Duplicated Gephyrin Genes Showing Distinct Tissue Distribution and Alternative Splicing Patterns Mediate Molybdenum Cofactor Biosynthesis,Glycine Receptor Clustering,and Escape Behavior in Zebrafish

Gephyrin mediates the postsynaptic clustering of glycine receptors (GlyRs) and GABA_A receptors at inhibitory synapses and molybdenum-dependent enzyme (molybdoenzyme) activity in non-neuronal tissues. Gephyrin knock-out mice show a phenotype resembling both defective glycinergic transmission and molybdenum cofactor (Moco) deficiency and die within 1 day of birth due to starvation and dyspnea resulting from deficits in motor and respiratory networks, respectively. To address whether gephyrin function is conserved among vertebrates and whether gephyrin deficiency affects molybdoenzyme activity and motor development, we cloned and characterized zebrafish gephyrin genes. We report here that zebrafish have two gephyrin genes, gphna and gphnb. The former is expressed in all tissues and has both C3 and C4 cassette exons, and the latter is expressed predominantly in the brain and spinal cord and harbors only C4 cassette exons. We confirmed that all of the gphna and gphnb splicing isoforms have Moco synthetic activity. Antisense morpholino knockdown of either gphna or gphnb alone did not disturb synaptic clusters of GlyRs in the spinal cord and did not affect touch-evoked escape behaviors. However, on knockdown of both gphna and gphnb, embryos showed impairments in GlyR clustering in the spinal cord and, as a consequence, demonstrated touch-evoked startle response behavior by contracting antagonistic muscles simultaneously, instead of displaying early coiling and late swimming behaviors, which are executed by side-to-side muscle contractions. These data indicate that duplicated gephyrin genes mediate Moco biosynthesis and control postsynaptic clustering of GlyRs, thereby mediating key escape behaviors in zebrafish.     

Deciphering the structural framework of glycine receptor anchoring by gephyrin

Glycine is the major inhibitory neurotransmitter in the spinal cord and brain stem. Gephyrin is required to achieve a high concentration of glycine receptors (GlyRs) in the postsynaptic membrane, which is crucial for efficient glycinergic signal transduction. The interaction between gephyrin and the GlyR involves the E-domain of gephyrin and a cytoplasmic loop located between transmembrane segments three and four of the GlyR beta subunit. Here, we present crystal structures of the gephyrin E-domain with and without the GlyR beta-loop at 2.4 and 2.7 A resolutions, respectively. The GlyR beta-loop is bound in a symmetric 'key and lock' fashion to each E-domain monomer in a pocket adjacent to the dimer interface. Structure-guided mutagenesis followed by in vitro binding and in vivo colocalization assays demonstrate that a hydrophobic interaction formed by Phe 330 of gephyrin and Phe 398 and Ile 400 of the GlyR beta-loop is crucial for binding.     

Reciprocal stabilization of glycine receptors and gephyrin scaffold proteins at inhibitory synapses

Postsynaptic scaffold proteins immobilize neurotransmitter receptors in the synaptic membrane opposite to presynaptic vesicle release sites, thus ensuring efficient synaptic transmission. At inhibitory synapses in the spinal cord, the main scaffold protein gephyrin assembles in dense molecule clusters that provide binding sites for glycine receptors (GlyRs). Gephyrin and GlyRs can also interact outside of synapses, where they form receptor-scaffold complexes. Although several models for the formation of postsynaptic scaffold domains in the presence of receptor-scaffold interactions have been advanced, a clear picture of the coupled dynamics of receptors and scaffold proteins at synapses is lacking. To characterize the GlyR and gephyrin dynamics at inhibitory synapses, we performed fluorescence time-lapse imaging after photoconversion to directly visualize the exchange kinetics of recombinant Dendra2-gephyrin in cultured spinal cord neurons. Immuno-immobilization of endogenous GlyRs with specific antibodies abolished their lateral diffusion in the plasma membrane, as judged by the lack of fluorescence recovery after photobleaching. Moreover, the cross-linking of GlyRs significantly reduced the exchange of Dendra2-gephyrin compared with control conditions, suggesting that the kinetics of the synaptic gephyrin pool is strongly dependent on GlyR-gephyrin interactions. We did not observe any change in the total synaptic gephyrin levels after GlyR cross-linking, however, indicating that the number of gephyrin molecules at synapses is not primarily dependent on the exchange of GlyR-gephyrin complexes. We further show that our experimental data can be quantitatively accounted for by a model of receptor-scaffold dynamics that includes a tightly interacting receptor-scaffold domain, as well as more loosely bound receptor and scaffold populations that exchange with extrasynaptic pools. The model can make predictions for single-molecule data such as typical dwell times of synaptic proteins. Taken together, our data demonstrate the reciprocal stabilization of GlyRs and gephyrin at inhibitory synapses and provide a quantitative understanding of their dynamic organization.     

