Direct binding of GABAA receptor ß2 and ß3 subunits to gephyrin.

GABAergic transmission is essential to brain function, and a large repertoire of GABA type A receptor (GABA(A) R) subunits is at a neuron's disposition to serve this function. The glycine receptor (GlyR)-associated protein gephyrin has been shown to be essential for the clustering of a subset of GABA(A) R. Despite recent progress in the field of gephyrin-dependent mechanisms of postsynaptic GABA(A) R stabilisation, the role of gephyrin in synaptic GABA(A) R localisation has remained a complex matter with many open questions. Here, we analysed comparatively the interaction of purified rat gephyrin and mouse brain gephyrin with the large cytoplasmic loops of GABA(A) R α1, α2, ß2 and ß3 subunits. Binding affinities were determined using surface plasmon resonance spectroscopy, and showed an ~ 20-fold lower affinity of the ß2 loop to gephyrin as compared to the GlyR ß loop-gephyrin interaction. We also probed in vivo binding in primary cortical neurons by the well-established use of chimaeras of GlyR α1 that harbour respective gephyrin-binding motifs derived from the different GABA(A) R subunits. These studies identify a novel gephyrin-binding motif in GABA(A) R ß2 and ß3 large cytoplasmic loops.

Splice-specific roles of glycine receptor alpha3 in the hippocampus.

Glycine receptor (GlyR) alpha3 is involved in vision, and processing of acoustic and nociceptive signals, and RNA editing of GLRA3 transcripts was associated with hippocampal pathophysiology of mesial temporal lobe epilepsy (TLE). However, neither the role of GlyR alpha3 splicing in hippocampal neurons nor the expression of splice variants have yet been elucidated. We report here that the long (L) splice variant of GlyR alpha3 predominates in the brain of rodents. Cellular analysis using primary hippocampal neurons and hippocampus cryosections revealed preferential association of synaptic alpha3L clusters with glutamatergic nerve endings in strata granulare and pyramidale. In primary hippocampal neurons GlyR alpha3L clusters also preferred glutamatergic nerve endings while alpha3K was mainly in a diffuse state. Co-expression of GlyR beta subunit with alpha3L or alpha3K produced heteromeric receptor clusters and favoured their association with GABAergic terminals. However, heteromeric alpha3L was still more efficient than heteromeric alpha3K in associating with glutamatergic nerve endings. To give physiological relevance to these results we have finally analysed GlyR alpha3 splicing in human hippocampus obtained from patients with intractable TLE. As up-regulation of alpha3K occurred at the expense of alpha3L in TLE patients with a severe course of disease and a high degree of hippocampal damage, our results again involve post-transcriptional processing of GLRA3 transcripts in the pathophysiology of TLE.

Changes in neural network homeostasis trigger neuropsychiatric symptoms.

The mechanisms that regulate the strength of synaptic transmission and intrinsic neuronal excitability are well characterized; however, the mechanisms that promote disease-causing neural network dysfunction are poorly defined. We generated mice with targeted neuron type-specific expression of a gain-of-function variant of the neurotransmitter receptor for glycine (GlyR) that is found in hippocampectomies from patients with temporal lobe epilepsy. In this mouse model, targeted expression of gain-of-function GlyR in terminals of glutamatergic cells or in parvalbumin-positive interneurons persistently altered neural network excitability. The increased network excitability associated with gain-of-function GlyR expression in glutamatergic neurons resulted in recurrent epileptiform discharge, which provoked cognitive dysfunction and memory deficits without affecting bidirectional synaptic plasticity. In contrast, decreased network excitability due to gain-of-function GlyR expression in parvalbumin-positive interneurons resulted in an anxiety phenotype, but did not affect cognitive performance or discriminative associative memory. Our animal model unveils neuron type-specific effects on cognition, formation of discriminative associative memory, and emotional behavior in vivo. Furthermore, our data identify a presynaptic disease-causing molecular mechanism that impairs homeostatic regulation of neural network excitability and triggers neuropsychiatric symptoms.

Splice-specific functions of gephyrin in molybdenum cofactor biosynthesis.

Gephyrin is a multifunctional protein involved in the clustering of inhibitory neuroreceptors. In addition, gephyrin catalyzes the last step in molybdenum cofactor (Moco) biosynthesis essential for the activities of Mo-dependent enzymes such as sulfite oxidase and xanthine oxidoreductase. Functional complexity and diversity of gephyrin is believed to be regulated by alternative splicing in a tissue-specific manner. Here, we investigated eight gephyrin variants with combinations of seven alternatively spliced exons located in the N-terminal G domain, the central domain, and the C-terminal E domain. Their activity in Moco synthesis was analyzed in vivo by reconstitution of gephyrin-deficient L929 cells, which were found to be defective in the G domain of gephyrin. Individual domain functions were assayed in addition and confirmed that variants containing either an additional C5 cassette or missing the C6 cassette are inactive in Moco synthesis. In contrast, different alterations within the central domain retained the Moco synthetic activity of gephyrin. The recombinant gephyrin G domain containing the C5 cassette forms dimers in solution, binds molybdopterin, but is unable to catalyze molybdopterin (MPT) adenylylation. Determination of Moco and MPT content in different tissues showed that besides liver and kidney, brain was capable of synthesizing Moco most efficiently. Subsequent analysis of cultured neurons and glia cells demonstrated glial Moco synthesis due to the expression of gephyrins containing the cassettes C2 and C6 with and without C3.1.

Vertebrate-specific sequences in the gephyrin E-domain regulate cytosolic aggregation and postsynaptic clustering.

Gephyrin is a multifunctional protein contributing to molybdenum cofactor (Moco) synthesis and postsynaptic clustering of glycine and GABA(A) receptors. It contains three major functional domains (G-C-E) and forms cytosolic aggregates and postsynaptic clusters by unknown mechanisms. Here, structural determinants of gephyrin aggregation and clustering were investigated by neuronal transfection of EGFP-tagged deletion and mutant gephyrin constructs. EGFP-gephyrin formed postsynaptic clusters containing endogenous gephyrin and GABA(A)-receptors. Isolated GC- or E-domains failed to aggregate and exerted dominant-negative effects on endogenous gephyrin clustering. A construct interfering with intermolecular E-domain dimerization readily auto-aggregated but showed impaired postsynaptic clustering. Finally, two mutant constructs with substitution of vertebrate-specific E-domain sequences with homologue bacterial MoeA sequences uncovered a region crucial for gephyrin clustering. One construct failed to aggregate, but retained Moco biosynthesis capacity, demonstrating the independence of gephyrin enzymatic activity and aggregation. Reinserting two vertebrate-specific residues restored gephyrin aggregation and increased formation of postsynaptic clusters containing GABA(A) receptors at the expense of PSD-95 clusters - a marker of glutamatergic synapses. These results underscore the key role of specific E-domain regions distinct from the known dimerization interface for controlling gephyrin aggregation and postsynaptic clustering and suggest that formation of gephyrin clusters influences the homeostatic balance between inhibitory and excitatory synapses.

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.