A Role for the V0 Sector of the V-ATPase in Neuroexocytosis: Exogenous V0d Blocks Complexin and SNARE Interactions with V0c.

V-ATPase is an important factor in synaptic vesicle acidification and is implicated in synaptic transmission. Rotation in the extra-membranous V1 sector drives proton transfer through the membrane-embedded multi-subunit V0 sector of the V-ATPase. Intra-vesicular protons are then used to drive neurotransmitter uptake by synaptic vesicles.

Patient-derived antibodies reveal the subcellular distribution and heterogeneous interactome of LGI1.

Autoantibodies against leucine-rich glioma-inactivated 1 (LGI1) occur in patients with encephalitis who present with frequent focal seizures and a pattern of amnesia consistent with focal hippocampal damage. To investigate whether the cellular and subcellular distribution of LGI1 may explain the localization of these features, and hence gain broader insights into LGI1's neurobiology, we analysed the detailed localization of LGI1 and the diversity of its protein interactome, in mouse brains using patient-derived recombinant monoclonal LGI1 antibodies. Combined immunofluorescence and mass spectrometry analyses showed that LGI1 is enriched in excitatory and inhibitory synaptic contact sites, most densely within CA3 regions of the hippocampus.

Gangliosides interact with synaptotagmin to form the high-affinity receptor complex for botulinum neurotoxin B.

Botulinum neurotoxin type B (BoNT/B) recognizes nerve terminals by binding to 2 receptor components: a polysialoganglioside, predominantly GT1b, and synaptotagmin 1/2. It is widely thought that BoNT/B initially binds to GT1b then diffuses in the plane of the membrane to interact with synaptotagmin. We have addressed the hypothesis that a GT1b-synaptotagmin complex forms the BoNT/B receptor.

Chromophore-Assisted Light Inactivation of the V-ATPase V0c Subunit Inhibits Neurotransmitter Release Downstream of Synaptic Vesicle Acidification.

Synaptic vesicle proton V-ATPase is an essential component in synaptic vesicle function. Active acidification of synaptic vesicles, triggered by the V-ATPase, is necessary for neurotransmitter storage. Independently from its proton transport activity, an additional important function of the membrane-embedded sector of the V-ATPase has been uncovered over recent years.

LGI1 tunes intrinsic excitability by regulating the density of axonal Kv1 channels.

Autosomal dominant epilepsy with auditory features results from mutations in leucine-rich glioma-inactivated 1 (LGI1), a soluble glycoprotein secreted by neurons. Animal models of LGI1 depletion display spontaneous seizures, however, the function of LGI1 and the mechanisms by which deficiency leads to epilepsy are unknown. We investigated the effects of pure recombinant LGI1 and genetic depletion on intrinsic excitability, in the absence of synaptic input, in hippocampal CA3 neurons, a classical focus for epileptogenesis.

An optical biosensor assay for rapid dual detection of Botulinum neurotoxins A and E.

The enzymatic activity of the pathogenic botulinum neurotoxins type A and E (BoNT/A and E) leads to potentially lethal paralytic symptoms in humans and their prompt detection is of crucial importance. A chip assay based on Surface Plasmon Resonance monitoring of the cleavage products is a simple method that we have previously established to detect BoNT/A activity. We have now developed a similar format assay to measure BoNT/E activity.

A chip-based assay for botulinum neurotoxin A activity in pharmaceutical preparations.

The production of botulinum neurotoxin A (BoNT/A) for therapeutic and cosmetic applications requires precise determination of batch potency, and the enzymatic activity of BoNT/A light chain is a crucial index that can be measured in vitro. We previously established a SNAP-25 chip-based assay using surface plasmon resonance (SPR) that is more sensitive than the standard mouse bioassay for the quantification of BoNT/A activity. We have now adapted this procedure for pharmaceutical preparations.

Direct biosensor detection of botulinum neurotoxin endopeptidase activity in sera from patients with type A botulism.