Neuronal cotransport of glycine receptor and the scaffold protein gephyrin

The dynamics of postsynaptic receptor scaffold formation and remodeling at inhibitory synapses remain largely unknown. Gephyrin, which is a multimeric scaffold protein, interacts with cytoskeletal elements and stabilizes glycine receptors (GlyRs) and individual subtypes of gamma-aminobutyric acid A receptors at inhibitory postsynaptic sites. We report intracellular mobility of gephyrin transports packets over time. Gephyrin units enter and exit active synapses within several minutes. In addition to previous reports of GlyR-gephyrin interactions at plasma membranes, we show cosedimentation and coimmunoprecipitation of both proteins from vesicular fractions. Moreover, GlyR and gephyrin are cotransported within neuronal dendrites and further coimmunoprecipitate and colocalize with the dynein motor complex. As a result, the blockade of dynein function or dynein-gephyrin interaction, as well as the depolymerization of microtubules, interferes with retrograde gephyrin recruitment. Our data suggest a GlyR-gephyrin-dynein transport complex and support the concept that gephyrin-motor interactions contribute to the dynamic and activity-dependent rearrangement of postsynaptic GlyRs, a process thought to underlie the regulation of synaptic strength.     

The trafficking proteins Vacuolar Protein Sorting 35 and Neurobeachin interact with the glycine receptor β-subunit

Inhibitory glycine receptors (GlyRs) are densely packed in the postsynaptic membrane due to a high-affinity interaction of their β-subunits with the scaffolding protein gephyrin. Here, we used an affinity-based proteomic approach to identify the trafficking proteins Vacuolar Protein Sorting 35 (Vps35) and Neurobeachin (Nbea) as novel GlyR β-subunit (GlyRβ) interacting proteins in rat brain. Recombinant Vps35 and a central fragment of Nbea bound to the large intracellular loop of GlyRβ in glutathione-S-transferase pull-downs; in addition, Vps35 displayed binding to gephyrin. Immunocytochemical staining of spinal cord sections revealed Nbea immunoreactivity apposed to and colocalizing with marker proteins of inhibitory synapses. Our data are consistent with roles of Vps35 and Nbea in the retrieval and post-Golgi trafficking of synaptic GlyRs and possibly other neurotransmitter receptors.     

Structural basis of dynamic glycine receptor clustering by gephyrin

Gephyrin is a bi-functional modular protein involved in molybdenum cofactor biosynthesis and in postsynaptic clustering of inhibitory glycine receptors (GlyRs). Here, we show that full-length gephyrin is a trimer and that its proteolysis in vitro causes the spontaneous dimerization of its C-terminal region (gephyrin-E), which binds a GlyR beta-subunit-derived peptide with high and low affinity. The crystal structure of the tetra-domain gephyrin-E in complex with the beta-peptide bound to domain IV indicates how membrane-embedded GlyRs may interact with subsynaptic gephyrin. In vitro, trimeric full-length gephyrin forms a network upon lowering the pH, and this process can be reversed to produce stable full-length dimeric gephyrin. Our data suggest a mechanism by which induced conformational transitions of trimeric gephyrin may generate a reversible postsynaptic scaffold for GlyR recruitment, which allows for dynamic receptor movement in and out of postsynaptic GlyR clusters, and thus for synaptic plasticity.     

Regulation of gephyrin assembly and glycine receptor synaptic stability

Gephyrin is required for the formation of clusters of the glycine receptor (GlyR) in the neuronal postsynaptic membrane. It can make trimers and dimers through its N- and C-terminal G and E domains, respectively. Gephyrin oligomerization could thus create a submembrane lattice providing GlyR-binding sites. We investigated the relationships between the stability of cell surface GlyR and the ability of gephyrin splice variants to form oligomers. Using truncated and full-length gephyrins we found that the 13-amino acid sequence (cassette 5) prevents G domain trimerization. Moreover, E domain dimerization is inhibited by the gephyrin central L domain. All of the gephyrin variants bind GlyR beta subunit cytoplasmic loop with high affinity regardless of their cassette composition. Coexpression experiments in COS-7 cells demonstrated that GlyR bound to gephyrin harboring cassette 5 cannot be stabilized at the cell surface. This gephyrin variant was found to deplete synapses from both GlyR and gephyrin in transfected neurons. These data suggest that the relative expression level of cellular variants influence the overall oligomerization pattern of gephyrin and thus the turnover of synaptic GlyR.     

