The open gate of the AMPA receptor forms a Ca2+ binding site critical in regulating ion transport.

Alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid receptors (AMPARs) are cation-selective ion channels that mediate most fast excitatory neurotransmission in the brain. Although their gating mechanism has been studied extensively, understanding how cations traverse the pore has remained elusive. Here we investigated putative ion and water densities in the open pore of Ca2+-permeable AMPARs (rat GRIA2 flip-Q isoform) at 2.3-2.6 Å resolution. We show that the ion permeation pathway attains an extracellular Ca2+ binding site (site-G) when the channel gate moves into the open configuration. Site-G is highly selective for Ca2+ over Na+, favoring the movement of Ca2+ into the selectivity filter of the pore. Seizure-related N619K mutation, adjacent to site-G, promotes channel opening but attenuates Ca2+ binding and thus diminishes Ca2+ permeability. Our work identifies the importance of site-G, which coordinates with the Q/R site of the selectivity filter to ensure the preferential transport of Ca2+ through the channel pore.

GSG1L-containing AMPA receptor complexes are defined by their spatiotemporal expression, native interactome and allosteric sites.

Transmembrane AMPA receptor regulatory proteins (TARPs) and germ cell-specific gene 1-like protein (GSG1L) are claudin-type AMPA receptor (AMPAR) auxiliary subunits that profoundly regulate glutamatergic synapse strength and plasticity. While AMPAR-TARP complexes have been extensively studied, less is known about GSG1L-containing AMPARs. Here, we show that GSG1L's spatiotemporal expression, native interactome and allosteric sites are distinct. GSG1L generally expresses late during brain development in a region-specific manner, constituting about 5% of all AMPAR complexes in adulthood. While GSG1L can co-assemble with TARPs or cornichons (CNIHs), it also assembles as the sole auxiliary subunit. Unexpectedly, GSG1L acts through two discrete evolutionarily-conserved sites on the agonist-binding domain with a weak allosteric interaction at the TARP/KGK site to slow desensitization, and a stronger interaction at a different site that slows recovery from desensitization. Together, these distinctions help explain GSG1L's evolutionary past and how it fulfills a unique signaling role within glutamatergic synapses.

CryoEM structure of a post-assembly MS-ring reveals plasticity in stoichiometry and conformation.

The flagellar motor supports bacterial chemotaxis, a process that allows bacteria to move in response to their environment. A central feature of this motor is the MS-ring, which is composed entirely of repeats of the FliF subunit. This MS-ring is critical for the assembly and stability of the flagellar switch and the entire flagellum. Despite multiple independent cryoEM structures of the MS-ring, there remains a debate about the stoichiometry and organization of the ring-building motifs (RBMs). Here, we report the cryoEM structure of a Salmonella MS-ring that was purified from the assembled flagellar switch complex (MSC-ring). We term this the 'post-assembly' state. Using 2D class averages, we show that under these conditions, the post-assembly MS-ring can contain 32, 33, or 34 FliF subunits, with 33 being the most common. RBM3 has a single location with C32, C33, or C34 symmetry. RBM2 is found in two locations with RBM2inner having C21 or C22 symmetry and an RBM2outer-RBM1 having C11 symmetry. Comparison to previously reported structures identifies several differences. Most strikingly, we find that the membrane domain forms 11 regions of discrete density at the base of the structure rather than a contiguous ring, although density could not be unambiguously interpreted. We further find density in some previously unresolved areas, and we assigned amino acids to those regions. Finally, we find differences in interdomain angles in RBM3 that affect the diameter of the ring. Together, these investigations support a model of the flagellum with structural plasticity, which may be important for flagellar assembly and function.

Modulatory mechanisms of TARP γ8-selective AMPA receptor therapeutics.

AMPA glutamate receptors (AMPARs) mediate excitatory neurotransmission throughout the brain. Their signalling is uniquely diversified by brain region-specific auxiliary subunits, providing an opportunity for the development of selective therapeutics. AMPARs associated with TARP γ8 are enriched in the hippocampus, and are targets of emerging anti-epileptic drugs. To understand their therapeutic activity, we determined cryo-EM structures of the GluA1/2-γ8 receptor associated with three potent, chemically diverse ligands. We find that despite sharing a lipid-exposed and water-accessible binding pocket, drug action is differentially affected by binding-site mutants. Together with patch-clamp recordings and MD simulations we also demonstrate that ligand-triggered reorganisation of the AMPAR-TARP interface contributes to modulation. Unexpectedly, one ligand (JNJ-61432059) acts bifunctionally, negatively affecting GluA1 but exerting positive modulatory action on GluA2-containing AMPARs, in a TARP stoichiometry-dependent manner. These results further illuminate the action of TARPs, demonstrate the sensitive balance between positive and negative modulatory action, and provide a mechanistic platform for development of both positive and negative selective AMPAR modulators.

