First published online September 9, 2005
doi: 10.1242/10.1242/jcs.02525
Journal of Cell Science 118, 4243-4252 (2005)
Published by The Company of Biologists 2005
Basolateral localization of native ClC-2 chloride channels in absorptive intestinal epithelial cells and basolateral sorting encoded by a CBS-2 domain di-leucine motif
Gaspar Peña-Münzenmayer1,*,
Marcelo Catalán1,*,
Isabel Cornejo1,2,*,
Carlos D. Figueroa2,
James E. Melvin3,
María I. Niemeyer1,
L. Pablo Cid1 and
Francisco V. Sepúlveda1,
1 Centro de Estudios Científicos (CECS), Av. Arturo Prat 514, Valdivia, Casilla 1469, Chile
2 Instituto de Histología y Patología, Facultad de Medicina, Universidad Austral de Chile, Valdivia, Chile
3 University of Rochester Medical Center, Rochester, 601 Elmwood Ave., NY 14627, USA
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Fig. 1. ClC-2 distribution in mouse duodenum and colon. (a-c) Immunoperoxidase labeling for ClC-2 in distal colon of wild-type mouse tissue; arrows in c indicate surface epithelial cell-to-cell junction. (d-f) similar tissue sections taken from a ClC-2 KO mouse. (g,h) Sections from duodenum villi from wild type and ClC-2 KO mice. Bars: a and d, 100 µm; b and e, 25 µm; c, f, g and h, 10 µm.
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Fig. 2. Comparison of ClC-2 and H+-K+-ATPase distribution in mouse colon. (a,c) Consecutive sections stained with anti-ClC-2 and anti-H+-K+-ATPase respectively. (b,d) Higher magnifications of the portions identified by the boxes in a and c are shown. Arrows in b indicate surface epithelial cell-to-cell junctions and the bottom of a surface cell. Bars: a and c, 25 µm; b and d, 10 µm.
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Fig. 3. Comparison of ClC-2 and NKCC1 distribution in mouse duodenum and colon. (a-c) Double immunostaining for ClC-2 (reddish) and NKCC1 (brown) in distal colon of wild-type mouse tissue. (b,c) Show higher magnification views of surface and crypt epithelium respectively. (d-f) Similar immunostaining obtained using mouse duodenum. (e,f) Higher magnification views of villus and crypt region, respectively. Arrows in e indicate villus epithelial cell-to-cell junctions. Bars, 20 µm except for d, 30 µm.
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Fig. 4. Sorting of ClC-2 Cl– channel in MDCK cells and Caco-2 cells. MDCK cells (a and b) were grown to confluence upon glass coverslips (a) or Transwell filters (b), transfected with ClC-2-GFP and observed after 24 hours. Specimens were analyzed by confocal microscopy. Representative X-Y and X-Z sections are shown. (c) Same MDCK monolayer as in (a), but stained with an anti-ZO-1 antibody. (d) MDCK monolayer grown on glass coverslip and transfected with c-myc-tagged hSlo K+ revealed with anti-c-myc antibody. (e,f) Experiments as in (a) and (b) but performed upon monolayers of Caco-2 cells. Arrows mark the filter position in (b) and (f). Bar: 10 µm, applies to all panels.
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Fig. 5. Sorting of ClC-2 Cl– channel in LLC-PK1 cells. Cells stably transfected with either the µ1A (LLC-PK1-µ1A, a and c) or the µ1B (LLC-PK1-µ1B, b and d) subunit of the AP-1 clathrin adaptor complex cells were grown to confluence upon class coverslips (a and b) or Transwell filters (c and d). Monolayers were transfected with ClC-2-GFP and observed after 24 hours. Specimens were analyzed by confocal microscopy. Representative X-Y and X-Z sections are shown. Arrows mark the filter position in (c) and (d). Bar: 10 µm, applies to all panels.
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Fig. 7. Sorting and functional assay of L812AL813A-ClC-2-HA mutant Cl– channel. (A) MDCK cells were grown to confluence upon class coverslips, transfected with ClC-2-HA (MDCK::ClC-2) or L812AL813A-ClC-2-HA. Cells were observed 24 hours post-transfection after staining with an anti-HA antibody. Specimens were analyzed by confocal microscopy. Representative X-Y and X-Z sections are shown. Calibration bar: 10 µm, applies to all panels. (B) Representative current traces in a HEK-293 cell transfected with L812AL813A-ClC-2. VH was 0 mV test pulses ranged from –170 to 0 mV as indicated. These were followed by a pulse to 30 mV. The duration of the main pulses was increased at more positive voltages in order to approximate full activation of the conductance. For illustration proposes, the beginning of the tail currents at 30 mV were set at the same time. (C) Apparent conductance calculated by taking the current at the beginning of the pulses at 30 mV given after various conditioning prepulses to the voltages in the abscissa. Results are means±s.e.m. of four separate experiments. Triangles, data from L812AL813A-ClC-2-HA-transfected cells. Circles, data for WT ClC-2, taken from Niemeyer et al. (Niemeyer et al., 2004 ).
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© The Company of Biologists Ltd 2005
300 aa) C-terminus of ClC-2. Portions labeled CBS are cystathionine beta synthase domains highly conserved in eukaryotic ClCs. The amino acids shown are potential tyrosine- or di-leucine-containing basolateral sorting signals.