Splice-specific glycine receptor binding, folding, and phosphorylation of the scaffolding protein gephyrin

The multimeric scaffolding protein gephyrin forms post-synaptic clusters at inhibitory sites, thereby anchoring inhibitory glycine (GlyR) and subsets of γ-aminobutyric acid type A (GABAA) receptors. Gephyrin is composed of three domains, the conserved N-terminal G- and C-terminal E-domain, connected by the central (C-) domain. In this study we investigated the oligomerization, folding and stability, GlyR β-loop binding, and phosphorylation of three gephyrin splice variants (Geph, Geph-C3, Geph-C4) after expression and purification from insect cells (Sf9). In contrast to Escherichia coli-derived trimeric gephyrin, we found that Sf9 gephyrins form hexamers as basic oligomeric form. In the case of Geph and Geph-C4, also high-oligomeric forms (～900 kDa) were isolated. Partial proteolysis revealed a compact folding of the Gephyrin G and C domain in one complex, whereas a much lower stability for the E domain was found. After GlyR β-loop binding, the stability of the E domain increased in Geph and Geph-C4 significantly. In contrast, the E domain in Geph-C3 is less stable and binds the GlyR β-loop with one order of magnitude lower affinity. Finally, we identified 18 novel phosphorylation sites in gephyrin, of which all except one are located within the C domain. We propose two models for the domain arrangement in hexameric gephyrin based on the oligomerization of either the E or C domains, with the latter being crucial for the regulation of gephyrin clustering.     

Rescue of molybdenum cofactor biosynthesis in gephyrin-deficient mice by a Cnx1 transgene

Gephyrin is a bifunctional protein which is essential for both synaptic clustering of inhibitory neurotransmitter receptors in the central nervous system and the biosynthesis of the molybdenum cofactor (MoCo) in peripheral tissues. Mice deficient in gephyrin die early postnatally and display a loss of glycine receptors (GlyRs) and many GABA(A) receptor (GABA(A)R) subtypes from postsynaptic sites. In addition, the activities of the MoCo-dependent enzymes xanthine dehydrogenase and sulfite oxidase are reduced to background levels in the liver and intestine of these animals. To genetically separate the different consequences of gephyrin deficiency, we expressed a transgene of the plant homolog Cnx1, known to rescue mammalian MoCo deficiency, on the background of gephyrin knockout mice. Cnx1 partially restored sulfite oxidase activity in the liver of the transgenic animals, whereas early lethality and the loss of GlyR clustering were unaltered. Our data suggest that the loss of neurotransmitter receptor clustering at inhibitory synapses causes the early lethality of gephyrin deficient mice.     

Proteomic Analysis of Glycine Receptor β Subunit (GlyRβ)-interacting Proteins: EVIDENCE FOR SYNDAPIN I REGULATING SYNAPTIC GLYCINE RECEPTORS*

Glycine receptors (GlyRs) mediate inhibitory neurotransmission in spinal cord and brainstem. They are clustered at inhibitory postsynapses via a tight interaction of their β subunits (GlyRβ) with the scaffolding protein gephyrin. In an attempt to isolate additional proteins interacting with GlyRβ, we performed pulldown experiments with rat brain extracts using a glutathione S-transferase fusion protein encompassing amino acids 378–455 of the large intracellular loop of GlyRβ as bait. This identified syndapin I (SdpI) as a novel interaction partner of GlyRβ that coimmunoprecipitates with native GlyRs from brainstem extracts. Both SdpI and SdpII bound efficiently to the intracellular loop of GlyRβ in vitro and colocalized with GlyRβ upon coexpression in COS-7 cells. The SdpI-binding site was mapped to a proline-rich sequence of 22 amino acids within the intracellular loop of GlyRβ. Deletion and point mutation analysis disclosed that SdpI binding to GlyRβ is Src homology 3 domain-dependent. In cultured rat spinal cord neurons, SdpI immunoreactivity was found to partially colocalize with marker proteins of inhibitory and excitatory synapses. When SdpI was acutely knocked down in cultured spinal cord neurons by viral miRNA expression, postsynaptic GlyR clusters were significantly reduced in both size and number. Similar changes in GlyR cluster properties were found in spinal cultures from SdpI-deficient mice. Our results are consistent with a role of SdpI in the trafficking and/or cytoskeletal anchoring of synaptic GlyRs.     

