Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Functional Validation of Heteromeric Kainate Receptor Models.

ABSTRACT: Kainate receptors require the presence of external ions for gating. Most work thus far has been performed on homomeric GluK2 but, in vivo, kainate receptors are likely heterotetramers. Agonists bind to the ligand-binding domain (LBD) which is arranged as a dimer of dimers as exemplified in homomeric structures, but no high-resolution structure currently exists of heteromeric kainate receptors. In a full-length heterotetramer, the LBDs could potentially be arranged either as a GluK2 homomer alongside a GluK5 homomer or as two GluK2/K5 heterodimers. We have constructed models of the LBD dimers based on the GluK2 LBD crystal structures and investigated their stability with molecular dynamics simulations. We have then used the models to make predictions about the functional behavior of the full-length GluK2/K5 receptor, which we confirmed via electrophysiological recordings. A key prediction and observation is that lithium ions bind to the dimer interface of GluK2/K5 heteromers and slow their desensitization.

SUBMITTER: Paramo T

PROVIDER: S-EPMC5700254 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Functional Validation of Heteromeric Kainate Receptor Models.

Paramo Teresa T Brown Patricia M G E PMGE Musgaard Maria M Bowie Derek D Biggin Philip C PC

Biophysical journal 20170919 10

Kainate receptors require the presence of external ions for gating. Most work thus far has been performed on homomeric GluK2 but, in vivo, kainate receptors are likely heterotetramers. Agonists bind to the ligand-binding domain (LBD) which is arranged as a dimer of dimers as exemplified in homomeric structures, but no high-resolution structure currently exists of heteromeric kainate receptors. In a full-length heterotetramer, the LBDs could potentially be arranged either as a GluK2 homomer along ...[more]

PMID: 28935133

Similar Datasets

Preferential assembly of heteromeric kainate and AMPA receptor amino terminal domains.

Project description:Ion conductivity and the gating characteristics of tetrameric glutamate receptor ion channels are determined by their subunit composition. Competitive homo- and hetero-dimerization of their amino-terminal domains (ATDs) is a key step controlling assembly. Here we measured systematically the thermodynamic stabilities of homodimers and heterodimers of kainate and AMPA receptors using fluorescence-detected sedimentation velocity analytical ultracentrifugation. Measured affinities span many orders of magnitude, and complexes show large differences in kinetic stabilities. The association of kainate receptor ATD dimers is generally weaker than the association of AMPA receptor ATD dimers, but both show a general pattern of increased heterodimer stability as compared to the homodimers of their constituents, matching well physiologically observed receptor combinations. The free energy maps of AMPA and kainate receptor ATD dimers provide a framework for the interpretation of observed receptor subtype combinations and possible assembly pathways.

| S-EPMC5665649 | biostudies-literature

Architecture and structural dynamics of the heteromeric GluK2/K5 kainate receptor.

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.

| S-EPMC7997659 | biostudies-literature

Structure and assembly mechanism for heteromeric kainate receptors.

Project description:Native glutamate receptor ion channels are tetrameric assemblies containing two or more different subunits. NMDA receptors are obligate heteromers formed by coassembly of two or three divergent gene families. While some AMPA and kainate receptors can form functional homomeric ion channels, the KA1 and KA2 subunits are obligate heteromers which function only in combination with GluR5-7. The mechanisms controlling glutamate receptor assembly involve an initial step in which the amino terminal domains (ATD) assemble as dimers. Here, we establish by sedimentation velocity that the ATDs of GluR6 and KA2 coassemble as a heterodimer of K(d) 11 nM, 32,000-fold lower than the K(d) for homodimer formation by KA2; we solve crystal structures for the GluR6/KA2 ATD heterodimer and heterotetramer assemblies. Using these structures as a guide, we perform a mutant cycle analysis to probe the energetics of assembly and show that high-affinity ATD interactions are required for biosynthesis of functional heteromeric receptors.

| S-EPMC3145919 | biostudies-literature

The structural arrangement and dynamics of the heteromeric GluK2/GluK5 kainate receptor as determined by smFRET.

