Caveolin-1 modulates the activity of the volume-regulated chloride channel - PubMed (original) (raw)

Caveolin-1 modulates the activity of the volume-regulated chloride channel

D Trouet et al. J Physiol. 1999.

Abstract

1. Caveolae are small invaginations of the plasma membrane that have recently been implicated in signal transduction. In the present study, we have investigated whether caveolins, the principal protein of caveolae, also modulate volume-regulated anion channels (VRACs). 2. ICl,swell, the cell swelling-induced chloride current through VRACs, was studied in three caveolin-1-deficient cell lines: Caco-2, MCF-7 and T47D. 3. Electrophysiological measurements showed that ICl, swell was very small in these cells and that transient expression of caveolin-1 restored ICl,swell. The caveolin-1 effect was isoform specific: caveolin-1beta but not caveolin-1alpha upregulated VRACs. This correlated with a different subcellular distribution of caveolin-1alpha (perinuclear location) from caveolin-1beta (perinuclear and peripheral). 4. To explain the modulation of ICl, swell by caveolin-1 we propose that caveolin increases the availability of VRACs in the plasma membrane or, alternatively, that it plays a crucial role in the signal transduction cascade of VRACs.

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Figures

Figure 1

Figure 1. Caveolin-1 is not expressed in Caco-2, MCF-7 and T47D cells

Fifty micrograms of Caco-2, MCF-7 and T47D cell lysates were separated on SDS-PAGE (12.5 % acrylamide), electroblotted and stained with a monoclonal antibody against caveolin-1. Human fibroblasts (left lane) and calf pulmonary artery endothelial cells (CPAE) were used as positive controls. The migration pattern of protein molecular mass standards (expressed in kDa) is shown on the right.

Figure 2

Figure 2. Transfection of caveolin-1 in Caco-2 cells restores _I_Cl,swell

Membrane currents before, during and after 25 % hypotonic stimulation (HTS) were recorded in a non-transfected Caco-2 cell (A-C) and in a Caco-2 cell transfected with caveolin-1 (D-F). A and D, time course of the current at +50 mV (upper trace) and at −100 mV (lower trace). During hypotonic stimulation (HTS), control cells developed only a small current with time (A), whereas in transfected cells a pronounced increase in membrane current is observed (D). The HTS-induced changes are reversible upon returning to isotonic conditions and completely disappear upon perfusion with a hypertonic solution (mann). B and E, I-V curves taken at the times marked by the filled symbols in A and D. The current-voltage relation of the membrane current at time zero in a typical control cell (a) and in a transfected cell (e) is compared to the respective I-V curves recorded during the plateau phase of the HTS-activated membrane current in the same control cell (b) and transfected cell (f). After switching back to isotonic conditions, the current returned to nearly control level (c and g). After application of a hypertonic mannitol solution (mann) the current reached its basal level (d and h). C and F, current traces during voltage steps applied at the time indicated by the asterisk in A and D. Note the different current scales. Voltage protocol: holding potential at −50 mV, steps between −100 and +100 mV, increment +20 mV.

Figure 3

Figure 3. Effect of caveolin-1 isoforms on _I_Cl,swell in caveolin-1-deficient cell lines

Caco-2, MCF-7 and T47D cells, either control (con) or transfected with caveolin-1, −1α or −1β as indicated, were subjected to a 25 % HTS and _I_Cl,swell was measured. Difference currents (maximal HTS-triggered current minus basal current in isotonic medium) are plotted for the different conditions either at +50 mV (Caco-2) or at +80 mV (MCF-7 and T47D). The boxes correspond to interquartile ranges (25-75 percentile; middle line is median) with error bars representing the 5th (lower) and 95th percentile (upper). Small open square denotes the mean. Asterisks above and below boxes represent the 0th and 100th percentile. The differences between transfected and control conditions were statistically significant.

Figure 4

Figure 4. Immunofluorescence of Caco-2 cells transfected with caveolin-1 isoforms

Caco-2 cells were transfected with the bicistronic GFP expression vector containing caveolin-1 (A and B), −1α (C and D) or −1β (E and F). The GFP fluorescent signal (B, D and F) was used to identify transfected cells and to define the cell border (white superimposed line in GFP panels). Caveolin-1 isoforms were visualised using a non-discriminating monoclonal antibody (A, C and E) and phycoerythrin-conjugated secondary antibodies. Images representative for the different conditions indicate that caveolin-1, −1α and −1β are expressed. In addition, the subcellular distribution of caveolin-1α seems to be restricted to the perinuclear region, whereas the caveolin-1 and caveolin-1β signals reach the cell periphery. Scale bar corresponds to 10 μm in all panels.

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