Homeostatic regulation of synaptic GlyR numbers driven by lateral diffusion

In the spinal cord, most inhibitory synapses have a mixed glycine-GABA phenotype. Using a pharmacological approach, we report an NMDAR activity-dependent regulation of the mobility of GlyRs but not GABA(A)Rs at inhibitory synapses in cultured rat spinal cord neurons. The NMDAR-induced decrease in GlyR lateral diffusion was correlated with an increase in receptor cluster number and glycinergic mIPSC amplitude. Changes in GlyR diffusion properties occurred rapidly and before the changes in the number of synaptic receptors. Regulation of synaptic GlyR content occurred without change in the amount of gephyrin. Moreover, NMDAR-dependent regulation of GlyR lateral diffusion required calcium influx and calcium release from stores. Therefore, excitation may increase GlyR levels at synapses by a calcium-mediated increase in postsynaptic GlyR trapping involving regulation of receptor-scaffold interactions. This provides a mechanism for a rapid homeostatic regulation of the inhibitory glycinergic component at mixed glycine-GABA synapses in response to increased NMDA excitatory transmission.     

Glycine receptors: recent insights into their structural organization and functional diversity

Strychnine-sensitive glycine receptors (GlyRs) are known to mediate synaptic inhibition in spinal cord, brainstem and other regions of the CNS. During the past 5 years, considerable progress has been made in delineating structural determinants of ligand binding and channel activation in recombinant GlyRs. Furthermore, immunohistochemical and gene inactivation studies have disclosed distinct distributions and functions of differentially expressed GlyR subtypes in retina, hippocampus and the dorsal horn of the spinal cord. Accordingly, GlyRs regulate not only the excitability of motor and sensory neurones, but are also essential for the processing of photoreceptor signals, neuronal development and inflammatory pain sensitization. Hence, these receptors constitute promising targets for the development of clinically useful compounds.     

Coexpression of the receptor-associated protein gephyrin changes the ligand binding affinities of alpha 2 glycine receptors.

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel protein, whose ligand binding alpha subunit occurs in several isoforms in the mammalian central nervous system. Here we show that coexpression of the GlyR-associated protein gephyrin changes the agonist and antagonist binding affinities of GlyRs generated by alpha 2 subunit expression in 293 kidney cells. Thus, a receptor-associated protein modifies the functional properties of a neurotransmitter receptor. This may contribute to an optimization of the postsynaptic neurotransmitter response.     

Gephyrin-mediated γ-aminobutyric acid type A and glycine receptor clustering relies on a common binding site

Gephyrin is the major protein determinant for the clustering of inhibitory neurotransmitter receptors. Earlier analyses revealed that gephyrin tightly binds to residues 398-410 of the glycine receptor β subunit (GlyR β) and, as demonstrated only recently, also interacts with GABA(A) receptors (GABA(A)Rs) containing the α1, α2, and α3 subunits. Here, we dissect the molecular basis underlying the interactions between gephyrin and GABA(A)Rs containing these α-subunits and compare them to the crystal structure of the gephyrin-GlyR β complex. Biophysical and biochemical assays revealed that, in contrast to its tight interaction with GlyR β, gephyrin only loosely interacts with GABA(A)R α2, whereas it has an intermediate affinity for the GABA(A)R α1 and α3 subunits. Despite the wide variation in affinities and the low overall sequence homology among the identified receptor subunits, competition assays confirmed the receptor-gephyrin interaction to be a mutually exclusive process. Selected gephyrin point mutants that critically weaken complex formation with GlyR β also abolished the GABA(A)R α1 and α3 interactions. Additionally, we identified a common binding motif with two conserved aromatic residues that are central for gephyrin binding. Consistent with the biochemical data, mutations of the corresponding residues within the cytoplasmic domain of α2 subunit-containing GABA(A)Rs attenuated clustering of these receptors at postsynaptic sites in hippocampal neurons. Taken together, our experiments provide key insights regarding similarities and differences in the complex formation between gephyrin and GABA(A)Rs compared with GlyRs and, hence, the accumulation of these receptors at postsynaptic sites.     

