Fusion complex formation protects synaptobrevin against proteolysis by tetanus toxin light chain

AbstractThe clostridial neurotoxin, tetanus toxin, is a Zn2+-dependent protease which inhibits neurotransmitter exocytosis by selective cleavage of the synaptic vesicle protein, synaptobrevin. Synaptobrevin is thought to serve as a receptor for two neuronal plasma membrane proteins, syntaxin and SNAP-25, which in the presence of non-hydrolyzable ATP analogs form a 20 S fusion complex with the soluble fusion proteins NSF and α-SNAP. Here we show that synaptobrevin, when in this 20 S complex, or its 7 S precursor, is protected against proteolysis by the enzymatically active tetanus toxin light chain. Our data define distinct pools of synaptobrevin, which provide markers of different steps of vesicle/plasma membrane interaction

Potentiation of Glycine-Gated NR1/NR3A NMDA Receptors Relieves Ca2+-Dependent Outward Rectification

Glycine has diverse functions within the mammalian central nervous system. It inhibits postsynaptic neurons via strychnine-sensitive glycine receptors (GlyRs) and enhances neuronal excitation through co-activation of N-methyl-D-aspartate (NMDA) receptors. Classical Ca2+-permeable NMDA receptors are composed of glycine-binding NR1 and glutamate-binding NR2 subunits, and hence require both glutamate and glycine for efficient activation. In contrast, recombinant receptors composed of NR1 and the glycine binding NR3A and/or NR3B subunits lack glutamate binding sites and can be activated by glycine alone. Therefore these receptors are also named “excitatory glycine receptors”. Co-application of antagonists of the NR1 glycine-binding site or of the divalent cation Zn2+ markedly enhances the glycine responses of these receptors. To gain further insight into the properties of these glycine-gated NMDA receptors, we investigated their current-voltage (I–V) dependence. Whole-cell current-voltage relations of glycine currents recorded from NR1/NR3B and NR1/NR3A/NR3B expressing oocytes were found to be linear under our recording conditions. In contrast, NR1/NR3A receptors displayed a strong outwardly rectifying I–V relation. Interestingly, the voltage-dependent inward current block was abolished in the presence of NR1 antagonists, Zn2+ or a combination of both. Further analysis revealed that Ca2+ (1.8 mM) present in our recording solutions was responsible for the voltage-dependent inhibition of ion flux through NR1/NR3A receptors. Since physiological concentrations of the divalent cation Mg2+ did not affect the I–V dependence, our data suggest that relief of the voltage-dependent Ca2+ block of NR1/NR3A receptors by Zn2+ may be important for the regulation of excitatory glycinergic transmission, according to the Mg2+-block of conventional NR1/NR2 NMDA receptors

Molecular Determinants of Agonist Discrimination by NMDA Receptor Subunits: Analysis of the Glutamate Binding Site on the NR2B Subunit

AbstractNMDA receptors require both L-glutamate and the coagonist glycine for efficient channel activation. The glycine binding site of these heteromeric receptor proteins is formed by regions of the NMDAR1 (NR1) subunit that display sequence similarity to bacterial amino acid binding proteins. Here, we demonstrate that the glutamate binding site is located on the homologous regions of the NR2B subunit. Mutation of residues within the N-terminal domain and the loop region between membrane segments M3 and M4 significantly reduced the efficacy of glutamate, but not glycine, in channel gating. Some of the mutations also decreased inhibition by the glutamate antagonists, D-AP5 and R-CPP. Homology-based molecular modeling of the glutamate and glycine binding domains indicates that the NR2 and NR1 subunits use similar residues to ligate the agonists' α-aminocarboxylic acid groups, whereas differences in side chain interactions and size of aromatic residues determine ligand selectivity

Characterization of an invertebrate nicotinic acetylcholine receptor gene: The ard gene of Drosophila melanogaster

AbstractThe ard gene encodes a neuronal nicotinic acetylcholine receptor (AChR) protein from Drosophila (ARD protein). Cytogenetically this gene maps at position 64B/C on the left arm of the 3rd chromosome. Five introns interrupt the protein coding region of the gene, and one is found upstream of the translation start site. The ard gene thus contains less introns than vertebrate muscle AChR genes, but, with one exception, the positions of the resident introns are precisely conserved. Implications for the evolution of AChR genes are discussed