Structure, Function, and Pharmacology of Glutamate Receptor Ion Channels.

Many physiologic effects of l-glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, are mediated via signaling by ionotropic glutamate receptors (iGluRs). These ligand-gated ion channels are critical to brain function and are centrally implicated in numerous psychiatric and neurologic disorders. There are different classes of iGluRs with a variety of receptor subtypes in each class that play distinct roles in neuronal functions. The diversity in iGluR subtypes, with their unique functional properties and physiologic roles, has motivated a large number of studies. Our understanding of receptor subtypes has advanced considerably since the first iGluR subunit gene was cloned in 1989, and the research focus has expanded to encompass facets of biology that have been recently discovered and to exploit experimental paradigms made possible by technological advances. Here, we review insights from more than 3 decades of iGluR studies with an emphasis on the progress that has occurred in the past decade. We cover structure, function, pharmacology, roles in neurophysiology, and therapeutic implications for all classes of receptors assembled from the subunits encoded by the 18 ionotropic glutamate receptor genes. SIGNIFICANCE STATEMENT: Glutamate receptors play important roles in virtually all aspects of brain function and are either involved in mediating some clinical features of neurological disease or represent a therapeutic target for treatment. Therefore, understanding the structure, function, and pharmacology of this class of receptors will advance our understanding of many aspects of brain function at molecular, cellular, and system levels and provide new opportunities to treat patients.

AMPA receptor structure and auxiliary subunits.

Fast excitatory synaptic transmission in the mammalian brain is largely mediated by AMPA-type ionotropic glutamate receptors (AMPARs), which are activated by the neurotransmitter glutamate. In synapses, the function of AMPARs is tuned by their auxiliary subunits, a diverse set of membrane proteins associated with the core pore-forming subunits of the AMPARs. Each auxiliary subunit provides distinct functional modulation of AMPARs, ranging from regulation of trafficking to shaping ion channel gating kinetics. Understanding the molecular mechanism of the function of these complexes is key to decoding synaptic modulation and their global roles in cognitive activities, such as learning and memory. Here, we review the structural and molecular complexity of AMPAR-auxiliary subunit complexes, as well as their functional diversity in different brain regions. We suggest that the recent structural information provides new insights into the molecular mechanisms underlying synaptic functions of AMPAR-auxiliary subunit complexes.

AMPA Receptor Auxiliary Subunit GSG1L Suppresses Short-Term Facilitation in Corticothalamic Synapses and Determines Seizure Susceptibility.

The anterior thalamus (AT) is critical for memory formation, processing navigational information, and seizure initiation. However, the molecular mechanisms that regulate synaptic function of AT neurons remain largely unexplored. We report that AMPA receptor auxiliary subunit GSG1L controls short-term plasticity in AT synapses that receive inputs from the cortex, but not in those receiving inputs from other pathways. A canonical auxiliary subunit stargazin co-exists in these neurons but is functionally absent from corticothalamic synapses. In GSG1L knockout mice, AT neurons exhibit hyperexcitability and the animals have increased susceptibility to seizures, consistent with a negative regulatory role of GSG1L. We hypothesize that negative regulation of synaptic function by GSG1L plays a critical role in maintaining optimal excitation in the AT.

Mammalian Retromer Is an Adaptable Scaffold for Cargo Sorting from Endosomes.

Metazoan retromer (VPS26/VPS35/VPS29) associates with sorting nexins on endosomal tubules to sort proteins to the trans-Golgi network or plasma membrane. Mechanisms of metazoan retromer assembly remain undefined. We combine single-particle cryoelectron microscopy with biophysical methods to uncover multiple oligomer structures. 2D class averages reveal mammalian heterotrimers; dimers of trimers; tetramers of trimers; and flat chains. These species are further supported by biophysical solution studies. We provide reconstructions of all species, including key sub-structures (∼5 Å resolution). Local resolution variation suggests that heterotrimers and dimers adopt multiple conformations. Our structures identify a flexible, highly conserved electrostatic dimeric interface formed by VPS35 subunits. We generate structure-based mutants to disrupt this interface in vitro. Equivalent mutations in yeast demonstrate a mild cargo-sorting defect. Our data suggest the metazoan retromer is an adaptable and plastic scaffold that accommodates interactions with different sorting nexins to sort multiple cargoes from endosomes their final destinations.

