Ingo H. Greger

RNA Editing at Arg607 Controls AMPA Receptor Exit from the Endoplasmic Reticulum

AMPA-receptor (AMPAR) transport to synapses plays a critical role in the modulation of synaptic strength. We show that the functionally critical GluR2 subunit stably resides in an intracellular pool in the endoplasmic reticulum (ER). GluR2 in this pool is extensively complexed with GluR3 but not with GluR1, which is mainly confined to the cell surface. Mutagenesis revealed that elements in the C terminus including the PDZ motif are required for GluR2 forward-transport from the ER. Surprisingly, ER retention of GluR2 is controlled by Arg607 at the Q/R-editing site. Reversion to Gln (R607Q) resulted in rapid release from the pool and elevated surface expression of GluR2 in neurons. Therefore, Arg607 is a central regulator. In addition to channel gating, it also controls ER exit and may thereby ensure the availability of GluR2 for assembly into AMPARs.

AMPA Receptor Tetramerization Is Mediated by Q/R Editing

AMPA-type glutamate receptors (AMPARs) play a major role in excitatory synaptic transmission and plasticity. Channel properties are largely dictated by their composition of the four subunits, GluR1-4 (or A-D). Here we show that AMPAR assembly and subunit stoichiometry are determined by RNA editing in the pore loop. We demonstrate that editing at the GluR2 Q/R site regulates AMPAR assembly at the step of tetramerization. Specifically, edited R subunits are largely unassembled and ER retained, whereas unedited Q subunits readily tetramerize and traffic to synapses. This assembly mechanism restricts the number of the functionally critical R subunits in AMPAR tetramers. Therefore, a single amino acid residue affects channel composition and, in turn, controls ion conduction through the majority of AMPARs in the brain.

Molecular determinants of AMPA receptor subunit assembly

AMPA-type (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) glutamate receptors (AMPARs) mediate post-synaptic depolarization and fast excitatory transmission in the central nervous system. AMPARs are tetrameric ion channels that assemble in the endoplasmic reticulum (ER) in a poorly understood process. The subunit composition determines channel conductance properties and gating kinetics, and also regulates vesicular traffic to and from synaptic sites, and is thus critical for synaptic function and plasticity. The distribution of functionally different AMPARs varies within and between neuronal circuits, and even within individual neurons. In addition, synapses employ channels with specific subunit stoichiometries, depending on the type of input and the frequency of stimulation. Taken together, it appears that assembly is not simply a stochastic process. Recently, progress has been made in understanding the molecular mechanisms underlying subunit assembly and receptor biogenesis in the ER. These processes ultimately determine the size and shape of the postsynaptic response, and are the subject of this review.

AMPA receptor biogenesis and trafficking

AMPA-type glutamate receptors mediate the majority of fast excitatory transmission in the central nervous system. The trafficking of AMPA receptors to and from synapses alters synaptic strength and has been recognized as a central mechanism underlying various forms of synaptic plasticity. Both secretory and endocytic trafficking events seem to be driven by the subunit composition of AMPA receptor tetramers. Moreover, recent work suggests that synapses employ different tetramer combinations in response to altered synaptic input, suggesting the existence of signalling pathways that mediate remodelling of AMPA receptors. These latest developments and recent progress in elucidating the mechanisms that underlie channel assembly and trafficking are the subject of this review.

Developmentally Regulated, Combinatorial RNA Processing Modulates AMPA Receptor Biogenesis

The subunit composition determines AMPA receptor (AMPA-R) function and trafficking. Mechanisms underlying channel assembly are thus central to the efficacy and plasticity of glutamatergic synapses. We previously showed that RNA editing at the Q/R site of the GluR2 subunit contributes to the assembly of AMPA-R heteromers by attenuating formation of GluR2 homotetramers. Here we report that this function of the Q/R site depends on subunit contacts between adjacent ligand binding domains (LBDs). Changes of LBD interface contacts alter GluR2 assembly properties, forward traffic, and expression at synapses. Interestingly, developmentally regulated RNA editing within the LBD (at the R/G site) produces analogous effects. Our data reveal that editing to glycine reduces the self-assembly competence of this critical subunit and slows GluR2 maturation in the endoplasmic reticulum (ER). Therefore, RNA editing sites, located at strategic subunit interfaces, shape AMPA-R assembly and trafficking in a developmentally regulated manner.

Gating motions underlie AMPA receptor secretion from the endoplasmic reticulum

Ion channel biogenesis involves an intricate interplay between subunit folding and assembly. Channel stoichiometries vary and give rise to diverse functions, which impacts on neuronal signalling. AMPA glutamate receptor (AMPAR) assembly is modulated by RNA processing. Here, we provide mechanistic insight into this process. First, we show that a single alternatively spliced residue within the ligand‐binding domain alters AMPAR secretion from the ER. Local contacts differ between Leu758 of the GluR2‐flop splice form as compared with the flip‐specific Val758, which is transmitted globally to alter resensitization kinetics. Detailed biochemical and functional analysis of mutants suggest that AMPARs sample the gating cascade prior to ER export. Irreversibly locking the receptor within various states of the cascade severely attenuates ER transit. Alternative RNA processing by contrast, shifts equilibria between transition states reversibly and thereby modulates secretion kinetics. These data reveal how RNA processing tunes AMPAR biogenesis, and imply that gating transitions in the ER determine iGluR secretory traffic.

Subunit-selective N-terminal domain associations organize the formation of AMPA receptor heteromers

The assembly of AMPA‐type glutamate receptors (AMPARs) into distinct ion channel tetramers ultimately governs the nature of information transfer at excitatory synapses. How cells regulate the formation of diverse homo‐ and heteromeric AMPARs is unknown. Using a sensitive biophysical approach, we show that the extracellular, membrane‐distal AMPAR N‐terminal domains (NTDs) orchestrate selective routes of heteromeric assembly via a surprisingly wide spectrum of subunit‐specific association affinities. Heteromerization is dominant, occurs at the level of the dimer, and results in a preferential incorporation of the functionally critical GluA2 subunit. Using a combination of structure‐guided mutagenesis and electrophysiology, we further map evolutionarily variable hotspots in the NTD dimer interface, which modulate heteromerization capacity. This ‘flexibility’ of the NTD not only explains why heteromers predominate but also how GluA2‐lacking, Ca2+‐permeable homomers could form, which are induced under specific physiological and pathological conditions. Our findings reveal that distinct NTD properties set the stage for the biogenesis of functionally diverse pools of homo‐ and heteromeric AMPAR tetramers.

Structural and Functional Architecture of AMPA-Type Glutamate Receptors and Their Auxiliary Proteins

AMPA receptors (AMPARs) are tetrameric ion channels that together with other ionotropic glutamate receptors (iGluRs), the NMDA and kainate receptors, mediate a majority of excitatory neurotransmission in the central nervous system. Whereas NMDA receptors gate channels with slow kinetics, responsible primarily for generating long-term synaptic potentiation and depression, AMPARs are the main fast transduction elements at synapses and are critical for the expression of plasticity. The kinetic and conductance properties of AMPARs are laid down during their biogenesis and are regulated by post-transcriptional RNA editing, splice variation, post-translational modification, and subunit composition. Furthermore, AMPAR assembly, trafficking, and functional heterogeneity depends on a large repertoire of auxiliary subunits-a feature that is particularly striking for this type of iGluR. Here, we discuss how the subunit structure, stoichiometry, and auxiliary subunits generate a heterogeneous plethora of receptors, each tailored to fulfill a vital role in fast synaptic signaling and plasticity.

Structure and organization of heteromeric AMPA-type glutamate receptors.

