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. 1999 Feb 15;515(Pt 1):75–85. doi: 10.1111/j.1469-7793.1999.075ad.x

Inhibition of volume-regulated anion channels by expression of the cystic fibrosis transmembrane conductance regulator

Rudi Vennekens *, Dominique Trouet *, Anne Vankeerberghen *, Thomas Voets *, Harry Cuppens *, Jan Eggermont *, Jean-Jacques Cassiman *, Guy Droogmans *, Bernd Nilius *
PMCID: PMC2269134  PMID: 9925879

Abstract

  1. To investigate whether the cystic fibrosis transmembrane conductance regulator (CFTR) interacts with volume regulated anion channels (VRACs), we measured the volume-activated chloride current (ICl,swell) using the whole-cell patch-clamp technique in calf pulmonary artery endothelial (CPAE) cells and in COS cells transiently transfected with wild-type (WT) CFTR and the deletion mutant ΔF508 CFTR.

  2. ICl,swell was significantly reduced in CPAE cells expressing WT CFTR to 66.5 ± 8.8% (n = 13; mean ±s.e.m.) of the control value (n = 11). This reduction was independent of activation of the CFTR channel.

  3. Expression of ΔF508 CFTR resulted in two groups of CPAE cells. In the first group IBMX and forskolin could activate a Cl− current. In these cells ICl,swell was reduced to 52.7 ± 18.8% (n = 5) of the control value (n = 21). In the second group IBMX and forskolin could not activate a current. The amplitude of ICl,swell in these cells was not significantly different from the control value (112.4 ± 13.7%, n = 11; 21 control cells).

  4. Using the same method we showed that expression of WT CFTR in COS cells reduced ICl,swell to 62.1 ± 11.9% (n = 14) of the control value (n = 12) without any changes in the kinetics of the current. Non-stationary noise analysis suggested that there is no significant difference in the single channel conductance of VRAC between CFTR expressing and non-expressing COS cells.

  5. We conclude that expression of WT CFTR down-regulates ICl,swell in CPAE and COS cells, suggesting an interaction between CFTR and VRAC independent of activation of CFTR.

Many cell types react upon cell swelling by activating a current, ICl,swell, through volume-regulated anion channels (VRACs). This current is involved in many processes such as cell volume regulation, transport of osmolytes, regulation of membrane potential and possibly also cell cycle regulation (for a review see Strange, 1996; Okada, 1997; Nilius et al. 1997). The electrophysiological and pharmacological properties of VRACs have been extensively described in endothelial cells and include a permeability sequence I− > Br− > Cl− >> gluconate, outward rectification, inactivation at strong positive potentials, and block by low micromolar concentrations of tamoxifen and mibefradil (Nilius et al. 1994, 1996, 1997).

The cystic fibrosis transmembrane conductance regulator (CFTR) is a non-rectifying, 8-10 pS, PKA-activated chloride channel (Cliff et al. 1992) with a halide-permeability sequence Br− > Cl− > I− > F−. Mutations in the gene for this channel cause cystic fibrosis, a disease characterized by severe lung dysfunction, pancreatic insufficiency and high salt concentrations in sweat (Boat & Cheng, 1989), due to impaired fluid secretion and reabsorption in epithelial tissues. The channel has a specific structure, with two nucleotide-binding folds, two membrane domains (each consisting of six transmembrane α-helices), and a regulatory domain. Structurally CFTR belongs to the family of ATP-binding cassette (ABC) proteins (Hyde et al. 1990). Besides its function as a Cl− channel, CFTR has been shown to regulate other ion channels including the amiloride-sensitive epithelial sodium channel (ENaC) (Grubb et al. 1994; Stutts et al. 1995; Ismailov et al. 1996), the outwardly rectifying Cl− channel (ORCC) (Jovov et al. 1995a,b; Schwiebert et al. 1995) and KATP channels like ROMK2 (McNicholas et al. 1996, 1997) and Kir6.1 (Ishida-Takahashi et al. 1998). In this work we investigated whether VRACs are regulated by CFTR. Previous observations already pointed indirectly to such a role for CFTR. Activation of ICl,swell was inhibited in T84 cells loaded with anti-CFTR505-511 antibodies (Chan et al. 1992). Moreover, cells isolated from CFTR-/- mice were unable to regulate their cell volume (Valverde et al. 1995). Finally, the regulatory volume decrease in cardiac myocytes only occurs under conditions of elevated cAMP level, e.g. during β-adrenergic stimulation which stimulates CFTR (Wang et al. 1997). In the present experiments we used a more direct approach to test this hypothesis, i.e. we expressed CFTR in cell lines that lack endogenous CFTR, namely CPAE cells and COS cells, and examined whether CFTR affected ICl,swell in these cells. We show that mere expression of CFTR significantly reduces the amplitude of ICl,swell in these cells.

