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First published online 25 April 2006
doi: 10.1242/jcs.02931

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Endocytosis of GABA_B receptors modulates membrane excitability in the single-celled organism Paramecium

¹ Department for the Study of the Territory and its Resources (DIP.TE.RIS.), University of Genoa, Corso Europa 26, 16132 Genova, Italy
² Department of Communication, Computer and System Sciences (DIST), University of Genoa, Viale Causa 13, 16145 Genova, Italy
³ INFM and Department of Physics, University of Genoa, Via Dodecaneso 33, 16146 Genova, Italy
⁴ IFOM Center of Cell Oncology and Ultrastructure, Department of Experimental Medicine, University of Genoa, Medical School, Via de Toni 14, 16132 Genova, Italy
⁵ Department of Experimental Medicine, Section of Pharmacology and Toxicology, University of Genoa, Viale Cembriano 4, 16148 Genova, Italy
⁶ Institute of Biophysics, CNR Genoa, Via De Marini 6, 16149 Genova, Italy

View larger version (29K): [in a new window]   Fig. 1. Interference of the duration of CCR evoked by 40 mM KCl. (a) Baclofen (100 µM) causes a 20% inhibition of CCR (P<0.01) but its effect is abolished after long exposures (60 minutes). (b) Baclofen-induced downregulation of GABAB receptors is counteracted by sucrose (150 mM) and filipin (0.1 µg/ml); P<0.01 compared with baclofen adapted cells. (c) Effect on CCR by 150 mM sucrose, cytosol acidification (pH 5), 0.1 µg/ml filipin and 50 µM P4 peptide. (d) The inhibitory effect of baclofen is enhanced when cells are treated with 150 mM sucrose, 10 mM acetic acid (pH 5.0), 0.1 µg/ml filipin and 50 µM P4 peptide; P<0.01 compared with baclofen non-adapted cells. Tests were carried out on 15 cells and were repeated on four different occasions over several weeks.

View larger version (69K): [in a new window]   Fig. 2. (a) Colocalization of GABAB receptors and clathrin. In cells labeled with a polyclonal antibody against GABAB receptor (red) and a monoclonal antibody against clathrin HC (green), a clustered distribution of fluorescence is detected on the plasma membrane and inside the cytoplasm. GABAB receptors and clathrin vesicles are partly colocalized (yellow fluorescence). Bar, 10 µm. (b) 2D cytofluorogram. Colocalized pixels are visualized in blue. (c) Ultrastructural localization of GABAB receptors in Paramecium cells using 10 nm gold particles in electron microscopy. GABAB receptor immuno-analogue is present in coated pits. Bar, 100 nm. (d) Immunogold labeling in the transmission electron microscopy confirms the colocalization of GABAB receptors (15 nm gold particles) and clathrin (10 nm gold particles) in coated pits. Bar, 100 nm.

View larger version (75K): [in a new window]   Fig. 3. z-Stack profile of fluorescence intensity of double-labeled vesicles. The left side of the figure shows three optical planes (a, first; b, middle; c, last) from a stack of 40 images (total thickness 3 µm). For each focal plane a sample of three vesicles that show colocalization (yellow) of GABAB receptor (red) and clathrin (green) is selected (white bars). The right side of the figure shows fluorescence-intensity distribution along the z-axis for each selected vesicle (green, ), (red, ).

View larger version (35K): [in a new window]   Fig. 4. GABAB-receptor internalization is mediated by clathrin-coated vesicles. In cells whose phagocytic activity is blocked by trifluoperazine, 20-minute treatment with 150 mM sucrose (b) or cytosol acidification (c) inhibits receptor internalization, which can be seen by receptor accumulation on the cell membrane and receptor reduction inside the cytoplasm. Controls (a) are cells incubated with the anti-GABAB receptor antibody in the absence of inhibitors; the antibody is localized in endosomes and phagosomes. Incubation temperature, 25°C. Bar, 20 µm.

View larger version (113K): [in a new window]   Fig. 5. GABAB receptors colocalize with the clathrin-adaptor protein AP2. (a) Cells labeled with an anti-GABAB receptor antibody (red) and an anti-adaptin ß antibody (green). Colocalization of GABAB receptors and AP2 proteins is seen as a yellow fluorescence (image acquired at cell-surface level). Bar, 10 µm. (b) 2D cytofluorogram. Colocalized pixels are visualized in blue. (c,d) Colocalization of GABAB receptors (15 nm gold particles) and ß2 adaptin (10 nm gold particles) in electron microscopy. (d) Proteins from Paramecium primaurelia (line 1) and Jurkat cells (line 2) were subjected to SDS-PAGE and western blotting. A protein band with an estimated molecular mass of 105 kDa was detected with an anti-adaptin ß antibody. The position of the molecular mass marker is shown on the right. Bars, 100 nm.

View larger version (110K): [in a new window]   Fig. 6. GABAB receptors interact with caveolin 1. (a) Cells labeled with an anti-GABAB receptor antibody (red) and an anti-caveolin 1 antibody (green). Colocalization is seen as a yellow fluorescence. Bar, 10 µm. (b) 2D cytofluorogram. Colocalized pixels are visualized in blue. (c,d) Immunogold labeling in the transmission electron microscopy confirms the colocalization of GABAB receptors (15 nm gold partcles) and caveolin 1 (10 nm gold particles). Bars, 50 nm. (e) Proteins from Paramecium primaurelia cells (line 1) and human endothelial lysate (line 2) were subjected to SDS-PAGE and western blotting. Two protein bands with estimated molecular masses of 24 and 21 kDa were detected with an anti-caveolin 1 antibody, corresponding to the and ß splicing forms, respectively. The position of the molecular mass marker is shown on the right.

View larger version (85K): [in a new window]   Fig. 7. GABAB receptors are internalized by clathrin-independent endocytosis. Cells blocked in their phagocytic activity by trifluoperazine (2.5 µg/ml for 15 minutes) were incubated for 5 minutes at 25°C with anti-GABAB receptor antibody (dilution 1:100) and dextran-coupled Texas-Red (0.02% w/v). Colocalization of GABAB receptors and dextran is seen inside the vesicles located on the cell membrane (yellow). Bar, 20 µm.

View larger version (36K): [in a new window]   Fig. 8. GABAB receptors are internalized by non-coated endocytosis. Cells preincubated at 4°C for 30 minutes and labeled with anti-GABAB receptor antibody (dilution 1:100) for 30 minutes (a) are fixed after a 20-minute chase at 25°C in the absence (b) or in the presence of non-coated-pit endocytosis inhibitors filipin (c) and nystatin (d). (e) Measurement of the internalization representatively shown in b-d. Constitutive receptor internalization is partially inhibited by filipin (0.1 µg/ml) and nystatin (2 µg/ml) (47% and 38%, respectively); P<0.01, Student's t-test). Data were normalized to cells before internalization at 25°C (shown in a). Bar, 20 µm.