Neuronal L-Type Calcium Channel Signaling to the Nucleus Requires a Novel CaMKIIα-Shank3 Interaction.

The activation of neuronal plasma membrane Ca2+ channels stimulates many intracellular responses. Scaffolding proteins can preferentially couple specific Ca2+ channels to distinct downstream outputs, such as increased gene expression, but the molecular mechanisms that underlie the exquisite specificity of these signaling pathways are incompletely understood. Here, we show that complexes containing CaMKII and Shank3, a postsynaptic scaffolding protein known to interact with L-type calcium channels (LTCCs), can be specifically coimmunoprecipitated from mouse forebrain extracts. Activated purified CaMKIIα also directly binds Shank3 between residues 829 and 1130. Mutation of Shank3 residues 949Arg-Arg-Lys951 to three alanines disrupts CaMKII binding in vitro and CaMKII association with Shank3 in heterologous cells. Our shRNA/rescue studies revealed that Shank3 binding to both CaMKII and LTCCs is important for increased phosphorylation of the nuclear CREB transcription factor and expression of c-Fos induced by depolarization of cultured hippocampal neurons. Thus, this novel CaMKII-Shank3 interaction is essential for the initiation of a specific long-range signal from LTCCs in the plasma membrane to the nucleus that is required for activity-dependent changes in neuronal gene expression during learning and memory.SIGNIFICANCE STATEMENT Precise neuronal expression of genes is essential for normal brain function. Proteins involved in signaling pathways that underlie activity-dependent gene expression, such as CaMKII, Shank3, and L-type calcium channels, are often mutated in multiple neuropsychiatric disorders. Shank3 and CaMKII were previously shown to bind L-type calcium channels, and we show here that Shank3 also binds to CaMKII. Our data show that each of these interactions is required for depolarization-induced phosphorylation of the CREB nuclear transcription factor, which stimulates the expression of c-Fos, a neuronal immediate early gene with key roles in synaptic plasticity, brain development, and behavior.

Cryo-EM structure of human type-3 inositol triphosphate receptor reveals the presence of a self-binding peptide that acts as an antagonist.

Calcium-mediated signaling through inositol 1,4,5-triphosphate receptors (IP3Rs) is essential for the regulation of numerous physiological processes, including fertilization, muscle contraction, apoptosis, secretion, and synaptic plasticity. Deregulation of IP3Rs leads to pathological calcium signaling and is implicated in many common diseases, including cancer and neurodegenerative, autoimmune, and metabolic diseases. Revealing the mechanism of activation and inhibition of this ion channel will be critical to an improved understanding of the biological processes that are controlled by IP3Rs. Here, we report structural findings of the human type-3 IP3R (IP3R-3) obtained by cryo-EM (at an overall resolution of 3.8 Å), revealing an unanticipated regulatory mechanism where a loop distantly located in the primary sequence occupies the IP3-binding site and competitively inhibits IP3 binding. We propose that this inhibitory mechanism must differ qualitatively among IP3R subtypes because of their diverse loop sequences, potentially serving as a key molecular determinant of subtype-specific calcium signaling in IP3Rs. In summary, our structural characterization of human IP3R-3 provides critical insights into the mechanistic function of IP3Rs and into subtype-specific regulation of these important calcium-regulatory channels.

Structures of the AMPA receptor in complex with its auxiliary subunit cornichon.

In the brain, AMPA-type glutamate receptors (AMPARs) form complexes with their auxiliary subunits and mediate the majority of fast excitatory neurotransmission. Signals transduced by these complexes are critical for synaptic plasticity, learning, and memory. The two major categories of AMPAR auxiliary subunits are transmembrane AMPAR regulatory proteins (TARPs) and cornichon homologs (CNIHs); these subunits share little homology and play distinct roles in controlling ion channel gating and trafficking of AMPAR. Here, I report high-resolution cryo-electron microscopy structures of AMPAR in complex with CNIH3. Contrary to its predicted membrane topology, CNIH3 lacks an extracellular domain and instead contains four membrane-spanning helices. The protein-protein interaction interface that dictates channel modulation and the lipids surrounding the complex are revealed. These structures provide insights into the molecular mechanism for ion channel modulation and assembly of AMPAR/CNIH3 complexes.

Structure-function analyses of the ion channel TRPC3 reveal that its cytoplasmic domain allosterically modulates channel gating.

The transient receptor potential ion channels support Ca2+ permeation in many organs, including the heart, brain, and kidney. Genetic mutations in transient receptor potential cation channel subfamily C member 3 (TRPC3) are associated with neurodegenerative diseases, memory loss, and hypertension. To better understand the conformational changes that regulate TRPC3 function, we solved the cryo-EM structures for the full-length human TRPC3 and its cytoplasmic domain (CPD) in the apo state at 5.8- and 4.0-Å resolution, respectively. These structures revealed that the TRPC3 transmembrane domain resembles those of other TRP channels and that the CPD is a stable module involved in channel assembly and gating. We observed the presence of a C-terminal domain swap at the center of the CPD where horizontal helices (HHs) transition into a coiled-coil bundle. Comparison of TRPC3 structures revealed that the HHs can reside in two distinct positions. Electrophysiological analyses disclosed that shortening the length of the C-terminal loop connecting the HH with the TRP helices increases TRPC3 activity and that elongating the length of the loop has the opposite effect. Our findings indicate that the C-terminal loop affects channel gating by altering the allosteric coupling between the cytoplasmic and transmembrane domains. We propose that molecules that target the HH may represent a promising strategy for controlling TRPC3-associated neurological disorders and hypertension.

Cryo-EM structure of the cytoplasmic domain of murine transient receptor potential cation channel subfamily C member 6 (TRPC6).

The kidney maintains the internal milieu by regulating the retention and excretion of proteins, ions, and small molecules. The glomerular podocyte forms the slit diaphragm of the ultrafiltration filter, whose damage leads to progressive kidney failure and focal segmental glomerulosclerosis (FSGS). The canonical transient receptor potential 6 (TRPC6) ion channel is expressed in the podocyte, and mutations in its cytoplasmic domain cause FSGS in humans. In vitro evaluation of disease-causing mutations in TRPC6 has revealed that these genetic alterations result in abnormal ion channel gating. However, the mechanism whereby the cytoplasmic domain modulates TRPC6 function is largely unknown. Here, we report a cryo-EM structure of the cytoplasmic domain of murine TRPC6 at 3.8 Å resolution. The cytoplasmic fold of TRPC6 is characterized by an inverted dome-like chamber pierced by four radial horizontal helices that converge into a vertical coiled-coil at the central axis. Unlike other TRP channels, TRPC6 displays a unique domain swap that occurs at the junction of the horizontal helices and coiled-coil. Multiple FSGS mutations converge at the buried interface between the vertical coiled-coil and the ankyrin repeats, which form the dome, suggesting these regions are critical for allosteric gating modulation. This functionally critical interface is a potential target for drug design. Importantly, dysfunction in other family members leads to learning deficits (TRPC1/4/5) and ataxia (TRPC3). Our data provide a structural framework for the mechanistic investigation of the TRPC family.

Amyloid Accumulation Drives Proteome-wide Alterations in Mouse Models of Alzheimer's Disease-like Pathology.

Amyloid beta (Aß) peptides impair multiple cellular pathways and play a causative role in Alzheimer's disease (AD) pathology, but how the brain proteome is remodeled by this process is unknown. To identify protein networks associated with AD-like pathology, we performed global quantitative proteomic analysis in three mouse models at young and old ages. Our analysis revealed a robust increase in Apolipoprotein E (ApoE) levels in nearly all brain regions with increased Aß levels. Taken together with prior findings on ApoE driving Aß accumulation, this analysis points to a pathological dysregulation of the ApoE-Aß axis. We also found dysregulation of protein networks involved in excitatory synaptic transmission. Analysis of the AMPA receptor (AMPAR) complex revealed specific loss of TARPγ-2, a key AMPAR-trafficking protein. Expression of TARPγ-2 in hAPP transgenic mice restored AMPA currents. This proteomic database represents a resource for the identification of protein alterations responsible for AD.

A novel mechanism for Ca2+/calmodulin-dependent protein kinase II targeting to L-type Ca2+ channels that initiates long-range signaling to the nucleus.

