PH-domain-driven targeting of collybistin but not Cdc42 activation is required for synaptic gephyrin clustering.

Collybistin (Cb) is a brain-specific guanine nucleotide exchange factor (GEF) that is essential for the synaptic clustering of gephyrin and GABAA receptors in selected regions of the mammalian central nervous system. It has been previously proposed that Cb regulates gephyrin clustering by activating Cdc42, and thus acts as a signal transducer in a membrane activation process which labels postsynaptic membrane domains for inhibitory synapse formation. Here, we dissected the functional roles of the Dbl-homology (DH) and pleckstrin homology (PH) domains of the constitutively active splice variant Cb II by substituting conserved amino acid residues that are required for GEF activity towards Cdc42 and phosphoinositide binding, respectively. A Cb II mutant lacking any detectable GEF activity towards Cdc42 was still fully active in inducing gephyrin scaffold formation, both in transfected NIH-3T3 cells and in cultured hippocampal neurons. Furthermore, mice with a forebrain-specific inactivation of the Cdc42 gene displayed normal densities of gephyrin and GABA(A) receptor clusters in the hippocampus. In contrast, substitution of Cb II PH-domain residues essential for phosphoinositide binding abolished gephyrin recruitment to synaptic sites. Our results provide evidence that the formation of gephyrin scaffolds at inhibitory synapses requires an intact Cb II PH-domain but is Cdc42-independent.

Influence of peritoneal dialysis solution on measurements of fluid status by bioimpedance spectroscopy.

PURPOSE AND METHODS: The accurate estimation of volume status is a central problem in dialysis patients. Recently, a bioimpedance spectroscopy (BIS) device (BCM Body Composition Monitor FMC, Germany) has attained growing interest in this regard. By processing the raw data for extracellular water (ECW) and intracellular water (ICW) by means of a validated body composition model, this device allows a quantification of the individual fluid overload (FO) compared to a representative healthy population. In this study, we addressed the issue whether the presence of peritoneal dialysate has an impact on measurements of FO by BIS in PD patients. RESULTS: Forty-two BIS measurements using the BCM device were performed both in the absence (D-) and presence (D+) of peritoneal dialysate in 17 stable PD patients. Data for ECW, ICW and FO (D+; D-) were analyzed by paired t test and linear regression. Mean FO was 0.99 ± 1.17 L in D- and 0.94 ± 1.27 in D+ (p = n.s. paired t test). Linear regression demonstrated an excellent degree of conformity between FO (D-) and FO (D+) (r (2) = 0.93). CONCLUSION: The presence of peritoneal fluid in PD patients has a negligible influence on measurements of FO by BIS. The BIS measurements can be therefore conveniently and reliably done without emptying the peritoneal cavity; this may facilitate the use of BIS in this particular group of patients.

Characterization of lamin mutation phenotypes in Drosophila and comparison to human laminopathies.

Lamins are intermediate filament proteins that make up the nuclear lamina, a matrix underlying the nuclear membrane in all metazoan cells that is important for nuclear form and function. Vertebrate A-type lamins are expressed in differentiating cells, while B-type lamins are expressed ubiquitously. Drosophila has two lamin genes that are expressed in A- and B-type patterns, and it is assumed that similarly expressed lamins perform similar functions. However, Drosophila and vertebrate lamins are not orthologous, and their expression patterns evolved independently. It is therefore of interest to examine the effects of mutations in lamin genes. Mutations in the mammalian lamin A/C gene cause a range of diseases, collectively called laminopathies, that include muscular dystrophies and premature aging disorders. We compared the sequences of lamin genes from different species, and we have characterized larval and adult phenotypes in Drosophila bearing mutations in the lam gene that is expressed in the B-type pattern. Larvae move less and show subtle muscle defects, and surviving lam adults are flightless and walk like aged wild-type flies, suggesting that lam phenotypes might result from neuromuscular defects, premature aging, or both. The resemblance of Drosophila lam phenotypes to human laminopathies suggests that some lamin functions may be performed by differently expressed genes in flies and mammals. Such still-unknown functions thus would not be dependent on lamin gene expression pattern, suggesting the presence of other lamin functions that are expression dependent. Our results illustrate a complex interplay between lamin gene expression and function through evolution.

Molecular basis of gephyrin clustering at inhibitory synapses: role of G- and E-domain interactions.

Gephyrin is a bifunctional modular protein that, in neurons, clusters glycine receptors and gamma-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 beta-subunit binding region prevented the localization of the clusters at synaptic sites. Together our findings show that domain interactions mediate gephyrin scaffold formation.

Mass spectrometric analysis of glycine receptor-associated gephyrin splice variants.

Gephyrin is an ubiquitously expressed protein that, in the nervous system, is essential for synaptic anchoring of glycine receptors (GlyRs) and major GABAA receptor subtypes. The binding of gephyrin to the GlyR depends on an amphipathic motif within the large intracellular loop of the GlyRbeta subunit. The mouse gephyrin gene consists of 30 exons. Ten of these exons, encoding cassettes of 5-40 amino acids, are subject to alternative splicing (C1-C7, C4'-C6'). Since one of the cassettes, C5', has recently been reported to exclude GlyRs from GABAergic synapses, we investigated which cassettes are found in gephyrin associated with the GlyR. Gephyrin variants were purified from rat spinal cord, brain, and liver by binding to the glutathione S-transferase-tagged GlyRbeta loop or copurified with native GlyR from spinal cord by affinity chromatography and analyzed by mass spectrometry. In addition to C2 and C6', already known to be prominent, C4 was found to be abundant in gephyrin from all tissues examined. The nonneuronal cassette C3 was easily detected in liver but not in GlyR-associated gephyrin from spinal cord. C5 was present in brain and spinal cord polypeptides, whereas C5' was coisolated mainly from liver. Notably C5'-containing gephyrin bound to the GlyRbeta loop, inconsistent with its proposed selectivity for GABAA receptors. Our data show that GlyR-associated gephyrin, lacking C3, but enriched in C4 without C5, differs from other neuronal and nonneuronal gephyrin isoforms.

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.