Seizure protein 6 controls glycosylation and trafficking of kainate receptor subunits GluK2 and GluK3
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ABSTRACT:
SUBMITTER: Mrs. Martina Pigoni
PROVIDER: S-SCDT-EMBOJ-2019-103457 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:Seizure protein 6 (SEZ6) is required for the development and maintenance of the nervous system, is a major substrate of the protease BACE1 and is linked to Alzheimer's disease (AD) and psychiatric disorders, but its molecular functions are not well understood. Here, we demonstrate that SEZ6 controls glycosylation and cell surface localization of kainate receptors composed of GluK2/3 subunits. Loss of SEZ6 reduced surface levels of GluK2/3 in primary neurons and reduced kainate-evoked currents in CA1 pyramidal neurons in acute hippocampal slices. Mechanistically, loss of SEZ6 in vitro and in vivo prevented modification of GluK2/3 with the human natural killer-1 (HNK-1) glycan, a modulator of GluK2/3 function. SEZ6 interacted with GluK2 through its ectodomain and promoted post-endoplasmic reticulum transport of GluK2 in the secretory pathway in heterologous cells and primary neurons. Taken together, SEZ6 acts as a new trafficking factor for GluK2/3. This novel function may help to better understand the role of SEZ6 in neurologic and psychiatric diseases.
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Project description:Ionotropic glutamate receptors assemble as homo- or heterotetramers. One well-studied heteromeric complex is formed by the kainate receptor subunits GluK2 and GluK5. Retention motifs prevent trafficking of GluK5 homomers to the plasma membrane, but coassembly with GluK2 yields functional heteromeric receptors. Additional control over GluK2/GluK5 assembly seems to be exerted by the aminoterminal domains, which preferentially assemble into heterodimers as isolated domains. However,the stoichiometry of the full-length GluK2/GluK5 receptor complex has yet to be determined, as is the case for all non-NMDA glutamate receptors. Here, we address this question, using a single-molecule imaging technique that enables direct counting of the number of each GluK subunit type in homomeric and heteromeric receptors in the plasma membranes of live cells. We show that GluK2 and GluK5 assemble with 2:2 stoichiometry. This is an important step toward understanding the assembly mechanism, architecture, and functional consequences of heteromer formation in ionotropic glutamate receptors.
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Project description:Glutamate receptors are the primary excitatory neurotransmitter receptors in vertebrate brain and are of critical importance to a wide variety of neurological processes. Recent reports suggest that ionotropic glutamate receptors may have a unique transmembrane topology not shared by other ligand-gated ion channels. We report here the cloning of cDNAs from goldfish brain encoding two homologous kainate receptors with protein molecular masses of 41 kDa. Using a cell-free translation/translocation system, we show that (i) a portion of these receptors previously thought to be a large intracellular loop is actually located extracellularly and (ii) the putative second transmembrane region of the receptor thought to line the ion channel may not be a true membrane-spanning domain. An alternative model for the transmembrane topology of kainate receptors is proposed that could potentially serve as a framework for future detailed study of the structure of this important class of neurotransmitter receptors.
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Project description:Kainate receptors (KARs) are L-glutamate-gated ion channels that regulate synaptic transmission and modulate neuronal circuits. KARs have strict assembly rules and primarily function as heteromeric receptors in the brain. A longstanding question is how KAR heteromer subunits organize and coordinate together to fulfill their signature physiological roles. Here we report structures of the GluK2/GluK5 heteromer in apo, antagonist-bound, and desensitized states. The receptor assembles with two copies of each subunit, ligand binding domains arranged as two heterodimers and GluK5 subunits proximal to the channel. Strikingly, during desensitization, GluK2, but not GluK5, subunits undergo major structural rearrangements to facilitate channel closure. We show how the large conformational differences between antagonist-bound and desensitized states are mediated by the linkers connecting the pore helices to the ligand binding domains. This work presents the first KAR heteromer structure, reveals how its subunits are organized, and resolves how the heteromer can accommodate functionally distinct closed channel structures.
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Project description:The surface expression and regulated endocytosis of kainate (KA) receptors (KARs) plays a critical role in neuronal function. PKC can modulate KAR trafficking, but the sites of action and molecular consequences have not been fully characterized. Small ubiquitin-like modifier (SUMO) modification of the KAR subunit GluK2 mediates agonist-evoked internalization, but how KAR activation leads to GluK2 SUMOylation is unclear. Here we show that KA stimulation causes rapid phosphorylation of GluK2 by PKC, and that PKC activation increases GluK2 SUMOylation both in vitro and in neurons. The intracellular C-terminal domain of GluK2 contains two predicted PKC phosphorylation sites, S846 and S868, both of which are phosphorylated in response to KA. Phosphomimetic mutagenesis of S868 increased GluK2 SUMOylation, and mutation of S868 to a nonphosphorylatable alanine prevented KA-induced SUMOylation and endocytosis in neurons. Infusion of SUMO-1 dramatically reduced KAR-mediated currents in HEK293 cells expressing WT GluK2 or nonphosphorylatable S846A mutant, but had no effect on currents mediated by the S868A mutant. These data demonstrate that agonist activation of GluK2 promotes PKC-dependent phosphorylation of S846 and S868, but that only S868 phosphorylation is required to enhance GluK2 SUMOylation and promote endocytosis. Thus, direct phosphorylation by PKC and GluK2 SUMOylation are intimately linked in regulating the surface expression and function of GluK2-containing KARs.
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Project description:The kainate family of ionotropic glutamate receptors (iGluRs) mediates pre- and postsynaptic neurotransmission. Previously computed conformational potentials of mean force (PMFs) for iGluR ligand-binding domains (LBDs) revealed subtype-dependent conformational differences between α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartic acid (NMDA) iGluR subfamilies. Here we report PMFs for the kainate receptor GluK2 in apo and glutamate-bound states. Apo and glutamate-bound GluK2 LBDs preferentially access closed-cleft conformations. Apo GluK2 exhibits a surprisingly high degree of conformational flexibility, accessing open and closed states. Comparing across iGluR subtypes, these results are similar to glycine-binding GluN1 and GluN3A NMDA subunits and differ from glutamate-binding GluA2 and GluN2A subunits. To test the contribution of cross-lobe interactions on closed-cleft LBD stability, we computed PMFs for two GluK2 mutants, D462A and D656S. D462A, but not D656S, weakens closed-cleft conformations of the glutamate-bound LBD. Theoretical Boltzmann-weighted small-angle X-ray scattering profiles improve agreement with experimental results compared with calculations from the LBD crystal structure alone.
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Project description:Kainate receptors (KAR) play a crucial role in the plasticity and functional maturation of glutamatergic synapses. However, how they regulate structural plasticity of dendritic spines is not known. The GluK2 subunit was recently shown to coexist in a functional complex with the neuronal K-Cl cotransporter KCC2. Apart from having a crucial role in the maturation of GABAergic transmission, KCC2 has a morphogenic role in the maturation of dendritic spines. Here, we show that in vivo local inactivation of GluK2 expression in CA3 hippocampal neurons induces altered morphology of dendritic spines and reduction in mEPSC frequency. GluK2 deficiency also resulted in a strong change in the subcellular distribution of KCC2 as well as a smaller somatodendritic gradient in the reversal potential of GABAA. Strikingly, the aberrant morphology of dendritic spines in GluK2-deficient CA3 pyramidal neurons was restored by overexpression of KCC2. GluK2 silencing in hippocampal neurons significantly reduced the expression of 4.1N and functional form of the actin filament severing protein cofilin. Consistently, assessment of actin dynamics using fluorescence recovery after photobleaching (FRAP) of β-actin showed a significant increase in the stability of F-actin filaments in dendritic spines. In conclusion, our results demonstrate that GluK2-KCC2 interaction plays an important role in the structural maturation of dendritic spines. This also provides novel insights into the connection between KAR dysfunction, structural plasticity, and developmental disorders.
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Project description:Glutamate receptor ion channels (iGluRs) are excitatory neurotransmitter receptors with a unique molecular architecture in which the extracellular domains assemble as a dimer of dimers. The structure of individual dimer assemblies has been established previously for both the isolated ligand-binding domain (LBD) and more recently for the larger amino terminal domain (ATD). How these dimers pack to form tetrameric assemblies in intact iGluRs has remained controversial. Using recently solved crystal structures for the GluK2 kainate receptor ATD as a guide, we performed cysteine mutant cross-linking experiments in full-length tetrameric GluK2 to establish how the ATD packs in a dimer of dimers assembly. A similar approach, using a full-length AMPA receptor GluA2 crystal structure as a guide, was used to design cysteine mutant cross-links for the GluK2 LBD dimer of dimers assembly. The formation of cross-linked tetramers in full-length GluK2 by combinations of ATD and LBD mutants which individually produce only cross-linked dimers suggests that subunits in the ATD and LBD layers swap dimer partners. Functional studies reveal that cross-linking either the ATD or the LBD inhibits activation of GluK2 and that, in the LBD, cross-links within and between dimers have different effects. These results establish that kainate and AMPA receptors have a conserved extracellular architecture and provide insight into the role of individual dimer assemblies in activation of ion channel gating.
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Project description:GluK2 is a kainate receptor subunit that is alternatively spliced at the C-terminus. Previous studies implicated GluK2 in autism. In particular, the methionine-to-isoleucine replacement at amino acid residue 867 (M867I) that can only occur in the longest isoform of the human GluK2 (hGluK2), as the disease (autism) mutation, is thought to cause gain-of-function. However, the kinetic properties of the wild-type hGluK2 and the functional consequence of this gain-of-function mutation at the molecular level are not well understood. To investigate whether the M867I mutation affects the channel properties of the human GluK2 kainate receptor, we have systematically characterized the rate and the equilibrium constants pertinent to channel opening and channel desensitization for this mutant and the wild-type hGluK2 receptor, along with the wild-type rat GluK2 kainate receptor (rGluK2) as the control. Our results show that the M867I mutation does not affect either the rate or the equilibrium constants of the channel opening but does slow down the channel desensitization rate by ~1.6-fold at saturating glutamate concentrations. It is possible that a consequence of this mutation on the desensitization rate is linked to facilitating the receptor trafficking and membrane expression, given the close proximity of M867 to the forward trafficking motif in the C-terminal sequence. By comparing the kinetic data of the wild-type human and rat GluK2 receptors, we also find that the human GluK2 has a ~3-fold smaller channel-opening rate constant but an identical channel-closing rate constant and thus a channel-opening probability of 0.85 vs 0.96 for rGluK2. Furthermore, the intrinsic equilibrium dissociation constant K(1) for hGluK2, like the EC(50) value, is ~2-fold lower than rGluK2. Our results therefore suggest that the human GluK2 is relatively a slowly activating channel but more sensitive to glutamate, as compared to the rat ortholog, despite the fact that the human and rat forms share 99% sequence homology.
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