Botulinum neurotoxin A (BoNT/A) has intrinsic endoprotease activity specific for SNAP-25, a key protein for presynaptic neurotransmitter release. The inactivation of SNAP-25 by BoNT/A underlies botulism, a rare but potentially fatal disease. There is a crucial need for a rapid and sensitive in vitro serological test for BoNT/A to replace the current in vivo mouse bioassay.

A substrate sensor chip to assay the enzymatic activity of Botulinum neurotoxin A.

Botulinum neurotoxin A (BoNT/A) induces muscle paralysis by enzymatically cleaving the presynaptic SNARE protein SNAP-25, which results in lasting inhibition of acetylcholine release at the neuromuscular junction. A rapid and sensitive in vitro assay for BoNT/A is required to replace the mouse lethality assay (LD50) in current use. We have developed a fully automated sensor to assay the endoprotease activity of BoNT/A.

Calcium-dependent regulation of SNARE-mediated membrane fusion by calmodulin.

Neuroexocytosis requires SNARE proteins, which assemble into trans complexes at the synaptic vesicle/plasma membrane interface and mediate bilayer fusion. Ca(2+) sensitivity is thought to be conferred by synaptotagmin, although the ubiquitous Ca(2+)-effector calmodulin has also been implicated in SNARE-dependent membrane fusion. To examine the molecular mechanisms involved, we examined the direct action of calmodulin and synaptotagmin in vitro, using fluorescence resonance energy transfer to assay lipid mixing between target- and vesicle-SNARE liposomes.

Oriented binding of the His6-tagged carboxyl-tail of the L-type Ca2+ channel alpha1-subunit to a new NTA-functionalized self-assembled monolayer.

Oriented stable binding of functional proteins on surfaces is of fundamental interest for receptor/ligand studies in atomic force microscopy (AFM) and surface plasmon resonance (SPR) experiments. Here we have chosen the His6-tagged carboxyl-tail (C-tail) of the alpha1c-subunit of the L-type Ca2+ channel and calmodulin (CaM) as its cognitive partner as a model system to develop a new functional surface. Covalently attached self-assembled monolayers on ultraflat gold containing NTA-thiols to which the His6-tagged C-tail was bound and thiols with triethylene-glycol groups as matrix-thiols represented the system of choice.

Atypical L-type channels are down-regulated in hypoxia.

One type of cellular response to hypoxia is an increase in cytosolic Ca2+. VDCCs (voltage-dependent calcium channels) open upon membrane depolarization allowing inward current of Ca2+ ions. Two of the so-called L-type VDCC alpha1 subunits, Ca(v)1.

Calcium-dependent translocation of synaptotagmin to the plasma membrane in the dendrites of developing neurones.

In neurones, the morphological complexity of the dendritic tree requires regulated growth and the appropriate targeting of membrane components. Accurate delivery of specific supplies depends on the translocation and fusion of transport vesicles. Vesicle SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) and target membrane SNAREs play a central role in the correct execution of fusion events, and mediate interactions with molecules that endow the system with appropriate regulation.

Skeletal and cardiac ryanodine receptors bind to the Ca(2+)-sensor region of dihydropyridine receptor alpha(1C) subunit.

In striated muscles, excitation-contraction coupling is mediated by the functional interplay between dihydropyridine receptor L-type calcium channels (DHPR) and ryanodine receptor calcium-release channel (RyR). Although significantly different molecular mechanisms are involved in skeletal and cardiac muscles, bidirectional cross-talk between the two channels has been described in both tissues. In the present study using surface plasmon resonance spectroscopy, we demonstrate that both RyR1 and RyR2 can bind to structural elements of the C-terminal cytoplasmic domain of alpha(1C).

Interactions of calmodulin with two peptides derived from the c-terminal cytoplasmic domain of the Ca(v)1.2 Ca2+ channel provide evidence for a molecular switch involved in Ca2+-induced inactivation.

When opened by depolarization, L-type calcium channels are rapidly inactivated by the elevation of Ca(2+) concentration on the cytoplasmic side. Recent studies have shown that the interaction of calmodulin with the proximal part of the cytoplasmic C-terminal tail of the channel plays a prominent role in this modulation. Two motifs interacting with calmodulin in a Ca(2+)-dependent manner have been described: the IQ sequence and more recently the neighboring CB sequence.