METHODS

Vector construction

We used the pCINeo/IRES-GFP plasmid (Trouet et al. 1997) for expressing WT CFTR in CPAE and COS cells. For insertion of WT CFTR the GFP vector was cut with EcoRI, dephosphorylated and thereupon blunt-ended with T4 DNA polymerase. The WT CFTR cDNA was obtained from a pcDNA-CFTR plasmid through SacI digestion. The fragment obtained was blunt-ended using T4 DNA polymerase. Ligation was performed using standard procedures. ΔF508 CFTR was isolated from the pcDNA3 vector through digestion with BstXI and XbaI. The GFP vector was digested with XbaI and partially with BstXI. Sticky-end ligation was performed using standard techniques.

Cell culture and transfection

Cultured bovine pulmonary artery endothelial cells (cell line CPAE, American Type Culture Collection, CCL 209) were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10 % human serum, 2 mmol l−1 L-glutamine, 2 U ml−1 penicillin and 2 mg ml−1 streptomycin. Cell cultures were maintained at 37°C in a fully humidified atmosphere of 10 % CO2 in air. They were then detached by exposure to 0.05 % trypsin in a Ca2+- and Mg2+-free solution, reseeded on gelatin-coated coverslips, and kept in culture for 2-4 days before use. Only non-confluent cells were used. The fibroblast-like cell line COS was grown in DMEM containing 10 % fetal calf serum at 37°C in a fully humidified atmosphere of 5 % CO2 in air.

CPAE and COS cells were transiently transfected with WT CFTR or ΔF508 CFTR in the pCINeo/IRES-GFP vector (Trouet et al. 1997) using the same method as described previously (Kamouchi et al. 1997). In short, 150000 cells were incubated with a transfection cocktail containing 3 μg DNA and 12 μl polycationic SuperFect Transfection Reagent (Qiagen, Hilden, Germany). Cells were then transferred to gelatin-coated coverslips 24 h after transfection and electrophysiological measurements were done during 2-4 days after transfection. Incorporation of WT or ΔF508 CFTR in the bicistronic unit allows coupled expression of the channels and GFP. Transfected cells, positive for GFP, could be identified in the patch-clamp set up. GFP was excited at a wavelength between 450 and 490 nm and the emitted light was passed through a 520 nm long pass filter.

Solutions

The standard extracellular solution was a Krebs solution, containing (mM): 150 NaCl, 6 KCl, 1 MgCl2, 1.5 CaCl2, 10 glucose, 10 Hepes; titrated with NaOH to pH 7.4. The osmolarity of this solution, as measured with a vapour pressure osmometer (Wescor 5500, Schlag, Gladbach, Germany), was 320 ± 5 mosmol l−1. To activate ICl,swell the cells were perfused with a hypotonic (25 %, 240 ± 5 mosmol l−1) solution. These solutions contained (mM): 105 NaCl, 6 CsCl, 1 MgCl2, 1.5 CaCl2, 10 glucose, 10 Hepes; titrated with NaOH to pH 7.4. This solution was made isotonic (320 ± 5 mosmol l−1) by addition of 70 mM mannitol. The CFTR channel was activated by a cocktail containing 100 μM IBMX (3-isobutyl-1-methylxanthine) and 1 μM forskolin (both from Sigma-Aldrich Chemie) dissolved in isotonic solution. In experiments where ICl,swell was activated on top of CFTR current, we used 70 μM IBMX and 0.7 μM forskolin to avoid the current going out of range. The normal pipette solution contained (mM): 40 CsCl, 100 caesium aspartate, 1 MgCl2, 5 EGTA, 1.9 CaCl2 (free Ca2+ = 100 nM), 4 Na2ATP, 10 Hepes; titrated to pH 7.2 with KOH. This solution is slightly hypotonic (290 ± 5 mosmol l−1) in comparison with the Krebs solution to prevent spontaneous activation of volume-sensitive Cl− currents. Non-symmetrical Cl− concentrations were used in order to discriminate between Cl− currents and background non-selective cation currents (Voets et al. 1996).