Project description:Kainate receptors, which are glutamate activated excitatory neurotransmitter receptors, predominantly exist as heteromers of GluK2 and GluK5 subunits in the mammalian central nervous system. There are currently no structures of the full-length heteromeric kainate receptors. Here, we have used single molecule FRET to determine the specific arrangement of the GluK2 and GluK5 subunits within the dimer of dimers configuration in a full-length receptor. Additionally, we have also studied the dynamics and conformational heterogeneity of the amino-terminal and agonist-binding domain interfaces associated with the resting and desensitized states of the full-length heteromeric kainate receptor using FRET-based methods. The smFRET data are compared to similar experiments performed on the homomeric kainate receptor to provide insight into the differences in conformational dynamics that distinguish the two functionally. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins.

| S-EPMC6899175 | biostudies-literature

Activation of group I metabotropic glutamate receptors potentiates heteromeric kainate receptors.

Project description:Kainate receptors (KARs), a family of ionotropic glutamate receptors, are widely expressed in the central nervous system and are critically involved in synaptic transmission. KAR activation is influenced by metabotropic glutamate receptor (mGlu) signaling, but the underlying mechanisms are not understood. We undertook studies to examine how mGlu modulation affects activation of KARs. Confocal immunohistochemistry of rat hippocampus and cultured rat cortex revealed colocalization of the high-affinity KAR subunits with group I mGlu receptors. In hippocampal and cortical cultures, the calcium signal caused by activation of native KARs was potentiated by activation of group I mGlu receptors. In Xenopus laevis oocytes, activation of group I mGlu receptors potentiated heteromeric but not homomeric KAR-mediated currents, with no change in agonist potency. The potentiation of heteromeric KARs by mGlu1 activation was attenuated by GDPβS, blocked by an inhibitor of phospholipase C or the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), prolonged by the phosphatase inhibitor okadaic acid, but unaffected by the tyrosine kinase inhibitor lavendustin A. Protein kinase C (PKC) inhibition reduced the potentiation by mGlu1 of GluK2/GluK5, and conversely, direct activation of PKC by phorbol 12-myristate,13-acetate potentiated GluK2/GluK5. Using site-directed mutagenesis, we identified three serines (Ser833, Ser836, and Ser840) within the membrane proximal region of the GluK5 C-terminal domain that, in combination, are required for mGlu1-mediated potentiation of KARs. Together, these data suggest that phosphorylation of key residues in the C-terminal domain changes the overall charge of this domain, resulting in potentiated agonist responses.

| S-EPMC3533475 | biostudies-literature

Structural and Functional Insights into GluK3-kainate Receptor Desensitization and Recovery.

Project description:GluK3-kainate receptors are atypical members of the iGluR family that reside at both the pre- and postsynapse and play a vital role in the regulation of synaptic transmission. For a better understanding of structural changes that underlie receptor functions, GluK3 receptors were trapped in desensitized and resting/closed states and structures analyzed using single particle cryo-electron microscopy. While the desensitized GluK3 has domain organization as seen earlier for another kainate receptor-GluK2, antagonist bound GluK3 trapped a resting state with only two LBD domains in dimeric arrangement necessary for receptor activation. Using structures as a guide, we show that the N-linked glycans at the interface of GluK3 ATD and LBD likely mediate inter-domain interactions and attune receptor-gating properties. The mutational analysis also identified putative N-glycan interacting residues. Our results provide a molecular framework for understanding gating properties unique to GluK3 and exploring the role of N-linked glycosylation in their modulation.

| S-EPMC6635489 | biostudies-literature

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

Project description: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.

| S-EPMC6420462 | biostudies-literature

Expression and purification of a functional heteromeric GABAA receptor for structural studies.

