Strychnine-Blocked Glycine Receptor Is Removed from Synapses by a Shift in Insertion/Degradation Equilibrium

Rasmussen, Hanne B.; Rasmussen, Trine N.; Triller, Antoine; Vannier, Christian

doi:10.1006/mcne.2001.1074

Cited by 34 publications

(26 citation statements)

References 74 publications

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“…For calibration, 0, 0.125, 0.25, and 0.5 ng of anti-myc antibody were loaded in the same gel. Electrotransfer was performed as described previously (Rasmussen et al, 2002). The blot was finally processed for enhanced chemiluminescence (ECL) detection (Amersham Biosciences, Europe GmbH, Saday, France) on Hyperfilm.…”

Section: Methodsmentioning

confidence: 99%

Intracellular Association of Glycine Receptor with Gephyrin Increases Its Plasma Membrane Accumulation Rate

Hanus

Vannier²,

Triller³

2004

J. Neurosci.

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Gephyrin, a tubulin-binding protein, is the core of inhibitory postsynaptic scaffolds stabilizing glycine receptors (GlyRs) and/or GABA A receptors. Previous ultrastructural studies in vivo and in vitro have reported a localization of gephyrin to intracellular cisternas during development or after glycinergic denervation (Seitanidou et al., 1992; Colin et al., 1996, 1998). These data were compatible with a traffic of this cytoplasmic, but membrane-associated, protein together with membrane proteins such as GlyR after exocytosis and/or endocytosis pathways. We have now investigated the consequences of a GlyR-gephyrin interaction on the localization and the dynamics of these two molecules in African green monkey kidney cells (COS-7) cells and in neurons transfected with green fluorescent protein-taggedgephyrin and myc-tagged GlyR ␣ 1 subunits. In these experiments, myc-tagged GlyR ␣ 1 contained, or did not contain, the gephyrinbinding sequence (␤gb) of the GlyR ␤ subunit. We report here that GlyR-gephyrin interaction localizes gephyrin to GlyR-containing organelles. Videomicroscopy and nocodazole treatment indicate that the movements of these vesicles are microtubule dependent. Expressing GlyR ␣ 1 with a thrombin cleavage site between the myc-tag and the N terminal of the GlyR ␣ 1 subunit (Rosenberg et al., 2001) allowed monitoring of newly inserted receptors in the cell surface. Using temperature changes to block GlyR in, and then release it from, the trans-Golgi network, we show that gephyrin accelerates the accumulation of GlyR at the cell surface. Therefore, our data strongly suggest that some GlyR clusters are associated with gephyrin on their way to the cell surface and that this association increases the accumulation of GlyR at the plasma membrane.

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Section: Methodsmentioning

confidence: 99%

Intracellular Association of Glycine Receptor with Gephyrin Increases Its Plasma Membrane Accumulation Rate

Hanus

Vannier²,

Triller³

2004

J. Neurosci.

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“…We tested for changes in stable retention of surface α3*nAChRs using standard cellsurface biotinylation assays of endocytosis (Mammen et al, 1997;Rasmussen et al, 2002). Surface α3*nAChRs showed reduced stabilization as indicated by their 1.6-fold faster turnover rates on dissociated APC::EB1-dn neurons relative to control uninfected neurons (p<0.04; Fig.…”

Section: Expression Of Apc::eb1-dn Decreases Insertion and Increases mentioning

confidence: 99%

“…The turnover rate of surface α3*nAChRs was measured using standard cell-surface biotinylation assays of endocytosis (Mammen et al, 1997;Rasmussen et al, 2002). Briefly, dissociated cells from control and APC::EB1-dn-infected embryos were plated at equal density of ~10 000 neurons per well.…”

Section: Receptor Insertion and Endocytosis Assaysmentioning

confidence: 99%

“…The cells were returned to 37°C, chased for various lengths of times to allow endocytosis and lysed with (in mM): 100 NaCl, 50 Tris-HCl pH 8.0, 10 EDTA, 50 NaF, 0.1 Na 3 VO 4 , 10% glycerol, 0.5% Igepal CA-630 (chemically equivalent to NP-40, SigmaAldrich), and protease inhibitor cocktail (Roche). Biotinylated proteins were precipitated as described (Rasmussen et al, 2002). Levels of biotinylated surface α3*nAChRs (mAb313) and N-cadherin, as another nicotinic synapse surface membrane protein, were assessed by standard immunoblot techniques (Temburni et al, 2004) using ECL plus detection system (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and exposure to X-ray film for 30 sec -5 min.…”

