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doi: 10.1242/10.1242/jcs.00053

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Functional involvement of VAMP/synaptobrevin-2 in cAMP-stimulated aquaporin 2 translocation in renal collecting duct cells

Sabine Gouraud^*,1, Antonia Laera^*,1, Giuseppe Calamita¹, Monica Carmosino¹, Giuseppe Procino¹, Ornella Rossetto², Roberta Mannucci³, Walter Rosenthal⁴, Maria Svelto¹ and Giovanna Valenti^1,

¹ Dipartimento di Fisiologia Generale ed Ambientale, University of Bari, Via Amendola 165/A, 70126 Bari, Italy
² Centro CNR Biomembrane and Dipartimento di Scienze Biomediche, Via G. Colombo 3, 35100 Padova, Italy
³ Sezione di Medicina Interna ed Oncologia, Dipartimento di Medicina Clinica e Sperimentale, Università degli Studi, Policlinico Monteluce, 06100 Perugia, Italy
⁴ Forschungsinstitut für Molekulare Pharmakologie, 13125 Berlin, Germany

View larger version (37K): [in a new window]   Fig. 1. Degenerate RT-PCR amplification of SNARE cDNAs expressed by CD8 cells. Total RNA from confluent CD8 cells was subjected to reverse transcription followed by PCR amplification using degenerate primers for the coding region of known SNARE isoforms (see Materials and Methods for experimental details). Bands of 266, 370, 379, 244 and 398 bp corresponding to syntaxin-1A, syntaxin-3, syntaxin-4, VAMP-2 and SNAP-23, respectively, were amplified. As a positive control, parallel RT-PCR experiments with total RNA extracted from the rat brain, lung or kidney were performed. The results shown are representative of at least three separate experiments.

View larger version (58K): [in a new window]   Fig. 2. An immunoblot of membrane fractions from different tissues or CD8 cells. Each lane was loaded with 60 µg of protein. Membrane fractions enriched in the plasma membrane (LS, 17,000 g pellet) or in intracellular vesicles (HS, 200,000 g pellet) were probed with VAMP-2 (1:100 dilution) or syntaxin-1A (1:300) or syntaxin-4 (1:300) or SNAP-25 (1:500) or SNAP-23 (1:300) antibodies. Immunoreactive bands were revealed with chemiluminescence ECL-plus (Amersham, USA). The results shown are representative of at least three separate experiments.

View larger version (32K): [in a new window]   Fig. 3. Cleavage of VAMP-2 by the clostridial neurotoxins TeNT: in vitro cleavage. Membrane fractions enriched in intracellular vesicles from CD8 cells and rabbit brain membranes were incubated with TeNT 500 nM for 1 hour at 37°C. TeNT was previously activated by incubation with 10 mM dithiothreitol (DTT) for 2 hours at 37°C. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL-plus). Cleavage of VAMP-2 in intact CD8 cells. CD8 cells were grown to confluency in 10 mm Petri dishes. Cells were incubated in the presence or in the absence of whole TeNT (100 nM, for 3 hours at 37°C in the medium). Proteins were transferred and subjected to western blotting using monoclonal antibodies (1:100 dilution) against human VAMP-2. The results shown are representative of at least three separate experiments. Internalization of TeNT-FITC was visualized by immunofluorescence analysis. Intact cells were exposed to TeNT-FITC, fixed and examined at the fluorescence microscope. TeNT-FITC was internalized in intracellular structures. Bar, 8 µm.

View larger version (65K): [in a new window]   Fig. 4. Characterization of the anti-AQP2 C-loop antibody. (A) Western blots of membrane fractions from CD8 cells and from rat kidney. LS, low-speed pellet; HS high-speed pellet. (B) Immunolocalization of AQP2 in CD8 cells using either the anti-AQP2 raised against the peptide reproducing the C-terminus (AQP2 C-term) or the anti-AQP2 raised against the peptide reproducing the loop C (AQP2 C-loop). In non-permeabilized (-Triton X-100) forskolin-stimulated cells (FK), the anti-AQP2 C-loop stained the apical surface of the CD8 cells. Under the same experimental conditions, no fluorescence signal was observed using the anti-AQP2 C-term antibody. By contrast, both antibodies stained AQP2-containing intracellular vesicles in permeabilized control CD8 cells (+Triton X-100). Bar, 5 µm.

View larger version (38K): [in a new window]   Fig. 5. Determination of cell-surface AQP2 immunoreactivity. The anti-AQP2 C-loop antibody was employed to monitor the AQP2 density on the plasma membrane in CD8 cells. (A) In non-permeabilized cells, the antibody is expected to cross-react only with AQP2 inserted into the plasma membrane. (B) After stimulation of untreated cells with forskolin, the immunodetectable AQP2 on the cell surface increased by approximately two-fold compared with that present in the plasma membrane in control cells (-TeNT). By contrast, TeNT pretreatment completely abolished forskolin-stimulated AQP2 targeting to the apical plasma membrane, as assessed by quantification of cell-surface immunoreactivity (Fig. 6B, +TeNT). The results shown represent the means±s.e.m. of three separate experiments in which about 4x106 cells (six separate wells) were tested for each experimental condition in each experiment. (C) Double labeling of AQP2 and VAMP-2 in cells transiently transfected with GFP-tagged AQP2 showed a partial colocalization of VAMP-2 in AQP2 bearing vesicles. Bar, 5 µm.