Neuronal excitation can induce new mRNA transcription, a phenomenon called excitation-transcription (E-T) coupling. Among several pathways implicated in E-T coupling, activation of voltage-gated L-type Ca2+ channels (LTCCs) in the plasma membrane can initiate a signaling pathway that ultimately increases nuclear CREB phosphorylation and, in most cases, expression of immediate early genes. Initiation of this long-range pathway has been shown to require recruitment of Ca2+-sensitive enzymes to a nanodomain in the immediate vicinity of the LTCC by an unknown mechanism. Here, we show that activated Ca2+/calmodulin-dependent protein kinase II (CaMKII) strongly interacts with a novel binding motif in the N-terminal domain of CaV1 LTCC α1 subunits that is not conserved in CaV2 or CaV3 voltage-gated Ca2+ channel subunits. Mutations in the CaV1.3 α1 subunit N-terminal domain or in the CaMKII catalytic domain that largely prevent the in vitro interaction also disrupt CaMKII association with intact LTCC complexes isolated by immunoprecipitation. Furthermore, these same mutations interfere with E-T coupling in cultured hippocampal neurons. Taken together, our findings define a novel molecular interaction with the neuronal LTCC that is required for the initiation of a long-range signal to the nucleus that is critical for learning and memory.

Engineering defined membrane-embedded elements of AMPA receptor induces opposing gating modulation by cornichon 3 and stargazin.

KEY POINTS: The AMPA-type ionotropic glutamate receptors (AMPARs) mediate the majority of excitatory synaptic transmission and their function impacts learning, cognition and behaviour. The gating of AMPARs occurs in milliseconds, precisely controlled by a variety of auxiliary subunits that are expressed differentially in the brain, but the difference in mechanisms underlying AMPAR gating modulation by auxiliary subunits remains elusive and is investigated. The elements of the AMPAR that are functionally recruited by auxiliary subunits, stargazin and cornichon 3, are located not only in the extracellular domains but also in the lipid-accessible surface of the AMPAR. We reveal that the two auxiliary subunits require a shared surface on the transmembrane domain of the AMPAR for their function, but the gating is influenced by this surface in opposing directions for each auxiliary subunit. Our results provide new insights into the mechanistic difference of AMPAR modulation by auxiliary subunits and a conceptual framework for functional engineering of the complex. ABSTRACT: During excitatory synaptic transmission, various structurally unrelated transmembrane auxiliary subunits control the function of AMPA receptors (AMPARs), but the underlying mechanisms remain unclear. We identified lipid-exposed residues in the transmembrane domain (TMD) of the GluA2 subunit of AMPARs that are critical for the function of AMPAR auxiliary subunits, stargazin (Stg) and cornichon 3 (CNIH3). These residues are essential for stabilizing the AMPAR-CNIH3 complex in detergents and overlap with the contacts made between GluA2 TMD and Stg in the cryoEM structures. Mutating these residues had opposite effects on gating modulation and complex stability when Stg- and CNIH3-bound AMPARs were compared. Specifically, in detergent the GluA2-A793F formed an unstable complex with CNIIH3 but in the membrane the GluA2-A793F-CNIH3 complex expressed a gain of function. In contrast, the GluA2-A793F-Stg complex was stable, but had diminished gating modulation. GluA2-C528L destabilized the AMPAR-CNIH3 complex but stabilized the AMPAR-Stg complex, with overall loss of function in gating modulation. Furthermore, loss-of-function mutations in this TMD region cancelled the effects of a gain-of-function Stg carrying mutation in its extracellular loop, demonstrating that both the extracellular and the TMD elements contribute independently to gating modulation. The elements of AMPAR functionally recruited by auxiliary subunits are, therefore, located not only in the extracellular domains but also in the lipid accessible surface of the AMPAR. The TMD surface we defined is a potential target for auxiliary subunit-specific compounds, because engineering of this hotspot induces opposing functional outcomes by Stg and CNIH3. The collection of mutant-phenotype mapping provides a framework for engineering AMPAR gating using auxiliary subunits.

Screening for AMPA receptor auxiliary subunit specific modulators.