Hydrophobic interactions mediate binding of the glycine receptor beta-subunit to gephyrin

Glycine receptors (GlyRs) are ligand-gated chloride channel proteins composed of alpha- and beta-subunits. GlyRs are located to and anchored at postsynaptic sites by the receptor-associated protein gephyrin. Previous work from our laboratory has identified a core motif for gephyrin binding in the cytoplasmic loop of the GlyR beta-subunit. Here, we localized amino acid residues implicated in gephyrin binding by site-directed mutagenesis. In a novel transfection assay, a green fluorescent protein-gephyrin binding motif fusion protein was used to monitor the consequences of amino acid substitutions for beta-subunit interaction with gephyrin. Only multiple, but not single, replacements of hydrophobic side chains abolished the interaction between the two proteins. Our data are consistent with gephyrin binding being mediated by the hydrophobic side of an imperfect amphipathic helix.     

Biochemical characterization of the high affinity binding between the glycine receptor and gephyrin

Gephyrin is an essential and instructive molecule for the formation of inhibitory synapses. Gephyrin binds directly to the large cytoplasmic loop located between transmembrane helices three and four of the beta-subunit of the glycine receptor and to microtubules, thus promoting glycine receptor (GlyR) anchoring to the cytoskeleton and clustering in the postsynaptic membrane. Besides its structural role, gephyrin is involved in the biosynthesis of the molybdenum cofactor that is essential for all molybdenum-dependent enzymes in mammals. Gephyrin can be divided into an N-terminal trimeric G domain and a C-terminal E domain, which are connected by a central linker region. Here we have studied the in vitro interaction of gephyrin and its domains with the large cytoplasmic loop of the GlyR beta-sub-unit (GlyRbeta-loop). Binding of gephyrin to the GlyR is exclusively mediated by the E domain, and the binding site was mapped to one of its sub-domains (residues 496-654). By using isothermal titration calorimetry, a high affinity (K(d) = 0.2-0.4 microm) and low affinity (K(d) = 11-30 microm) binding site for the GlyRbeta-loop was found on holo-gephyrin and the E domain, respectively, with a binding stoichiometry of two GlyRbeta-loops per E domain in both cases. Binding of the GlyRbeta-loop does not change the oligomeric state of either full-length gephyrin or the isolated E domain.     

Regulation of glycine receptor diffusion properties and gephyrin interactions by protein kinase C

Glycine receptors (GlyRs) can dynamically exchange between synaptic and extrasynaptic locations through lateral diffusion within the plasma membrane. Their accumulation at inhibitory synapses depends on the interaction of the β-subunit of the GlyR with the synaptic scaffold protein gephyrin. An alteration of receptor-gephyrin binding could thus shift the equilibrium between synaptic and extrasynaptic GlyRs and modulate the strength of inhibitory neurotransmission. Using a combination of dynamic imaging and biochemical approaches, we have characterised the molecular mechanism that links the GlyR-gephyrin interaction with GlyR diffusion and synaptic localisation. We have identified a protein kinase C (PKC) phosphorylation site within the cytoplasmic domain of the β-subunit of the GlyR (residue S403) that causes a reduction of the binding affinity between the receptor and gephyrin. In consequence, the receptor's diffusion in the plasma membrane is accelerated and GlyRs accumulate less strongly at synapses. We propose that the regulation of GlyR dynamics by PKC thus contributes to the plasticity of inhibitory synapses and may be involved in maladaptive forms of synaptic plasticity.     

Post-phosphorylation prolyl isomerisation of gephyrin represents a mechanism to modulate glycine receptors function

The microtubule binding protein gephyrin plays a prominent role in establishing and maintaining a high concentration of inhibitory glycine receptors juxtaposed to presynaptic releasing sites. Here, we show that endogenous gephyrin undergoes proline-directed phosphorylation, which is followed by the recruitment of the peptidyl-prolyl isomerase Pin1. The interaction between gephyrin and Pin1 is strictly dependent on gephyrin phosphorylation and requires serine-proline consensus sites encompassing the gephyrin proline-rich domain. Upon binding, Pin1 triggers conformational changes in the gephyrin molecule, thus enhancing its ability to bind the beta subunit of GlyRs. Consistently, a downregulation of GlyR clusters was detected in hippocampal neurons derived from Pin1 knockout mice, which was paralleled by a reduction in the amplitude of glycine-evoked currents. Our results suggest that phosphorylation-dependent prolyl isomerisation of gephyrin represents a mechanism for regulating GlyRs function.