The β-bungarotoxin-binding protein from chick brain: binding sites for different neuronal K+ channel ligands co-fractionate upon partial purification

Abstractβ-Bungarotoxin (β-Butx) is a presynaptically active neurotoxin which blocks neuronal A-type K+ channels. Here, the efficient solubilisation and about 300-fold purification of the β-Butx-binding protein from chick brain were achieved by detergent extraction at high ionic strength followed by chromatography on DEAE Affigel Blue, β-Butx Affigel 102 and wheat germ agglutinin Sepharose. Binding of 125I-labelled β-Butx to the purified protein was inhibited by two other K+ channel ligands, dendrotoxin I and mast cell-degranulating peptide. It is concluded that the β-Butx-binding protein is a member of a family of voltage-gated K+ channels which exhibit varying affinities for different polypeptide ligands

Expression of renal and intestinal Na/Pi cotransporters in the absence of GABARAP

We have recently shown that the abundance of the renal sodium (Na)/inorganic phosphate (Pi) cotransporter NaPi-IIa is increased in the absence of the GABAA receptor-associated protein (GABARAP). Accordingly, GABARAP-deficient mice have a reduced urinary excretion of Pi. However, their circulating levels of Pi do not differ from wild-type animals, suggesting the presence of a compensatory mechanism responsible for keeping serum Pi values constant. Here, we aimed first to identify the molecular basis of this compensation by analyzing the expression of Na/Pi cotransporters known to be expressed in the kidney and intestine. We found that, in the kidney, the upregulation of NaPi-IIa is not accompanied by changes on the expression of either NaPi-IIc or PiT2, the other cotransporters known to participate in renal Pi reabsorption. In contrast, the intestinal expression of NaPi-IIb is downregulated in mutant animals, suggesting that a reduced intestinal absorption of Pi could contribute to maintain a normophosphatemic status despite the increased renal retention. The second goal of this work was to study whether the alterations on the expression of NaPi-IIa induced by chronic dietary Pi are impaired in the absence of GABARAP. Our data indicate that, in response to high Pi diets, GABARAP-deficient mice downregulate the expression of NaPi-IIa to levels comparable to those seen in wild-type animals. However, in response to low Pi diets, the upregulation of NaPi-IIa is greater in the mutant mice. Thus, both the basal expression and the dietary-induced upregulation of NaPi-IIa are increased in the absence of GABARA

Роль метода электрофоретического осаждения в создании биокомпозита на основе слоев гидроксиапатити и наночастиц серебра

Работа посвящена созданию многофункционального биокомпозита, состоящего из покрытия на основе гидроксиапатита (ГА) и наночастиц серебра с использованием высокотехнологичных методов обработки поверхности. Высокочастотное магнетронное распыление использовалось для получения слоев ГА покрытия с различной толщиной и структурой на титане и наночастицах серебра. Для получения антибактериального слоя наночастиц серебра использовался метод электрофоретического осаждения. Наночастицы серебра имели сферическую форму с диаметром 70±20 нм и[zeta] -потенциалом -20 мВ. Дифракционные картины биокомпозитов выявили пики кристаллического ГА и серебра (Ag). Так же установлено, что наночастицы серебра являются кристаллическими с размером кристаллитов 14 нм

Frequency of silent brain infarction in transient global amnesia

Background: and purpose To determine the frequency and distribution pattern of acute DWI lesions outside the hippocampus in patients clinically presenting with Transient Global Amnesia (TGA). Methods: Consecutive patients clinically presenting with TGA between January 2010 and January 2017 admitted to our hospital were retrospectively evaluated. All patients fulfilled diagnostic criteria of TGA. We analyzed imaging and clinical data of all patients undergoing MRI with high-resolution diffusion-weighted imaging within 72 h from symptom onset. Results: A total of 126 cases were included into the study. Fifty-three percent (n = 71/126) presented with one or more acute lesions in hippocampal CA1-area. Additional acute DWI lesions in other cortical regions were found in 11% (n = 14/126). All patients with DWI lesions outside the hippocampus presented with neurological symptoms typical for TGA (without additional symptoms.) Conclusions: In a relevant proportion of clinical TGA patients, MRI reveals acute ischemic cerebral lesions. Therefore, cerebral MRI should be performed in patients with TGA to identify a possible cardiac involvement and to detect stroke chameleons