AMPA receptors (AMPAR) are ligand gated ion channels critical for synaptic transmission and plasticity. Their dysfunction is implicated in a variety of psychiatric and neurological diseases ranging from major depressive disorder to amyotrophic lateral sclerosis. Attempting to potentiate or depress AMPAR activity is an inherently difficult balancing act between effective treatments and debilitating side effects. A newly explored strategy to target subsets of AMPARs in the central nervous system is to identify compounds that affect specific AMPAR-auxiliary subunit complexes. This exploits diverse spatio-temporal expression patterns of known AMPAR auxiliary subunits, providing means for designing brain region-selective compounds. Here we report a high-throughput screening-based pipeline that can identify compounds that are selective for GluA2-CNIH3 and GluA2-stargazin complexes. These compounds will help us build upon the growing library of AMPAR-auxiliary subunit specific inhibitors, which have thus far all been targeted to TARP γ-8. We used a cell-based assay combined with a voltage-sensitive dye (VSD) to identify changes in glutamate-gated cation flow across the membranes of HEK cells co-expressing GluA2 and an auxiliary subunit. We then used a calcium flux assay to further validate hits picked from the VSD assay. VU0612951 and VU0627849 are candidate compounds from the initial screen that were identified as negative and positive allosteric modulators (NAM and PAM), respectively. They both have lower IC50/EC50s on complexes containing stargazin and CNIH3 than GSG1L or the AMPAR alone. We have also identified a candidate compound, VU0539491, that has NAM activity in GluA2(R)-CNIH3 and GluA2(Q) complexes and PAM activity in GluA2(Q)-GSG1L complexes.

A Novel Human CAMK2A Mutation Disrupts Dendritic Morphology and Synaptic Transmission, and Causes ASD-Related Behaviors.

Characterizing the functional impact of novel mutations linked to autism spectrum disorder (ASD) provides a deeper mechanistic understanding of the underlying pathophysiological mechanisms. Here we show that a de novo Glu183 to Val (E183V) mutation in the CaMKIIα catalytic domain, identified in a proband diagnosed with ASD, decreases both CaMKIIα substrate phosphorylation and regulatory autophosphorylation, and that the mutated kinase acts in a dominant-negative manner to reduce CaMKIIα-WT autophosphorylation. The E183V mutation also reduces CaMKIIα binding to established ASD-linked proteins, such as Shank3 and subunits of l-type calcium channels and NMDA receptors, and increases CaMKIIα turnover in intact cells. In cultured neurons, the E183V mutation reduces CaMKIIα targeting to dendritic spines. Moreover, neuronal expression of CaMKIIα-E183V increases dendritic arborization and decreases both dendritic spine density and excitatory synaptic transmission. Mice with a knock-in CaMKIIα-E183V mutation have lower total forebrain CaMKIIα levels, with reduced targeting to synaptic subcellular fractions. The CaMKIIα-E183V mice also display aberrant behavioral phenotypes, including hyperactivity, social interaction deficits, and increased repetitive behaviors. Together, these data suggest that CaMKIIα plays a previously unappreciated role in ASD-related synaptic and behavioral phenotypes.SIGNIFICANCE STATEMENT Many autism spectrum disorder (ASD)-linked mutations disrupt the function of synaptic proteins, but no single gene accounts for >1% of total ASD cases. The molecular networks and mechanisms that couple the primary deficits caused by these individual mutations to core behavioral symptoms of ASD remain poorly understood. Here, we provide the first characterization of a mutation in the gene encoding CaMKIIα linked to a specific neuropsychiatric disorder. Our findings demonstrate that this ASD-linked de novo CAMK2A mutation disrupts multiple CaMKII functions, induces synaptic deficits, and causes ASD-related behavioral alterations, providing novel insights into the synaptic mechanisms contributing to ASD.

Structural basis for integration of GluD receptors within synaptic organizer complexes.

Ionotropic glutamate receptor (iGluR) family members are integrated into supramolecular complexes that modulate their location and function at excitatory synapses. However, a lack of structural information beyond isolated receptors or fragments thereof currently limits the mechanistic understanding of physiological iGluR signaling. Here, we report structural and functional analyses of the prototypical molecular bridge linking postsynaptic iGluR δ2 (GluD2) and presynaptic ß-neurexin 1 (ß-NRX1) via Cbln1, a C1q-like synaptic organizer. We show how Cbln1 hexamers "anchor" GluD2 amino-terminal domain dimers to monomeric ß-NRX1. This arrangement promotes synaptogenesis and is essential for D: -serine-dependent GluD2 signaling in vivo, which underlies long-term depression of cerebellar parallel fiber-Purkinje cell (PF-PC) synapses and motor coordination in developing mice. These results lead to a model where protein and small-molecule ligands synergistically control synaptic iGluR function.