Metabolic cholesterol depletion hinders cell-surface trafficking of the nicotinic acetylcholine receptor

Pediconi, M. F.; Gallegos, Cristina Eugenia; Santos, Eugenio; Barrantes, Francisco J.

doi:10.1016/j.neuroscience.2004.06.007

Cited by 51 publications

(41 citation statements)

References 64 publications

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“…In previous works by us and others (22,37,38), lipid rafts have been shown to be required for intracellular trafficking and surface transport of AChRs. To ascertain the role of lipid rafts on agrin-elicited AChR clustering, rather than the effect of the depletion of cholesterol on AChR biosynthesis and/or surface expression, we quantified surface, intracellular, and total AChRs in control and MbCDtreated myotubes by means of 125 I-a-Bgtx binding.…”

Section: Cholesterol Is Required For Achr Clustering In C2c12 Myotubesmentioning

confidence: 84%

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

Stetzkowski‐Marden

Gaus

Recouvreur

et al. 2006

Journal of Lipid Research

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The formation of the neuromuscular junction is characterized by the progressive accumulation of nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane facing the nerve terminal, induced predominantly through the agrin/muscle-specific kinase (MuSK) signaling cascade. However, the cellular mechanisms linking MuSK activation to AChR clustering are still poorly understood. Here, we investigate whether lipid rafts are involved in agrinelicited AChR clustering in a mouse C2C12 cell line. We observed that in C2C12 myotubes, both AChR clustering and cluster stability were dependent on cholesterol, because depletion by methyl-b-cyclodextrin inhibited cluster formation or dispersed established clusters. Importantly, AChR clusters resided in ordered membrane domains, a biophysical property of rafts, as probed by Laurdan two-photon fluorescence microscopy. We isolated detergent-resistant membranes (DRMs) by three different biochemical procedures, all of which generate membranes with similar cholesterol/ GM1 ganglioside contents, and these were enriched in several postsynaptic components, notably AChR, syntrophin, and raft markers flotillin-2 and caveolin-3. Agrin did not recruit AChRs into DRMs, suggesting that they are present in rafts independently of agrin activation. Consequently, in C2C12 myotubes, agrin likely triggers AChR clustering or maintains clusters through the coalescence of lipid rafts. These data led us to propose a model in which lipid rafts play a pivotal role in the assembly of the postsynaptic membrane at the neuromuscular junction upon agrin signaling.-StetzkowskiMarden, F., K. Gaus, M. Recouvreur, A. Cartaud, and J. Cartaud. Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes.

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Section: Cholesterol Is Required For Achr Clustering In C2c12 Myotubesmentioning

confidence: 84%

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

Stetzkowski‐Marden

Gaus

Recouvreur

et al. 2006

Journal of Lipid Research

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“…Indeed, such a mechanism is implicated in postsynaptic localization of nicotinic acetylcholine (nACh), GABA A , and AMPA receptors (Bruses et al, 2001;Hering et al, 2003;Pediconi et al, 2004). For the nACh receptor, which is structurally closely related to GABA A receptors, the TM4 domain of ␣ and ␥ subunits is believed to interact directly with the lipid bilayer (Blanton and Cohen, 1994;de Almeida et al, 2004;Pediconi et al, 2004). Interestingly, the few amino acid residues that are uniquely present in the TM4 domain of the GABA A receptor ␥2 subunit but not the ␣1,2 and ␤2,3 subunits, map to two separate surfaces of the presumed TM4 ␣-helix (Fig.…”

Section: Discussionmentioning

confidence: 99%

Distinct γ2 Subunit Domains Mediate Clustering and Synaptic Function of Postsynaptic GABA_AReceptors and Gephyrin

Alldred¹,

Mulder-Rosi²,

et al. 2005

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Modulation of the concentration of postsynaptic GABA A receptors contributes to functional plasticity of inhibitory synapses. The ␥2 subunit of GABA A receptor is specifically required for clustering of these receptors, for recruitment of the submembrane scaffold protein gephyrin to postsynaptic sites, and for postsynaptic function of GABAergic inhibitory synapses. To elucidate this mechanism, we here have mapped the ␥2 subunit domains required for restoration of postsynaptic clustering and function of GABA A receptors in ␥2 subunit mutant neurons. Transfection of ␥2 Ϫ/Ϫ neurons with the ␥2 subunit but not the ␣2 subunit rescues postsynaptic clustering of GABA A receptors, results in recruitment of gephyrin to postsynaptic sites, and restores the amplitude and frequency of miniature inhibitory postsynaptic currents to wild-type levels. Analogous analyses of chimeric ␥2/␣2 subunit constructs indicate, unexpectedly, that the fourth transmembrane domain of the ␥2 subunit is required and sufficient for postsynaptic clustering of GABA A receptors, whereas cytoplasmic ␥2 subunit domains are dispensable. In contrast, both the major cytoplasmic loop and the fourth transmembrane domain of the ␥2 subunit contribute to efficient recruitment of gephyrin to postsynaptic receptor clusters and are essential for restoration of miniature IPSCs. Our study points to a novel mechanism involved in targeting of GABA A receptors and gephyrin to inhibitory synapses.