Section: Receptor Insertion and Endocytosis Assaysmentioning

confidence: 99%

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Adenomatous polyposis coli plays a key role, in vivo, in coordinating assembly of the neuronal nicotinic postsynaptic complex

Rosenberg

Yang

Giovanni

et al. 2008

Molecular and Cellular Neuroscience

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The neuronal nicotinic synapse plays a central role in normal cognitive and autonomic function. Molecular mechanisms that direct the assembly of this synapse remain poorly defined, however. We show here that adenomatous polyposis coli (APC) organizes a multi-molecular complex that is essential for targeting α3*nAChRs to synapses. APC interaction with microtubule plus-end binding protein EB1 is required for α3*nAChR surface membrane insertion and stabilization. APC brings together EB1, the key cytoskeletal regulators macrophin and IQGAP1, and 14-3-3 adapter protein at nicotinic synapses. 14-3-3, in turn, links the α3-subunit to APC. This multi-molecular APC complex stabilizes the local microtubule and F-actin cytoskeleton and links postsynaptic components to the cytoskeleton-essential functions for controlling the molecular composition and stability of synapses. This work identifies macrophin, IQGAP1 and 14-3-3 as novel nicotinic synapse components and defines a new role for APC as an in vivo coordinator of nicotinic postsynaptic assembly in vertebrate neurons.

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“…These results suggest that the activation of the receptors by the neurotransmitter glycine is required to initiate or stabilize glycine receptor clusters. However, the effect of strychnine might be attributable to a block of glycine receptor recycling after endocytosis, resulting in a subsequent depletion of receptors at postsynaptic domains (Rasmussen et al, 2002). In contrast to the glycine receptor clustering studies, initial reports indicate that GABA A receptor expression and clustering do not require neurotransmitter release.…”

Section: Introductionmentioning

confidence: 97%

GABA Is Dispensable for the Formation of Junctional GABA Receptor Clusters inCaenorhabditis elegans

Gally

Bessereau

2003

J. Neurosci.

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At GABAergic synapses, GABA receptors form high-density clusters opposite GABA release sites. Whether GABA release per se plays a role in the formation of GABA receptor clusters remains uncertain. To address this question in vivo, we characterized GABA receptor clustering in the nematode Caenorhabditis elegans. In C. elegans, body wall muscles receive excitatory inputs from cholinergic motor neurons and inhibitory inputs from GABAergic neurons. Using immunohistochemistry and green fluorescent protein-tagged proteins, we observed that the muscle GABA receptor UNC-49 is precisely clustered opposite GABA release sites. During development, these clusters appear slightly after the detection of presynaptic vesicles. If motor axons are mislocalized as in unc-5 mutants, GABA receptors cluster opposite ectopic axons at GABA release sites. Together, these data imply that a motor neuron-derived factor is instructing GABA receptor clustering. Presynaptic localization of this clustering activity requires the neuronal kinesin UNC-104, suggesting that release of GABA from synaptic vesicles may represent the clustering signal. However, unc-25 mutants do not synthesize GABA but do cluster postsynaptic GABA receptors indistinguishably from the wild type. Therefore, at GABAergic neuromuscular junctions, GABA receptor clustering requires nerve-muscle interaction but not GABA neurotransmission.

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Strychnine-Blocked Glycine Receptor Is Removed from Synapses by a Shift in Insertion/Degradation Equilibrium

Cited by 34 publications

References 74 publications

Intracellular Association of Glycine Receptor with Gephyrin Increases Its Plasma Membrane Accumulation Rate

Intracellular Association of Glycine Receptor with Gephyrin Increases Its Plasma Membrane Accumulation Rate

Adenomatous polyposis coli plays a key role, in vivo, in coordinating assembly of the neuronal nicotinic postsynaptic complex

GABA Is Dispensable for the Formation of Junctional GABA Receptor Clusters inCaenorhabditis elegans

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