Patch-clamp technique

Coverslips containing the seeded cells were placed in a recording chamber mounted on the stage of an Axiovert10 inverted microscope (Zeiss). Rapid solution exchange and extracellular application of drugs occurred via a multi-barrelled pipette connected to solution reservoirs, and was controlled by a set of magnetic valves. Patch electrodes were pulled from Vitrex capillary tubes (Modulohm, Herlev, Denmark) on a DMZ-Universal puller (Zeitz-instruments, Augsburg, Germany). When filled with pipette solution they had a DC resistance between 2 and 5 MΩ. An Ag-AgCl wire was used as reference electrode. Ag-AgCl electrodes of sintered pellets (IVM Systems, Healdsburg, CA, USA) were used to avoid contamination of the bath and pipette solutions. Membrane currents were recorded using an EPC-7 patch-clamp amplifier (List Electronic) and filtered with an eight-pole Bessel filter (Kemo, Bekenham, UK). For control of voltage-clamp protocols and data acquisition, we used the pCLAMP 6 software (Axon Instruments) run on an IBM-compatible PC which was connected to the amplifier via a TL-1 DMA interface (Axon Instruments). Membrane currents were measured in the whole-cell mode of the patch-clamp technique (Hamill et al. 1981). The cell capacitance and series resistance were assessed using the analog compensation circuit of the EPC-7 amplifier. Generally, between 50 and 80 % of the series resistance was electronically compensated to minimize voltage errors. A ramp protocol, consisting of a step to -80 mV for 0.6 s, followed by a step to -150 mV for 0.2 s and a 2.6 s linear voltage ramp to +100 mV, was applied every 15 s from a holding potential of 0 mV. In a similar protocol the initial step to -80 mV for 0.6 s was followed by a step to -100 mV for 0.2 s and a 2.6 s linear voltage ramp to +50 mV. Time courses of the whole-cell current were obtained by averaging the current at -80 mV during the first voltage step and at +50 mV. In all experiments, time zero corresponds to the rupture of the membrane. Current-voltage (I-V) relations were obtained from the currents measured during the linear voltage ramp. Other voltage protocols are mentioned in the legends of the figures. Currents were sampled at 1 or 2 ms intervals and filtered at 500 or 200 Hz. In order to estimate the single channel conductance (γ) of VRACs, non-stationary noise analysis was carried out on whole-cell currents during depolarization induced inactivation. Membrane potential was held at -80 mV. After attainment of stable swelling-induced current levels, channel inactivation was initiated by 1 s voltage steps to depolarizing membrane potentials (+80 to +120 mV). Traces were sampled at 1 kHz and filtered at 10 kHz. Mean current and current variance were calculated from 50 to 80 traces using Origin v. 5.0 (Microcal Software, Northhampton, MA, USA). For analysis, background current and variance were subtracted and the current variance- mean current plots were fitted with the following equation (Sigworth, 1985):

graphic file with name tjp0515-0075-m1.jpg (1)

where i is the single channel current amplitude and N the total number of active channels in the membrane. The single channel conductance was calculated from the current level at +100 mV obtained from noise analysis and the theoretical Cl− equilibrium potential (-23 mV). To quantify the inactivation of ICl,swell at positive potentials in transfected and non-transfected COS cells, the above mentioned traces were fitted to the sum of three exponentials using the WinASCD software package (G. Droogmans, Laboratory of Physiology, KU Leuven).