Molecular interaction of dihydropyridine receptors with type-1 ryanodine receptors in rat brain.

In striated muscles, Ca2+ release from internal stores through ryanodine receptor (RyR) channels is triggered by functional coupling to voltage-activated Ca2+ channels known as dihydropyridine receptors (DHPRs) located in the plasma membrane. In skeletal muscle, this occurs by a direct conformational link between the tissue-specific DHPR (Ca(v)1.1) and RyR(1), whereas in the heart the signal is carried from the cardiac-type DHPR (Ca(v)1.

Expression and targeting to the plasma membrane of xClC-K, a chloride channel specifically expressed in distinct tubule segments of Xenopus laevis kidney.

ClC-K channels are Cl- channels specifically expressed in vertebrate kidneys. Although their heterologous functional expression is still controversial, indirect evidence points to them as major factors involved in Cl- reabsorption in the nephron. We cloned xClC-K, an amphibian (Xenopus) homologue of mammalian ClC-K.

Low-voltage-activated Ca2+ currents are generated by members of the CavT subunit family (alpha1G/H) in rat primary sensory neurons.

Recently, two members of a new family of Ca2+ channel alpha1 subunits, alpha1G (or CavT.1) and alpha1H (or CavT.2), have been cloned and expressed.

T-type Ca2+ current properties are not modified by Ca2+ channel beta subunit depletion in nodosus ganglion neurons.

At the molecular level, our knowledge of the low voltage-activated Ca2+ channel (T-type) has made little progress. Using an antisense strategy, we investigated the possibility that the T-type channels have a structure similar to high voltage-activated Ca2+ channels. It is assumed that high voltage-activated channels are made of at least three components: a pore forming alpha1 subunit combined with a cytoplasmic modulatory beta subunit and a primarily extracellular alpha2delta subunit.

Polyethylenimine-mediated DNA transfection of peripheral and central neurons in primary culture: probing Ca2+ channel structure and function with antisense oligonucleotides.

To study neuronal ion channel function with antisense oligonucleotides, a reliable method is needed which allows different neuronal cell types to be transfected without artifactual disruptive effects on their electrical properties. Here we report that use of the recently introduced transfecting agent, polyethylenimine, fulfills this requirement. Four days after transfection, in both central and peripheral neurons, an antisense designed to block the synthesis of the Ca2+ channel beta subunits induced a maximal decrease of the Ca2- current amplitude and modification of their kinetics and voltage-dependence.

Evolution of acetylcholinesterase transcripts and molecular forms during development in the central nervous system of the quail.

We studied the expression of acetylcholinesterase (AChE) in the nervous system (cerebellum, optic lobes and neuroretina) of the quail at different stages of development, from embryonic day 10 (E10) to the adult. Analyzing AChE mRNAs and AChE molecular forms, we observed variations in the following: (a) production of multiple mRNA species (4.5 kb, 5.

Structural analysis of mouse glycine receptor alpha subunit genes. Identification and chromosomal localization of a novel variant.

The inhibitory glycine receptor is a ligand-gated ion channel protein that occurs in different developmentally regulated isoforms in the mammalian central nervous system. Here, we have analyzed genomic clones covering the coding regions of the murine glycine receptor alpha 1 and alpha 2 subunit genes. Both genes contain eight intronic regions with precisely conserved boundaries.

Primary structure and alternative splice variants of gephyrin, a putative glycine receptor-tubulin linker protein.

A 93 kd polypeptide associated with the mammalian inhibitory glycine receptor (GlyR) is localized at central synapses and binds with high affinity to polymerized tubulin. This protein, named gephyrin (from the Greek gamma epsilon phi upsilon rho alpha, bridge), is thought to anchor the GlyR to subsynaptic microtubules. Here we report its primary structure deduced from cDNA and show that corresponding transcripts are found in all rat tissues examined.