such receptors in the rat LSO by means of whole-cell patch-clamp recordings in combination with pharmacology. Current responses evoked by 200 microM AMPA were completely blocked by the specific AMPAR antagonist GYKI 52466 (100 microM). Properties of the AMPAR-mediated currents (latency, activation time constant, and peak amplitude) remained constant between postnatal day 3 (P3) and P10. Current responses evoked by 100 microM KA were not completely blocked by 100 microM GYKI 52466, indicating that the residual component was mediated by KARs. Throughout development, two groups of KAR-mediated currents (fast I(KA) and slow I(KA)) were distinguished because they had significantly different mean activation time constants. Moreover, the mean peak amplitude of fast I(KA) was significantly higher than that of slow I(KA). The differentiation into fast I(KA) and slow I(KA) can be explained by the existence of two groups of LSO neurons displaying different KAR densities, distributions, and/or diverse types with differences in conductance. Application of the specific KAR subunit agonists SYM 2081 (10 microM), ATPA (10 microM), or iodowillardiine (1 microM) evoked currents in almost all cells tested, showing that GluR5 subunits are a component of functional KARs in LSO neurons. Electrical stimulation of ipsilateral input fibers in the presence of KAR antagonists (NS-102 and GAMS), modulators (WGA), or GYKI 52466 revealed the presence of synaptic KARs in LSO neurons.     

Kainate receptor-mediated modulation of hippocampal fast spiking interneurons in a rat model of schizophrenia

Kainate receptor (KAR) subunits are believed to be involved in abnormal GABAergic neurotransmission in the hippocampus (HIPP) in schizophrenia (SZ) and bipolar disorder. Postmortem studies have shown changes in the expression of the GluR5/6 subunits of KARs in the stratum oriens (SO) of sectors CA2/3, where the basolateral amygdala (BLA) sends a robust projection. Previous work using a rat model of SZ demonstrated that BLA activation leads to electrophysiological changes in fast-spiking interneurons in SO of CA2/3. The present study explores KAR modulation of interneurons in CA2/3 in response to BLA activation. Intrinsic firing properties of these interneurons through KAR-mediated activity were measured with patch-clamp recordings from rats that received 15 days of picrotoxin infusion into the BLA. Chronic BLA activation induced changes in the firing properties of CA2/3 interneurons associated with modifications in the function of KARs. Specifically, the responsiveness of these interneurons to activation of KARs was diminished in picrotoxin-treated rats, while the after-hyperpolarization (AHP) amplitude was increased. In addition, we tested blockers of KAR subunits which have been shown to have altered gene expression in SO sector CA2/3 of SZ subjects. The GluR5 antagonist UBP296 further decreased AP frequency and increased AHP amplitude in picrotoxin-treated rats. Application of the GluR6/7 antagonist NS102 suggested that activation of GluR6/7 KARs may be required to maintain the high firing rates in SO interneurons in the presence of KA. Moreover, the GluR6/7 KAR-mediated signaling may be suppressed in PICRO-treated rats. Our findings indicate that glutamatergic activity from the BLA may modulate the firing properties of CA2/3 interneurons through GluR5 and GluR6/7 KARs. These receptors are expressed in GABAergic interneurons and play a key role in the synchronization of gamma oscillations. Modulation of interneuronal activity through KARs in response to amygdala activation may lead to abnormal oscillatory rhythms reported in SZ subjects.     

Activity-dependent endocytic sorting of kainate receptors to recycling or degradation pathways

Kainate receptors (KARs) play important roles in the modulation of neurotransmission and plasticity, but the mechanisms that regulate their surface expression and endocytic sorting remain largely unknown. Here, we show that in cultured hippocampal neurons the surface expression of GluR6-containing KARs is dynamically regulated. Furthermore, internalized KARs are sorted into recycling or degradative pathways depending on the endocytotic stimulus. Kainate activation causes a Ca2+- and PKA-independent but PKC-dependent internalization of KARs that are targeted to lysosomes for degradation. In contrast, NMDAR activation evokes a Ca2+-, PKA- and PKC-dependent endocytosis of KARs to early endosomes with subsequent reinsertion back into the plasma membrane. These results demonstrate that GluR6-containing KARs are subject to activity-dependent endocytic sorting, a process that provides a mechanism for both rapid and chronic changes in the number of functional receptors.     

Ligand binding is a critical requirement for plasma membrane expression of heteromeric kainate receptors

Intracellular trafficking of ionotropic glutamate receptors is controlled by multiple discrete determinants in receptor subunits. Most such determinants have been localized to the cytoplasmic carboxyl-terminal domain, but other domains in the subunit proteins can play roles in modulating receptor surface expression. Here we demonstrate that formation of an intact glutamate binding site also acts as an additional quality-control check for surface expression of homomeric and heteromeric kainate receptors. A key ligand-binding residue in the KA2 subunit, threonine 675, was mutated to either alanine or glutamate, which eliminated affinity for the receptor ligands kainate and glutamate. We found that plasma membrane expression of heteromeric GluR6/KA2(T675A) or GluR6/KA2(T675E) kainate receptors was markedly reduced compared with wild-type GluR6/KA2 receptors in transfected HEK 293 and COS-7 cells and in cultured neurons. Surface expression of homomeric KA2 receptors lacking a retention/retrieval determinant (KA2-R/A) was also reduced upon mutation of Thr-675 and elimination of the ligand binding site. KA2 Thr-675 mutant subunits were able to co-assemble with GluR5 and GluR6 subunits and were degraded at the same rate as wild-type KA2 subunit protein. These results suggest that glutamate binding and associated conformational changes are prerequisites for forward trafficking of intracellular kainate receptors following multimeric assembly.     