Experiments were performed at room temperature (20-25°C). Importantly, control (non-fluorescent) cells and transfected (green fluorescent) cells were analysed on the same coverslip and had undergone the same transfection procedure.

Data analysis

Electrophysiological data were analysed using the WinASCD software package. For statistical analysis and graphical presentation of the data we used Origin v. 5.0. For the statistical evaluation we have always compared currents measured at +50 mV in green fluorescent cells and non-fluorescent cells of the same culture. To account for variations in cell size, we have expressed the current amplitudes per unit membrane capacitance. In order to compensate for variations in measurements from day to day, we normalized the current values of the green fluorescent cells to the current values of control cells of the same day. These numbers were compared with the normalized current values of control cells. Pooled data are given as means ±s.e.m. from n cells. Significance was tested using Student's paired t tests using P < 0.05 as the level of significance.

Cell thickness measurements

Cells seeded on a coverslip were incubated for about 30 min with Red Neutravidin-labelled microspheres (F-8775, Molecular Probes; 5 μl beads in 1 ml normal Krebs solution). The microspheres were visualized using the XF40/E filter set (Omega Filter, Inc., Brattleboro, VT, USA) with a broad excitation band (maximal at 560 nm) and a 600 nm long pass emission filter. Cell height (Tc) taken as a measure for cell volume, was determined as the vertical distance between the beads on the gelatin coated surface of the coverslip and the beads on the cell surface, analogous to the method described in Van Driessche et al. (1993). To take into account the variability in cell height all data are expressed as a percentage of the cell height recorded before application of HTS. The time course of Tc is presented as the mean ±s.e.m. of several experiments.

RESULTS

Volume-activated currents in CPAE cells expressing WT CFTR

In a first approach we examined whether expression of WT CFTR would change the properties of ICl,swell in CPAE cells, which normally lack CFTR. The electrophysiological properties of the volume-activated current, inactivation at positive potentials and t½ for activation, did not differ significantly between transfected and non-transfected cells (not shown). However, the maximal current induced by application of 25 % HTS was significantly reduced in cells expressing WT CFTR (Fig 1 and Fig 2; for pooled data see also Fig. 9). At +50 mV the maximal current in transfected cells was on average 66.5 ± 8.8 % (n = 13) of that in control cells (76.5 ± 9.2 pA pF−1; n = 11). As a positive control for expression of WT CFTR in GFP-expressing (green) cells we applied, before or after activation of ICl,swell, IBMX and forskolin to activate the CFTR channel. This procedure had no effect in control cells. In order to exclude an effect of GFP, CPAE cells were transfected with an empty vector to express solely GFP. This had no significant effect on volume-activated current density (i.e. 44.5 ± 4.5 pA pF−1 (n = 9) in green cells compared with 44.2 ± 4.2 pA pF−1 (n = 10) in control cells at +50 mV).

Figure 1. Volume-activated current in CPAE cells.

Figure 1

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A, time course of activation of ICl,swell in a non-transfected CPAE cell, measured at +50 mV (○) and -80 mV (▵), during superfusion with 25 % hypotonic solution (HTS; horizontal bar). I-V relations were obtained at indicated time points. B, time course of activation of ICl,swell in a CPAE cell expressing WT CFTR. As a positive control for successful transfection, CFTR was activated with a cocktail of IBMX and forskolin (I/F, horizontal bar) dissolved in isotonic solution before or after application of HTS.

Figure 2. Representative current traces and I-V curves of WT CFTR and ICl,swell in transfected and control cells.

