Inhibitory glycine receptors (GlyRs) regulate motor coordination and sensory signal processing in spinal cord and other brain regions. GlyRs are pentameric proteins composed of membrane-spanning alpha and beta subunits. Here, site-directed mutagenesis combined with homology modeling based on the crystal structure of the acetylcholine binding protein identified key ligand binding residues of recombinant homooligomeric alpha1 and heterooligomeric alpha1beta GlyRs. This disclosed two highly conserved, oppositely charged residues located on adjacent subunit interfaces as being crucial for agonist binding. In addition, the beta subunit was found to determine the ligand binding properties of heterooligomeric GlyRs. Expression of an alpha1beta tandem construct and affinity purification of metabolically labeled GlyRs confirmed a subunit stoichiometry of 2alpha3beta. Because the beta subunit anchors GlyRs at synaptic sites, our results have important implications for the biosynthesis, clustering, and pharmacology of synaptic GlyRs.

Cys-loop receptors are pentameric ligand-gated ion channels (pLGICs) that mediate fast synaptic transmission. Here functional pentameric assembly of truncated fragments comprising the ligand-binding N-terminal ectodomains and the first three transmembrane helices, M1-M3, of both the inhibitory glycine receptor (GlyR) alpha1 and the 5HT(3)A receptor subunits was found to be rescued by coexpressing the complementary fourth transmembrane helix, M4. Alanine scanning identified multiple aromatic residues in M1, M3 and M4 as key determinants of GlyR assembly. Homology modeling and molecular dynamics simulations revealed that these residues define an interhelical aromatic network, which we propose determines the geometry of M1-M4 tetrahelical packing such that nascent pLGIC subunits must adopt a closed fivefold symmetry. Because pLGIC ectodomains form random nonstoichiometric oligomers, proper pentameric assembly apparently depends on intersubunit interactions between extracellular domains and intrasubunit interactions between transmembrane segments.

The ligand-gated ion channel superfamily plays a critical role in neuronal excitability. The functions of glycine receptor (GlyR) and nicotinic acetylcholine receptor are modulated by G protein ␤␥ subunits. The molecular determinants for this functional modulation, however, are still unknown. Studying mutant receptors, we identified two basic amino acid motifs within the large intracellular loop of the GlyR ␣ 1 subunit that are critical for binding and functional modulation by G␤␥. Mutations within these sequences demonstrated that all of the residues detected are important for G␤␥ modulation, although both motifs are necessary for full binding. Molecular modeling predicts that these sites are ␣-helixes near transmembrane domains 3 and 4, near to the lipid bilayer and highly electropositive. Our results demonstrate for the first time the sites for G protein ␤␥ subunit modulation on GlyRs and provide a new framework regarding the ligand-gated ion channel superfamily regulation by intracellular signaling.The ionotropic glycine receptors (GlyRs) 2 are members of the ligand-gated ion receptor superfamily, which includes inhibitory ␥-aminobutyric acid type A receptors and excitatory nAChR and 5-HT 3 receptors. These homologous receptors mediate fast synaptic transmission in the central nervous system (1, 2). Inhibitory GlyRs are critical for the control of excitability in the mammalian spinal cord and brain stem. Binding of glycine to the extracellular region induces a rapid increase in Cl Ϫ ion conductance, generating a hyperpolarization of the cell membrane (3-5). In neurons, the inhibitory action of GlyRs regulate several important physiological functions, like pain transmission, respiratory rhythms, motor coordination, and development (3-7). Like all members of the LGIC superfamily, GlyRs are pentamers composed of five subunits in which each subunit possesses four transmembrane domains arranged to form the ion pore. In this structure, the individual subunits provide extracellular and intracellular domains that play roles in ligand binding and intracellular modulation, respectively (1-3, 5).The function of GlyRs can be effectively modulated by extracellularly acting compounds like strychnine, picrotoxin, zinc ions, and ethanol (3-5, 8, 9). Furthermore, the receptor can also be modulated by intracellular signaling. One of the most studied and recognized pathways involved in regulation of ligandgated ion channel function are phosphorylation processes through protein kinases. Indeed, GlyR and other members of the LGIC superfamily are modulated by activation of cAMPdependent kinases and protein kinase C (10). This involves specific serine residues in the loop between transmembrane domains 3 and 4 (6, 10 -14). On the other hand, recent reports have shown that the activity of GlyRs and nAChRs can be modulated by G protein ␤␥ subunits in a phosphorylation-independent manner (15, 16). In both cases, activation of G proteins, using nonhydrolyzable GTP analogs or by application of purified G␤␥ dimers, generates a strong e...