Signaling at the synapse Neurons signal to each other at synapses using neurotransmitters. Glutamate is a key neurotransmitter, and AMPA-type glutamate receptors (AMPARs) mediate rapid responses to glutamate release. These receptors mainly occur as heteromers comprising GluA1-4 subunits. Herguedas et al. used electron microscopy and x-ray crystallography to determine the structure of GluA2/3 and GluA2/4 heteromers. The structures differ from those determined previously for GluA2 homomers but emphasize how signals may be transmitted through these dynamic receptors. Science, this issue p. 10.1126/science.aad3873 Crystal structures reveal organizational features of the central mediators of rapid neurotransmission and synaptic plasticity. INTRODUCTION Ionotropic glutamate receptors (iGluRs) are cation channels that mediate signal transmission by depolarizing the postsynaptic membrane in response to L-glutamate release from the presynaptic neuron. There are three major iGluR subtypes, and together their activity is pivotal to learning and memory. The first subtype, the AMPA-type receptor (AMPAR), initiates signaling and activates the second subtype, the N-methyl-d-aspartate receptor (NMDAR), thereby admitting Ca2+ ions that drive synaptic plasticity and ultimately enabling learning. The rapid submillisecond response of the AMPAR permits accurate transmission of high-frequency presynaptic impulses. In the brain, AMPARs preferentially exist as heterotetramers composed of the GluA1 to GluA4 subunits in various combinations. Their signaling properties are dominated by the GluA2 subunit, which is present in the majority of AMPARs, yet the architecture and subunit arrangement of GluA2-containing AMPAR heteromers has thus far been elusive. RATIONALE So far, AMPAR structures have been limited to GluA2 homomers. We used a combination of x-ray crystallography and cryogenic electron microscopy (cryo-EM) to obtain structural information for AMPAR heteromers. We first solved crystal structures of the N-terminal domain (NTD) layer, which constitutes the more sequence-diverse upper half of the receptor. Our structures of the GluA2/3 and GluA2/4 NTDs provide atomic resolution of the interface that initiates subunit assembly and reveal tetrameric arrangements in both of these heteromeric combinations that are distinct from known homomers. Cysteine cross-linking confirmed this architecture in full-length receptors and permitted us to determine cryo-EM structures of an intact GluA2/3 heteromer, which is a prominent variety in neural tissue. RESULTS In both GluA2/3 and GluA2/4 NTD crystal structures, the four subunits alternate around a central axis to create a compact O shape that deviates from the loose N shape commonly found in GluA2 homomers. Simulations suggest that the receptor can transition between these two states. Structure-guided cysteine mutants allowed us to trap receptors in the O state and facilitated further analysis by cryo-EM. The GluA2/3 receptor was captured in a ligand-free state and resulted in two models, determined at 8.2 and 10.3 Å. Both deviate from existing GluA2 homomer structures. Associated with the compact conformation of the NTD layer was a substantial vertical compression of the receptor heteromer, forming distinct interlayer interfaces. Contact points between the NTD and the ligand-binding domain (LBD) suggest that the NTD can contribute to signaling. We also observed a rearrangement of the gating machinery at the level of the LBD layer, and our results provide a structural snapshot of a desensitized receptor in the absence of agonist. Our data indicate that subunit placement in AMPAR tetramers is not strict, unlike in NMDARs, a finding that may extend the functional spectrum of heteromeric AMPARs. CONCLUSION The GluA2/3 structure and our simulations emphasize that AMPARs are dynamic complexes. The observed rearrangements of the extracellular region are expected to be of functional consequence in synaptic environments, where the NTD engages various interaction partners. Transitioning between N and O states could break existing contacts and make new ones, thereby affecting AMPAR clustering, a prerequisite for synaptic plasticity. Moreover, the vertical compression observed in GluA2/3 would permit crosstalk between the NTD and LBD, thus incorporating the NTD in allosteric regulation, as has been observed in NMDARs. The sequence-diverse AMPAR NTD, like that of NMDARs, may therefore offer a target for drug development—one that is as yet unexplored. Structure of a GluA2/3 AMPAR heteromer. A GluA2/3 heteromer (left) is shown with a GluA2 homomer (right) for comparison. The top panel shows the NTD layers of the compact O-shaped GluA2/3 and the loose N-shaped GluA2 [Protein Data Bank identifier (PDB ID) 3H5V], viewed from the top. The bottom panel shows full-length cryo-EM structures of GluA2/3 (ligand-free) and a GluA2 homomer (antagonist-bound; PDB ID 4UQJ). The difference in vertical packing between the NTD and LBD is indicated by arrows. AMPA-type glutamate receptors (AMPARs), which are central mediators of rapid neurotransmission and synaptic plasticity, predominantly exist as heteromers of the subunits GluA1 to GluA4. Here we report the first AMPAR heteromer structures, which deviate substantially from existing GluA2 homomer structures. Crystal structures of the GluA2/3 and GluA2/4 N-terminal domains reveal a novel compact conformation with an alternating arrangement of the four subunits around a central axis. This organization is confirmed by cysteine cross-linking in full-length receptors, and it permitted us to determine the structure of an intact GluA2/3 receptor by cryogenic electron microscopy. Two models in the ligand-free state, at resolutions of 8.25 and 10.3 angstroms, exhibit substantial vertical compression and close associations between domain layers, reminiscent of N-methyl-d-aspartate receptors. Model 1 resembles a resting state and model 2 a desensitized state, thus providing snapshots of gating transitions in the nominal absence of ligand. Our data reveal organizational features of heteromeric AMPARs and provide a framework to decipher AMPAR architecture and signaling.

Dynamics and allosteric potential of the AMPA receptor N-terminal domain

Glutamate‐gated ion channels (ionotropic glutamate receptors, iGluRs) sense the extracellular milieu via an extensive extracellular portion, comprised of two clamshell‐shaped segments. The distal, N‐terminal domain (NTD) has allosteric potential in NMDA‐type iGluRs, which has not been ascribed to the analogous domain in AMPA receptors (AMPARs). In this study, we present new structural data uncovering dynamic properties of the GluA2 and GluA3 AMPAR NTDs. GluA3 features a zipped‐open dimer interface with unconstrained lower clamshell lobes, reminiscent of metabotropic GluRs (mGluRs). The resulting labile interface supports interprotomer rotations, which can be transmitted to downstream receptor segments. Normal mode analysis reveals two dominant mechanisms of AMPAR NTD motion: intraprotomer clamshell motions and interprotomer counter‐rotations, as well as accessible interconversion between AMPAR and mGluR conformations. In addition, we detect electron density for a potential ligand in the GluA2 interlobe cleft, which may trigger lobe motions. Together, these data support a dynamic role for the AMPAR NTDs, which widens the allosteric landscape of the receptor and could provide a novel target for ligand development.