Figure 2

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A, current traces and I-V curve for ICl,swell in a non-transfected CPAE cell, after application of 25 % hypotonic solution (HTS). Step protocol consisted of 1 s voltage steps ranging from -100 to +120 mV from a holding potential of 0 mV. Arrows indicate zero current level. B, current traces and I-V curve for CFTR in a WT CFTR transfected CPAE cell, after application of 100 μM IBMX and 1 μM forskolin dissolved in isotonic solution. Step protocol analogous to A, except for a pre-step of 300 ms to 0 mV. C, current traces and I-V curve for ICl,swell in a WT CFTR transfected CPAE cell, after application of 25 % HTS. Step protocol analogous to A.

Figure 9. Overview of the results.

Figure 9

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Comparison of maximal volume-activated current in transfected and non-transfected cells, for the different experimental protocols described in the text. Columns represent means ±s.e.m. (n) of the current measured at +50 mV, expressed as a percentage of that in control cells. All columns refer to data obtained in CPAE cells except when mentioned otherwise. Grey columns represent control cells. *P < 0.05; n.s., non-significant difference.

In a different approach, applying IBMX and forskolin to the bath activated the WT CFTR channel (Fig. 3). When this current reached a maximum, 25 % HTS was added in the presence of IBMX and forskolin. As seen in Fig. 3 the WT CFTR current significantly runs down after about 2-3 min of stimulation. Under these conditions, ICl,swell in transfected cells reached on average a value of 67.4 ± 15.2 % (n = 14) of that in control cells (55.3 ± 5.1 pA pF−1; n = 18). Data are also shown in an overview (see Fig. 9).

Figure 3. Volume-activated current co-activated with WT CFTR.

Figure 3

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A, time course of activation of WT CFTR expressed in a CPAE cell, at +50 mV (○) and -80 mV (▵). Forskolin (0.7 μM) and (IBMX) 70 μM were applied (I/F, horizontal bar). B, time course of co-activation of WT CFTR and ICl,swell as in A. When the CFTR current reached it's maximum, 25 % hypotonic solution (HTS, horizontal bar) was applied. I-V relations were obtained at the time points marked with filled symbols, from a linear ramp protocol from -100 to +50 mV. Holding potential was 0 mV.

Volume-activated current in CPAE cells expressing ΔF508 CFTR

ΔF508, the deletion of a phenylalanine residue at position 508 in the NBF1, is the most widespread mutation causing severe cystic fibrosis. Due to this mutation the protein does not mature and is degraded in the endoplasmatic reticulum (for a review see Welsh & Ostedgaard, 1998). However, in some cases a small amount of the mutant protein is able to reach the membrane where it forms a functional channel (Haws et al. 1996). In our experiments, cells transiently transfected with ΔF508 CFTR could be subdivided into two groups (Fig. 4). Five out of 16 green cells showed a marked response upon application of IBMX and forskolin. In these cells the maximal volume-activated current amounted on average to 52.7 ± 18.8 % (n = 5) of the control value (55.14 ± 5.1 pA pF−1; n = 21). In the remaining 11 green cells IBMX and forskolin did not evoke a current. Moreover, in these cells the maximal volume-activated current was not significantly different from that in control cells (112.4 ± 13.7 %, n = 11; 21 control cells, see also Fig. 9).

Figure 4. Volume-activated current in a CPAE cell transfected with ΔF508 CFTR.

Figure 4

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A, time course of activation of ICl,swell at +50 (○) and -80 mV (▵) in a transfected cell that did not respond to IBMX and forskolin (I/F, horizontal bar). Twenty-five per cent hypotonic solution was applied (HTS, horizontal bar). I-V relations were obtained at the indicated time points. B, time course of activation of ICl,swell in a transfected cell that responds to application of IBMX and forskolin (horizontal bar). Twenty-five per cent HTS was applied before or after activation of ΔF508 CFTR (horizontal bar).