Project description:The GABA-gated chloride channels of the Cys-loop receptor family, known as GABAA receptors, function as the primary gatekeepers of fast inhibitory neurotransmission in the central nervous system. Formed by the pentameric arrangement of five identical or homologous subunits, GABAA receptor subtypes are defined by the subunit composition that shape ion channel properties. An understanding of the structural basis of distinct receptor properties has been hindered by the absence of high resolution structural information for heteromeric assemblies. Robust heterologous expression and purification protocols of high expressing receptor constructs are vital for structural studies. Here, we describe a unique approach to screen for well-behaving and functional GABAA receptor subunit assemblies by using the Xenopus oocyte as an expression host in combination with fluorescence detection size exclusion chromatography (FSEC). To detect receptor expression, GFP fusions were introduced into the ?1 subunit isoform. In contrast to expression of ?1 alone, co-expression with the ? subunit promoted formation of monodisperse assemblies. Mutagenesis experiments suggest that the ? and ? subunits can tolerate large truncations in the non-conserved M3/M4 cytoplasmic loop without compromising oligomeric assembly or GABA-gated channel activity, although removal of N-linked glycosylation sites is negatively correlated with expression level. Additionally, we report methods to improve GABAA receptor expression in mammalian cell culture that employ recombinant baculovirus transduction. From these methods we have identified a well-behaving minimal functional construct for the ?1/?1 GABAA receptor subtype that can be purified in milligram quantities while retaining high affinity agonist binding activity.

| S-EPMC6054424 | biostudies-literature

A nondesensitizing kainate receptor point mutant.

Project description:Ionotropic glutamate receptor (iGluR) desensitization can be modulated by mutations that change the stability of a dimer formed by the agonist binding domain. Desensitization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors can be blocked by a single point mutation (e.g., GluR2 L483Y) that stabilizes this dimer in an active conformation. In contrast, desensitization of kainate receptors can be slowed, but not blocked, by similar dimer interface mutations. Only covalent cross-linking via introduced disulfides has been previously shown to block kainate receptor desensitization completely. We have now identified an apparently nondesensitizing GluR6 point mutant (D776K) located at the apex of the ligand binding (S1S2) domain dimer interface. Asp776 is one of a cluster of four charged residues in this region that together mediate direct dimer interactions and contribute to the binding sites for one chloride and two sodium ions. Despite the localized +4 change in the net charge of the S1S2 dimer, the D776K mutation actually increased the thermodynamic stability of the dimer. Unlike GluR6 wild type, the D776K mutant is insensitive to external cations but retains sensitivity to external anions. We therefore hypothesize that the unexpected phenotype of this charge reversal mutation results from the substitution of the sodium ions bound within the dimer interface by the introduced lysine NH(3)(+) groups. The nondesensitizing D776K mutant provides insights into kainate receptor gating and represents a potentially useful new tool for dissecting kainate receptor function.

| S-EPMC2730386 | biostudies-literature

A kainate receptor-selective RNA aptamer.

Project description:Kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are two major, closely related receptor subtypes in the glutamate ion channel family. Excessive activities of these receptors have been implicated in a number of central nervous system diseases. Designing potent and selective antagonists of these receptors, especially of kainate receptors, is useful for developing potential treatment strategies for these neurological diseases. Here, we report on two RNA aptamers designed to individually inhibit kainate and AMPA receptors. To improve the biostability of these aptamers, we also chemically modified these aptamers by substituting their 2'-OH group with 2'-fluorine. These 2'-fluoro aptamers, FB9s-b and FB9s-r, were markedly resistant to RNase-catalyzed degradation, with a half-life of ∼5 days in rat cerebrospinal fluid or serum-containing medium. Furthermore, FB9s-r blocked AMPA receptor activity. Aptamer FB9s-b selectively inhibited GluK1 and GluK2 kainate receptor subunits, and also GluK1/GluK5 and GluK2/GluK5 heteromeric kainate receptors with equal potency. This inhibitory profile makes FB9s-b a powerful template for developing tool molecules and drug candidates for treatment of neurological diseases involving excessive activities of the GluK1 and GluK2 subunits.

| S-EPMC7212664 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data