Gephyrin is a bifunctional modular protein that, in neurons, clusters glycine receptors and ␥-aminobutyric acid, type A receptors in the postsynaptic membrane of inhibitory synapses. By x-ray crystallography and cross-linking, the N-terminal G-domain of gephyrin has been shown to form trimers and the C-terminal E-domain dimers, respectively. Gephyrin therefore has been proposed to form a hexagonal submembranous lattice onto which inhibitory receptors are anchored. Here, crystal structure-based substitutions at oligomerization interfaces revealed that both G-domain trimerization and E-domain dimerization are essential for the formation of higher order gephyrin oligomers and postsynaptic gephyrin clusters. Insertion of the alternatively spliced C5 cassette into the G-domain inhibited clustering by interfering with trimerization, and mutation of the glycine receptor ␤-subunit binding region prevented the localization of the clusters at synaptic sites. Together our findings show that domain interactions mediate gephyrin scaffold formation.The precise localization and a high density of neurotransmitter receptors at postsynaptic sites is a prerequisite for proper synaptic transmission. During the development of inhibitory synapses, the peripheral membrane protein gephyrin accumulates beneath the postsynaptic plasma membrane and plays a key role in recruiting inhibitory receptors under the contacting nerve terminals (1, 2). Both attenuation of gephyrin expression by antisense oligonucleotides and targeted disruption of the gephyrin gene prevent the synaptic clustering of glycine receptors (GlyRs) 4 (3, 4) and ␥2-subunit-containing GABA A R subtypes (5-7). Although a direct interaction with GABA A Rs has not yet been demonstrated, gephyrin binding to the large intracellular loop of GlyR␤ has been shown to be of high affinity (8, 9). Additional interaction partners of gephyrin include proteins implicated in the regulation of the cytoskeleton, intracellular trafficking, and protein synthesis (1, 10). Gephyrin is a modular protein consisting of an N-terminal G-domain, a C-terminal E-domain, and a connecting linker region (1, 11). The G-and E-domains of gephyrin show significant homology to Escherichia coli, Drosophila, and plant proteins and are involved in the synthesis of a coenzyme of oxidoreductases, the molybdenum cofactor (4,11,12). This enzymatic activity explains the widespread expression of the gephyrin gene also in non-neuronal tissues (11). Crystallographic analysis of the isolated G-and E-domains indicates that they have trimeric and dimeric structures, respectively (13-16). Bacterially expressed full-length gephyrin forms trimers that can assemble into higher order structures (15). This oligomerization behavior of gephyrin and its subdomains is thought to provide the basis for the formation of submembranous hexagonal gephyrin scaffolds that cluster inhibitory neurotransmitter receptors at postsynaptic membrane specializations (1, 15) by reducing their lateral mobility (17,18).In this study, we investigated whe...

Zusammenfassung. Hintergrund: In der Behandlung der persistierenden depressiven Störung (PDD) werden Psychotherapien mit interpersonellem Schwerpunkt empfohlen. Ein Beispiel dafür ist das Cognitive Behavioral Analysis System of Psychotherapy (CBASP), was davon ausgeht, dass Menschen mit PDD zu einem präoperatorischen Denkstil neigen. Das Ziel dieser Arbeit ist herauszufinden, ob dieser Denkstil auch bei Menschen mit einer Panikstörung oder einem chronischen Rückenschmerz beobachtet werden kann. Methodik: In einer Studie wurden Patient_innen mit einer Panikstörung ( n = 20) verglichen mit Patient_innen mit einer depressiven Störung ( n = 20) und gesunden Kontrollen ( n = 20). In einer weiteren Studie wurden Patient_innen mit chronischem Rückenschmerz ( n = 30) verglichen mit gesunden Kontrollen ( n = 32). Alle Proband_innen wurden mit dem Lübecker Fragebogen Präoperatorisches Denken (LFPD) befragt. Daneben wurden die Angehörigen der Proband_innen gebeten, das Interaktionsverhalten der Probandin bzw. des Probanden mit dem Interpersonal Message Inventory (IMI) zu beurteilen. Ergebnis: Die erste Studie kam zu dem Ergebnis, dass sich nur die depressiven Patient_innen im LFPD signifikant von den gesunden Kontrollen unterschieden ( p < .001). In der Subskala „feindselig“ des IMI unterschieden sich die Kontrollen von beiden Patient_innengruppen, allerdings war nur der Befund für die Panikstörung signifikant ( p = .008). In der zweiten Studie unterschieden sich die Patient_innen mit der Schmerzstörung von den gesunden Kontrollen nur im Bezug auf die IMI Subskala „feindselig“ ( p = .005), aber nicht im LFPD. Schlussfolgerung: Das präoperatorische Denken wurde nur bei Patient_innen mit depressiver Störung beobachtet, obwohl die anderen beiden Patient_innengruppen von ihren Angehörigen ebenfalls als feindselig wahrgenommen wurden. Diese Befunde sprechen dafür, dass interpersonelle Defizite bei psychischen Störungen nicht immer auf das dem CBASP zugrundeliegende Modell des präoperatorischen Denkens zurückzuführen sind.