Cell thickness measurements of WT CFTR transfected and non-transfected cells

The obvious differences in VRACs in the absence or presence of CFTR could be due to a changed response of the cells to the hypotonic stimulus. To exclude this possibility, we measured the changes of cell height in response to changes in extracellular hypotonicity (Van Driessche et al. 1993). Data are presented in Fig. 5. The thickness of the non-stimulated transfected and non-transfected cells was not significantly different (CFTR cells: 2.8 ± 0.4 μm, n = 10; control cells: 2.1 ± 0.3 μm, n = 10). Also the changes in cell thickness in response to 25 % HTS were similar both in the absence and presence of CFTR activation (0.7 μM forskolin and 70 μM IBMX). In the absence of this cocktail the cell height increased on average by 21.4 ± 3.5 % (n = 5) in WT CFTR transfected cells and 26.1 ± 5.2 % (n = 5) in non-transfected in cells. The changes in cell height in response to a combined VRAC activation by 25 % HTS were, respectively, 28.3 ± 10.3 % (n = 4) and 29.6 ± 8.2 %%(n = 4). The seeming time-dependent effect of IBMX and forskolin on cell height in transfected and control cells was not significant. From Fig. 5 it is clear that CPAE cells do not show regulatory volume decrease (RVD). This is probably due to the lack of activation of K+ channels in response to swelling in this cell line (De Smet et al. 1994).

Figure 5. Measurement of cell height changes in WT CFTR transfected and non-transfected CPAE cells during a 25 % hypotonic stimulus.

Figure 5

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A, cell height measurement from WT CFTR transfected (n = 5) and non-transfected CPAE cells (n = 5) during application of 25 % hypotonic solution (HTS). B, cell height measurement from WT CFTR transfected (n = 4) and non-transfected CPAE cells (n = 4) during application of 25 % HTS plus CFTR activation cocktail (70 μM IBMX and 0.7 μM forskolin, I/F). ▵, non-transfected cells; ○, WT CFTR transfected cells. Values were normalized to the mean cell height in isotonic solution.

Volume-activated current in COS cells expressing WT CFTR

To confirm the data obtained in CPAE cells, we repeated the same experiment in COS cells, which like CPAE cells lack endogenous cAMP-activated currents. ICl,swell in these cells is similar to that in CPAE cells, except for its more pronounced inactivation at positive potentials (Fig 6 and Fig 7). To check whether the GFP-expressing cells indeed expressed functional CFTR, the channels were activated with IBMX and forskolin before or after the application of hypotonic solution (Fig. 6). We found that ICl,swell in COS cells expressing WT CFTR was significantly lower than in control cells. On average the maximum induced current in transfected cells was reduced to 62 ± 11.9 % (n = 14) of the control value (124.1 ± 14.7 pA pF−1; n = 12) (see also Fig. 9). To quantify kinetic properties of ICl,swell, inactivation was fitted to the sum of three exponentials. Time constants and relative amplitudes of inactivation were not significantly different between transfected and non-transfected COS cells. Mean time constants were as follows (ms): 7 ± 2.7, 267 ± 54.9 and 57.6 ± 6.2 for control cells (n = 4) and 5.4 ± 0.9, 169 ± 24.1 and 68.8 ± 7.6 for CFTR transfected cells (n = 5). Mean relative amplitudes of inactivation were as follows (%): 10.1 ± 4.1, 32.1 ± 2.8 and 45.3 ± 2.9 in control cells (n = 4) and 4.3 ± 1.9, 37.8 ± 13.7 and 48.2 ± 12.1 for transfected cells (n = 5). To evaluate a possible mechanism of CFTR action on VRACs, we have estimated the single channel conductance (γ) of the VRAC from non-stationary noise analysis in transfected and non-transfected cells (Fig. 8). It was shown previously that non-stationary noise analysis during depolarization-induced inactivation of ICl,swell gives a reliable estimate of γ (Jackson & Strange, 1995). From this analysis we obtained a value of 36.7 ± 11.4 pS (n = 6) in control cells and 29.9 ± 5.9 pS (n = 7) in CFTR expressing cells. These values are not significantly different.

Figure 6. Volume-activated current in a COS cells overexpressing WT CFTR.

