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.

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...

Human P2X7 receptors were expressed in Xenopus laevis oocytes and single channels were recorded using the patch-clamp technique in the outside-out configuration. ATP4- evoked two types of P2X7 receptor-mediated single channel currents characterized by short-lived and long-lived openings. The short- and long-lasting open states had mean open times of approximately 5 and approximately 20 ms and slope conductances near -60 mV of 9 and 13 pS, respectively. The open probabilities of the short and long openings were strongly [ATP4-]-dependent with EC50 values of approximately 0.3 mM and approximately 0.1 mM ATP4-, respectively. The channel kinetics did not change significantly during sustained P2X7 receptor activation for several minutes, as was also observed in recordings in the cell-attached patch-clamp configuration. Activation and deactivation of the short openings followed exponential time courses with time constants in the range of 20 ms, and displayed a shallow [ATP4-] dependence of the activation process. The kinetics of the short channel openings at negative membrane potentials fitted well to a linear C-C-C-O model with two ATP4- binding steps at equal binding sites with a dissociation constant Kd of 139 microM.

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The voltage-gated sodium channel Nav1.7 is essential for an adequate perception of painful stimuli. Its mutations cause various pain syndromes in human patients. The hNav1.7/A1632E mutation induces symptoms of erythromelalgia and paroxysmal extreme pain disorder (PEPD), and its main gating change is a strongly enhanced persistent current.Using molecular simulations, we demonstrate that the disease causing persistent current of hNav1.7/A1632E is due to impaired binding of the IFM motif, thus affecting proper function of the recently proposed allosteric fast inactivation mechanism. By using native polyacrylamide gel electrophoresis (PAGE) gels, we show that hNav1.7 dimerizes. The disease-linked persistent current depends on the channel’s functional dimerization status: Using difopein, a 14-3-3 inhibitor known to uncouple dimerization of hNav1.5, we detect a significant decrease in hNav1.7/A1632E induced persistent currents.Our work identifies that functional uncoupling of hNav1.7/A1632E dimers rescues the pain-causing molecular phenotype by interferes with an allosteric fast inactivation mechanism, which we link for the first time to channel dimerization. Our work supports the concept of sodium channel dimerization and reveals its relevance to human pain syndromes.