Kainate receptor-mediated depression of glutamatergic transmission involving protein kinase A in the lateral amygdala

Kainate receptors (KARs) have been described as modulators of synaptic transmission at different synapses. However, this role of KARs has not been well characterized in the amygdala. We have explored the effect of kainate receptor activation at the synapse established between fibers originating at medial geniculate nucleus and the principal cells in the lateral amygdala. We have observed an inhibition of evoked excitatory postsynaptic currents (eEPSCs) amplitude after a brief application of KARs agonists KA and ATPA. Paired-pulse recordings showed a clear pair pulse facilitation that was enhanced after KA or ATPA application. When postsynaptic cells were loaded with BAPTA, the depression of eEPSC amplitude observed after the perfusion of KAR agonists was not prevented. We have also observed that the inhibition of the eEPSCs by KARs agonists was prevented by protein kinase A but not by protein kinase C inhibitors. Taken together our results indicate that KARs present at this synapse are pre-synaptic and their activation mediate the inhibition of glutamate release through a mechanism that involves the activation of protein kinase A.     

Kainate receptors differentially regulate release at two parallel fiber synapses

Presynaptic kainate receptors (KARs) facilitate or depress transmitter release at several synapses in the CNS. Here, we report that synaptically activated KARs presynaptically facilitate and depress transmission at parallel fiber synapses in the cerebellar cortex. Low-frequency stimulation of parallel fibers facilitates synapses onto both stellate cells and Purkinje cells, whereas high-frequency stimulation depresses stellate cell synapses but continues to facilitate Purkinje cell synapses. These effects are mimicked by exogenous KAR agonists and eliminated by blocking KARs. This differential frequency-dependent sensitivity of these two synapses regulates the balance of excitatory and inhibitory input to Purkinje cells and therefore their excitability.     

Presynaptic kainate receptor-mediated facilitation of glutamate release involves Ca(2+) -calmodulin at mossy fiber-CA3 synapses

J. Neurochem. (2012) 122, 891-899. ABSTRACT: Presynaptic kainate receptors (KARs) modulate the release of glutamate at synapses established between mossy fibers (MF) and CA3 pyramidal cells in the hippocampus. The activation of KAR by low, nanomolar, kainate concentrations facilitates glutamate release. KAR-mediated facilitation of glutamate release involves the activation of an adenylate cyclase/cyclic adenosine monophosphate/protein kinase A cascade at MF-CA3 synapses. Here, we studied the mechanisms by which KAR activation produces this facilitation of glutamate release in slices and synaptosomes. We find that the facilitation of glutamate release mediated by KAR activation requires an increase in Ca(2+) levels in the cytosol and the formation of a Ca(2+) -calmodulin complex to activate adenylate cyclase. The increase in cytosolic Ca(2+) underpinning this modulation is achieved, both, by Ca(2+) entering via Ca(2+) -permeable KARs and, by the mobilization of intraterminal Ca(2+) stores. Finally, we find that, congruent with the Ca(2+) -calmodulin support of KAR-mediated facilitation of glutamate release, induction of long-term potentiation at MF-CA3 synapses has an obligate requirement for Ca(2+) -calmodulin activity.     

Rapid and differential regulation of AMPA and kainate receptors at hippocampal mossy fibre synapses by PICK1 and GRIP

We identified four PDZ domain-containing proteins, syntenin, PICK1, GRIP, and PSD95, as interactors with the kainate receptor (KAR) subunits GluR5(2b,) GluR5(2c), and GluR6. Of these, we show that both GRIP and PICK1 interactions are required to maintain KAR-mediated synaptic function at mossy fiber-CA3 synapses. In addition, PKC alpha can phosphorylate ct-GluR5(2b) at residues S880 and S886, and PKC activity is required to maintain KAR-mediated synaptic responses. We propose that PICK1 targets PKC alpha to phosphorylate KARs, causing their stabilization at the synapse by an interaction with GRIP. Importantly, this mechanism is not involved in the constitutive recycling of AMPA receptors since blockade of PDZ interactions can simultaneously increase AMPAR- and decrease KAR-mediated synaptic transmission at the same population of synapses.     

Lateral entorhinal cortex lesions rearrange afferents, glutamate receptors, increase seizure latency and suppress seizure-induced c-fos expression in the hippocampus of adult rat

The entorhinal cortex (EC) provides the predominant excitatory drive to the hippocampal CA1 and subicular neurones in chronic epilepsy. Here we analysed the effects of one-sided lateral EC (LEC) and temporoammonic (alvear) path lesion on the development and properties of 4-aminopyridine-induced seizures. Electroencephalography (EEG) analysis of freely moving rats identified that the lesion increased the latency of the hippocampal seizure significantly and decreased the number of brief convulsions. Seizure-induced neuronal c-fos expression was reduced in every hippocampal area following LEC lesion. Immunocytochemical analysis 40 days after the ablation of the LEC identified sprouting of cholinergic and calretinin-containing axons into the dentate molecular layer. Region and subunit specific changes in the expression of ionotropic glutamate receptors (iGluRs) were identified. Although the total amount of AMPA receptor subunits remained unchanged, GluR1(flop) displayed a significant decrease in the CA1 region. An increase in NR1 and NR2B N-methyl-d-aspartate (NMDA) receptor subunits and KA-2 kainate receptor subunit was identified in the deafferented layers of the hippocampus. These results further emphasize the importance of the lateral entorhinal area in the spread and regulation of hippocampal seizures and highlight the potential role of the rewiring of afferents and rearrangement of iGluRs in the dentate gyrus in hippocampal convulsive activity.