Figure 6

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A, time course of the activation of ICl,swell at +50 mV (○) and -80 mV (▵) in a non-transfected COS cell, during a superfusion with 25 % hypotonic solution (HTS). I-V relations were obtained at the time points indicated with filled symbols. B, time course of activation of ICl,swell in a COS cell expressing CFTR, during superfusion with 25 % HTS. As a positive control for successful transfection, CFTR was activated with a cocktail of IBMX and forskolin dissolved in isotonic solution (I/F, horizontal bar) before or after application of HTS. I-V relations were obtained at time points indicated with filled symbols.

Figure 7. Representative current tracings and I-V curves of ICl,swell in transfected and control COS cells.

Figure 7

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A, current tracing and I-V curve for ICl,swell in a non-transfected COS cell, after application of 25 % hypotonic solution (HTS). Step protocol consisted of 1 s voltage steps ranging from -100 to +100 mV from a holding potential of -80 mV. Arrows indicate zero current level. B, current tracing and I-V curve for ICl,swell in a COS cell expressing WT CFTR, after application of 25 % HTS. Step protocol consisted of 1 s voltage steps ranging from -100 to +100 mV from a holding potential of-60 mV. Arrows indicate zero current level.

Figure 8. Non-stationary noise analysis of ICl,swell in CFTR expressing COS cells.

Figure 8

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Representative example of non-stationary noise analysis of VRAC in a CFTR expressing COS cell. When ICl,swell reached a stable maximal current value depolarizing steps to +100 mV were applied from a holding potential of -80 mV. Sixty subsequent traces were used for analysis. A and B show respectively the mean current and current variance as a function of time. C, the matching current variance-mean current plot. The single channel conductance of VRAC was derived by fitting this plot to eqn (1).

DISCUSSION

It is now well established that CFTR is a cAMP-activated chloride channel (Anderson & Welsh, 1991; Bear et al. 1992). In addition, its functional role as a channel regulator seems to be equally important. From our experiments it is obvious that expression of WT CFTR in CPAE cells and COS cells significantly reduces the amplitude of ICl,swell. This reduction is not dependent on the activation of the CFTR channel, and therefore suggests an interaction between the CFTR and VRAC proteins. Remarkably, in the human colonic adenocarcinoma cell line Caco-2, which endogenously expresses high levels of CFTR (Sood et al. 1992), ICl,swell has a very low current density (24.1 ± 3.5 pA pF−1, n = 12, at +50 mV; B. Nilius & J. Prenen, unpublished results) in comparison with CPAE (76.5 ± 9.2 pA pF−1, n = 11) and COS cells (103.9 ± 13.2 pA pF−1, n = 18) at the same potential. Furthermore, the current density of ICl,swell T84 cells, a CFTR expressing human colonic cell line (Cliff & Frizzell, 1990), is also low compared with CPAE and COS cells, i.e. 15.7 ± 7 pA pF−1 at +50 mV (n = 12, B. Nilius & J. Prenen, unpublished results). This suggests that CFTR- VRAC interaction is not exclusive for CPAE and COS cells. In this regard, however, it is necessary to consider that volume-activated channels in different tissues do not necessarily have the same molecular identity and a general occurrence of a CFTR-VRAC interaction can therefore not be anticipated. As summarized in the introduction, a possible interaction between CFTR and the VRAC was anticipated (Chan et al. 1992; Valverde et al. 1995; Wang et al. 1997). Moreover, several examples concerning interactions of CFTR with other ion channels are known, although the mechanism of these interactions remains unclear. A well-documented example is the outwardly rectifying chloride channel (ORCC) in epithelia. Bilayer experiments showed a diminished activation of ORCC in the absence of a functional CFTR protein (Jovov et al. 1995a,b) or when a mutant CFTR channel (G551D) was inserted. Another well-studied example is ENaC, the amiloride sensitive epithelial Na+ channel, which is down-regulated by CFTR. It was shown that Na+ reabsorption was abnormally elevated in CF epithelial tissues from humans and mice (Boucher et al. 1986; Grubb et al. 1994). Heterologous expression studies in MDCK epithelial cells and bilayer experiments showed that this was due to a loss of negative modulation of Na+ channels in CF epithelial cells (Stutts et al. 1995; Ismailov et al. 1996), exerted by CFTR. Basically four types of mechanisms for interaction are possible, as reviewed in Higgins (1995). (i) CFTR could interact directly through protein-protein interactions, or (ii) indirectly through an intermediate protein; (iii) CFTR could mediate the transport of a regulatory molecule; (iv) finally CFTR could influence the membrane insertion of the channel. In the case of CFTR-ORCC interaction a mechanism was proposed involving autocrine secretion of ATP mediated through CFTR which would interact with P2U-receptors that are coupled with ORCC (Schwiebert et al. 1995). In a recent paper the same group concluded from results obtained with several CFTR mutants that the regulatory and the Cl− conductance domain of CFTR can be separated. TMD1, and especially the predicted α-helices 5 and 6, would form the Cl− conductance domain, whereas the NBF1 and the R-domain would be essential for the ability of CFTR to interact with ORCC (Schwiebert et al. 1998). Interestingly it was previously shown that CFTR interacts with ENaC probably through protein-protein interactions between the NBF1 and the R-domain of CFTR and the α-subunit of ENaC (Kunzelmann et al. 1997), although it seems more complex in this case. A recent report shows that Cl− conductance of CFTR is very important for interaction with ENaC, since the interaction between the two channels could be abolished by poorly permeant anions like SCN− and gluconate or when CFTR was blocked with 1 mM DPC (Briel et al. 1998).

From our data, we cannot address the question of how CFTR interacts with VRACs in CPAE cells and COS cells. A change in the ability to respond to a hypotonic stimulus in transfected and control cells could be excluded. In addition, the fact that activation of the CFTR channel is not a prerequisite to down-regulate VRACs makes the necessity for transport of a regulatory molecule, like ATP, unlikely. This could point to a more direct interaction between CFTR and VRACs or the signalling cascade between cell swelling and channel activation. Moreover, the observation that ICl,swell is not influenced in the non-responding group of ΔF508 CFTR expressing cells suggests that the CFTR-VRAC interaction occurs at the plasma membrane level. The observation that transfection of cells with WT CFTR or ΔF508 CFTR results in the same reduction of ICl,swell might indicate that only a minute amount of CFTR protein is necessary for the interaction with VRACs. In order to down-regulate VRACs, CFTR can either affect the number of channels in the membrane (N), the single channel conductance (γ) of the VRAC or its open probability (Po). Through non-stationary noise analysis of ICl,swell we can probably rule out a change in the single channel conductance of the VRAC by expression of CFTR. This then leaves N and Po. From our data neither of these possibilities can be excluded, but it seems to us that a change in the amount of functional channels in the membrane is the most conceivable mechanism for CFTR-VRAC interaction. A change in the number of functional channels could result from an increase of the number of non-available channels in the membrane, a reduction of the total amount of recruitable channels or a decrease of the number of recruited channels during hypotonic challenge. However, since, the molecular identity of the VRAC is still not resolved (for an overview: Okada, 1997; Nilius et al. 1997), direct protein-protein interaction studies are not possible. Furthermore, the signal transduction cascade between cell swelling and channel activation is still elusive. To characterize further the interaction between CFTR and the VRAC we therefore have to await the molecular identification of the VRAC and the further elucidation of the signalling cascade activating VRACs in response to cell swelling.

Acknowledgments

We thank Drs M. Kamouchi, G. Buyse and A. Mamim for helpful discussions and J. Prenen, D. Hermans, A. Florizoone and M. Crabbe for expert technical assistance. A. V. was supported by Het Vlaams Instituut voor de bevordering van het Wetenschappelijk-Technologisch onderzoek in de industrie (IWT), H. C. by Het Onderzoeksfonds KULeuven. J. E. is a research associate of the Flemish Fund for Scientific Research (FWO-Vlaanderen). This work was supported by grants form the Federal Belgian and Flemish Government (N.F.W.O. G.0237.95, IUAP No. 3P4/23, and C.O.F./96/22-A0659), a grant from the Alphonse and Jean Forton - Koning Boudewijn Stichting and by the European Commission (concerted action BMH4-CT96-0602).

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