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“…The association kinetics of αBTX to α7 AChRs in SHE-P1-hα7-Ric-3 cells is much faster (apparent k on =32.3±1.32 h -1 ) than for adult muscle-type AChRs expressed in CHO-K1/A5 cells [33] (k on =1.2±0.12 h -1 ). The half-life of the α7 AChRs was also much shorter (~7 min) than that of adult muscle AChRs expressed in CHO-K1/A5 cells [34] (~5.0 h), BC3H-1 cells [34] (~7.7 h), or Q-A33 quail fibroblasts transfected with the adult AChR [35] (~9.8 h).…”

Section: Discussionmentioning

confidence: 91%

“…The ST-7 CCD camera was driven by the CCDOPS software package (SBIG Astronomical Instruments, version 5.02). Appropriate excitation, dichroic and emission filters were employed to avoid crossover of fluorescence emission following previously established conditions [32,33] . Sixteen-bit TIFF images were exported for further offline analysis.…”

Section: Methodsmentioning

confidence: 99%

Ric-3 chaperone-mediated stable cell-surface expression of the neuronal α7 nicotinic acetylcholine receptor in mammalian cells

2009

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Aim: Studies of the α7-type neuronal nicotinic acetylcholine receptor (AChR), one of the receptor forms involved in many physiologically relevant processes in the central nervous system, have been hampered by the inability of this homomeric protein to assemble in most heterologous expression systems. In a recent study, it was shown that the chaperone Ric-3 is necessary for the maturation and functional expression of α7-type AChRs [1] . The current work aims at obtaining and characterizing a cell line with high functional expression of the human α7 AChR. Methods: Ric-3 cDNA was incorporated into SHE-P1-hα7 cells expressing the α7-type AChR. Functional studies were undertaken using single-channel patch-clamp recordings. Equilibrium and kinetic [ 125 I]α-bungarotoxin binding assays, as well as fluorescence microscopy using fluorescent α-bungarotoxin, anti-α7 antibody, and GFP-α7 were performed on the new clone. Results: The human α7-type AChR was stably expressed in a new cell line, which we coined SHE-P1-hα7-Ric-3, by co-expression of the chaperone Ric-3. Cell-surface AChRs exhibited [ 125 I]αBTX saturable binding with an apparent K D of about 55 nmol/L. Fluorescence microscopy revealed dispersed and micro-clustered AChR aggregates at the surface of SHE-P1-hα7-Ric-3 cells. Larger micron-sized clusters were observed in the absence of receptor-clustering proteins or upon aggregation with anti-α7 antibodies. In contrast, chaperone-less SHE-P1-hα7 cells expressed only intracellular α7 AChRs and failed to produce detectable single-channel currents. Conclusion:The production of a stable and functional cell line of neuroepithelial lineage with robust cell-surface expression of neuronal α7-type AChR, as reported here, constitutes an important advance in the study of homomeric receptors in mammalian cells.

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Metabolic cholesterol depletion hinders cell-surface trafficking of the nicotinic acetylcholine receptor

Cited by 51 publications

References 64 publications

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

Distinct γ2 Subunit Domains Mediate Clustering and Synaptic Function of Postsynaptic GABA_AReceptors and Gephyrin

Ric-3 chaperone-mediated stable cell-surface expression of the neuronal α7 nicotinic acetylcholine receptor in mammalian cells

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Metabolic cholesterol depletion hinders cell-surface trafficking of the nicotinic acetylcholine receptor

Cited by 51 publications

References 64 publications

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

Distinct γ2 Subunit Domains Mediate Clustering and Synaptic Function of Postsynaptic GABAAReceptors and Gephyrin

Ric-3 chaperone-mediated stable cell-surface expression of the neuronal α7 nicotinic acetylcholine receptor in mammalian cells

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Distinct γ2 Subunit Domains Mediate Clustering and Synaptic Function of Postsynaptic GABA_AReceptors and Gephyrin