Cholesterol depletion activates rapid internalization of submicron-sized acetylcholine receptor domains at the cell membrane

Novel effects of cholesterol (Chol) on nicotinic acetylcholine receptor (AChR) cell-surface stability, internalization and function are reported. AChRs are shown to occur in the form of submicron-sized (240–280 nm) domains that remain stable at the cell-surface membrane of CHO-K1/A5 cells over a period of hours. Acute (30 min, 37°C) exposure to methyl-β-cyclodextrin (CDx), commonly used as a diagnostic tool of endocytic mechanisms, is shown here to enhance AChR internalization kinetics in the receptor-expressing clonal cell line. This treatment drastically reduced (∼50%) the number of receptor domains by accelerating the rate of endocytosis (t1/2 decreased from 1.5–0.5 h). In addition, Chol depletion produced ion channel gain-of-function of the remaining cell-surface AChR, whereas Chol enrichment had the opposite effect. Fluorescence measurements under conditions of direct excitation of the probe Laurdan and of Förster-type resonance energy transfer (FRET) using the intrinsic protein fluorescence as donor both indicated an increase in membrane fluidity in the bulk membrane and in the immediate environment of the AChR protein upon Chol depletion. Homeostatic control of Chol content at the plasmalemma may thus modulate cell-surface organization and stability of receptor domains, and fine tune receptor channel function to temporarily compensate for acute AChR loss from the cell surface.Fil: Borroni, Maria Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Unesco; ArgentinaFil: Baier, Carlos Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Lang, T. Max-Planck-Institut für biophysikalische Chemie; Alemania. Institut Max Planck fuer Bioanorganische Chemie; AlemaniaFil: Bonini, Ida Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: White, M. M. Drexel University; Estados UnidosFil: Garbus, Ingrid. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Barrantes, Francisco Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Unesco; Argentin

Agonist and cholesterol modulate the alpha7 acetylcholine receptor in non-neural endothelial cells

The "neuronal" α7-type nicotinic acetylcholine receptor (α7AChR) is found in various non-neural tissues, including vascular 2+ endothelium, where its peculiar ionotropic (high Ca permeability) 2+ and metabotropic (Ca -mediated intracellular cascades) properties may play important roles in angiogenesis, inflammation and atherogenesis. Molecular properties of the α7AChR, its response to nicotine stimulation, and its cellular distribution were studied here using a combination of pharmacological, biochemical and fluorescence microscopy tools. α7AChRs in rat arterial endothelial cells (RAEC) were found to undergo agonist (nicotine)-induced upregulation (from 53±16 to 385.2±46.8 fmol/mg protein with 50 µM nicotine), increasing their cell-surface exposure. α7AChRs occurred predominantly in the “non-raft" subcellular fractions, yet cholesterol depletion mediated by cyclodextrin treatment reduced the number of cell-surface α7AChRs. Nicotine was found to increase the affinity of the α7AChR for crystal violet, an openchannel blocker. Under basal conditions, α7AChRs in endothelial cells displayed a high-affinity, presumably desensitized conformation (Kd ~0.76 nM), and both nicotine and cyclodextrin affected their cell-surface expression.Fil: Ayala Peña, Victoria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Unesco; ArgentinaFil: Bonini, Ida Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Unesco; ArgentinaFil: Barrantes, Francisco Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Unesco; ArgentinaXLVI Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología MolecularPuerto MadrynArgentinaSociedad Argentina de Investigación Bioquímica y Biología Molecula

Sphingomyelin composition and physical asymmetries in native acetylcholine receptor-rich membranes

Selective enzymatic hydrolysis, lipid compositional analyses, and fluorescence studies have been carried out on acetylcholine receptor (AChR)-rich membranes from Torpedinidae to investigate the topology of sphingomyelin (SM) in the native membrane and its relationship with the AChR protein. Controlled sphingomyelinase hydrolysis of native membranes showed that SM is predominantly (∼60%) localized in the outer half of the lipid bilayer. Differences were also observed in the distribution of SM fatty acid molecular species in the two bilayer leaflets. A fluorescent SM derivative (N-[10-(1-pyrenyl)decanoyl]sphingomyelin; Py-SM) was used to study protein-lipid interactions in the AChR-rich membrane and in affinity-purified Torpedo AChR reconstituted in liposomes made from Torpedo electrocyte lipid extracts. The efficiency of Förster resonance energy transfer (FRET) from the protein to the pyrenyl-labeled lipid as a function of acceptor surface density was used to estimate distances and topography of the SM derivative relative to the protein. The dynamics of the lipid acyl chains were explored by measuring the thermal dependence of Py-SM excimer formation, sensitive to the fluidity of the membrane. Differences were observed in the concentration dependence of excimer/monomer pyrenyl fluorescence when measured by direct excitation of the probe as against under FRET conditions, indicating differences in the intermolecular collisional frequency of the fluorophores between bulk and protein-vicinal lipid environments, respectively. Py-SM exhibited a moderate selectivity for the protein-vicinal lipid domain, with a calculated relative affinity Kr≈0.55. Upon sphingomyelinase digestion of the membrane, FRET efficiency increased by about 50%, indicating that the resulting pyrenylceramide species have higher affinity for the protein than the parental SM derivative.Fil: Bonini, Ida Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Unesco; ArgentinaFil: Antollini, Silvia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Unesco; ArgentinaFil: Gutiérrez Merino, Carlos. Universidad de Extremadura; EspañaFil: Barrantes, Francisco Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Unesco; Argentin

Functional nicotinic acetylcholine receptor reconstitution in Au(111)-supported Thiolipid Monolayers

The insertion and function of the muscle-type nicotinic acetylcholine receptor (nAChR) in Au(111)- supported thiolipid self-assembled monolayers have been studied by atomic force microscopy (AFM), surface plasmon resonance (SPR), and electrochemical techniques. It was possible for the first time to resolve the supramolecular arrangement of the protein spontaneously inserted in a thiolipid monolayer in an aqueous solution. Geometric supramolecular arrays of nAChRs were observed, most commonly in a triangular form compatible with three nAChR dimers of ∼20 nm each. Addition of the full agonist carbamoylcholine activated and opened the nAChR ion channel, as revealed by the increase in capacitance relative to that of the nAChR?thiolipid system under basal conditions. Thus, the self-assembled system appears to be a viable biomimetic model to measure ionic conductance mediated by ion-gated ion channels under different experimental conditions, with potential applications in biotechnology and pharmacologyFil: Pissinis, Diego Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; ArgentinaFil: Diaz, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; ArgentinaFil: Maza, Eliana María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; ArgentinaFil: Bonini, Ida Clara. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); ArgentinaFil: Barrantes, Francisco Jose. Pontificia Universidad Católica Argentina "Santa María de Los Buenos Aires"; ArgentinaFil: Salvarezza, Roberto Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; ArgentinaFil: Schilardi, Patricia Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentin