Osmosensitive Cl⁻ currents and their relevance to regulatory volume decrease in human intestinal T₈₄ cells: outwardly vs. inwardly rectifying currents

Abstract

1. The swelling-activated outwardly rectifying Cl⁻ current (I_Cl(swell)) recorded in T₈₄ human intestinal cells was completely blocked by 10 μm tamoxifen, while 300 μm Cd²⁺ had no effect.

2. A ClC-2-like, inwardly rectifying Cl⁻ current was activated after strong hyperpolarization in T₈₄ cells. This current was completely inhibited by 300 μm Cd²⁺, unaffected by 10 μm tamoxifen, and its magnitude increased slightly in response to cell swelling under hyposmotic conditions. However, the swelling-dependent modulation occurred only after prior activation by hyperpolarizing voltages.

3. T₈₄ cells behaved initially close to perfect osmometers in response to changes in external osmolalities between +20 and -30 %. The cells underwent full regulatory volume decrease (RVD) within 16 min when exposed to 30 or 10 % hyposmotic shocks.

4. Pharmacological tools were used to determine the anionic pathway(s) involved in RVD in T₈₄ cells. Tamoxifen (10 μm), 1,9-dideoxyforskolin (DDFSK; 100 μm) and 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS; 100 μm) blocked RVD while 300 μm Cd²⁺ had no effect upon RVD following a 30 % hyposmotic shock. The RVD response was similarly unaffected by Cd²⁺ when cells were exposed to a smaller (10 %) hyposmotic shock.

5. In conclusion, these data show that the anionic pathway primarily activated by cell swelling and relevant to RVD in T₈₄ cells is the tamoxifen-, DDFSK- and DIDS-sensitive I_Cl(swell) and not the hyperpolarization-activated, Cd²⁺-sensitive Cl⁻ current associated with the ClC-2 Cl⁻ channel.

In most cell types volume regulatory mechanisms involve the activation of ionic pathways in order to restore the original volume of the cells. In response to a hyposmotic external solution the cell will activate pathways which will result in the net efflux of K⁺ and Cl⁻ (Hoffmann et al. 1993). This volume regulatory mechanism is referred to as regulatory volume decrease (RVD). Although many studies have documented and characterized swelling-activated ionic pathways (for reviews see Hoffmann et al. 1993; Strange et al. 1996), very little is known about which pathway(s) is important in RVD. Furthermore, since the mechanism for sensing volume changes is also unknown it may be that different stimuli which result in cell swelling will be sensed by different mechanisms and hence activate different RVD pathways. It has been difficult to resolve these possibilities, not least because the molecular identity of many of the membrane channels and transporters involved is still unknown.

An outwardly rectifying swelling-activated Cl⁻ current has been well characterized in many cell types including the T₈₄ human adenocarcinoma cell line (Worrell et al. 1989; Solc & Wine, 1991; Valverde et al. 1993). This current has been termed volume-sensitive osmolyte and anionic current (VSOAC; Strange et al. 1996) or I_Cl(swell) (Sheppard & Valverde, 1997). These currents are typically characterized by a time-dependent decay at depolarizing voltages and the anion permeability sequence I⁻ > Br⁻ > Cl⁻ > F⁻ (Díaz et al. 1993). Pharmacological studies have found this current to be sensitive to 1,9-dideoxyforskolin (DDFSK) and 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) (Díaz et al. 1993), and tamoxifen (Valverde et al. 1993). It has been suggested that this current is important in RVD and, in a recent report by Valverde et al. (1996), it was demonstrated that the activity of this current can be correlated with the ability to effect RVD in Chinese hamster ovary (CHO) cells. However, no demonstration of the association of this current with RVD in epithelial cells has been presented as yet.

ClC-2, a member of the ClC family of voltage-gated Cl⁻ channels, has also been suggested to play a role in volume regulation (Gründer et al. 1992). ClC-2 currents are characterized by an inward rectification and a time-dependent activation at hyperpolarizing voltages. Pharmacological studies have shown ClC-2 currents to be partially blocked by diphenylamine-2-carboxyl acid and DIDS (Thiemann et al. 1992). Ubiquitous expression in all cell types tested implies that ClC-2 must play a role in a universal and necessary function for all cells (Thiemann et al. 1992). ClC-2 channels expressed in Xenopus oocytes generate inwardly rectifying Cl⁻-selective currents which have been shown to increase with large hyperpolarizing voltages as well as after exposure to hyposmotic shock (Gründer et al. 1992).

The human homologue hClC-2 has been identified and cloned from the human T₈₄ adenocarcinoma cell line (Cid et al. 1995), and a hyperpolarization-activated ClC-2-like current has been characterized (Fritsch & Edelman, 1996) in this cell line. This current showed the same biophysical characteristics and pharmacology as had been previously described for rat ClC-2 expressed in Xenopus oocytes (Thiemann et al. 1992). In addition, the currents were shown to be inhibited by Cd²⁺ (Fritsch & Edelman, 1996) and modulated by cell swelling (Fritsch & Edelman, 1997).

Although both I_Cl(swell) and ClC-2 currents can be activated or modulated, respectively, by cell swelling it is unclear whether they play a role in the mechanisms involved in RVD after hyposmotic shock in epithelial cells. Moreover, the fact that hyposmotic stress elicits Cl⁻ secretion in T₈₄ cells (McEwan et al. 1993) makes the channels involved in such responses good candidates for manipulation in those epithelia affected in cystic fibrosis. To determine the Cl⁻ conductance(s) involved in RVD after hyposmotic stimulation we monitored RVD in T₈₄ cells in the presence of selective blockers of I_Cl(swell) (tamoxifen, DDFSK) or ClC-2 currents (Cd²⁺).

METHODS

Cells and cell culture

The T₈₄ colonic carcinoma cell line was maintained in Dulbecco's modified Eagle's medium and Ham's F12 medium (1 : 1 mixture; Sigma) supplemented with 10 % fetal calf serum (Sigma), 50 μg ml⁻¹ gentamicin (Sigma) and 2 mm L-glutamine (Gibco). Cells were incubated at 37°C in an atmosphere of 95 % air-5 % CO₂. All experiments were performed at room temperature (15-25°C).

Solutions and chemicals

Cells were bathed in isotonic Hanks’ solution containing (mm): 140 NaCl, 2.5 KCl, 0.5 MgCl₂, 1.2 CaCl₂, 10 Hepes, 5 glucose; pH 7.2, 305 mosmol kg⁻¹. The osmolality of the 20 % hyperosmotic solution was increased to 365 mosmol kg⁻¹ with the addition of 30 mm NaCl while the 10 and 30 % hyposmotic solutions were reduced to 270 and 205 mosmol kg⁻¹ with the removal of 18 and 50 mm NaCl, respectively. Tamoxifen, DDFSK, DIDS and CdCl₂ were all purchased from Sigma.

Morphometric analysis

Volume measurements were performed as described previously (Park et al. 1994). Briefly, cells were observed with an inverted microscope (World Precision Instruments; total magnification, × 40). Cell images were digitized using a CCD Sony camera which was connected to a 7100AV Apple Macintosh computer. Individual cells were selected and images taken at various time intervals. Cell images were subsequently analysed with the aid of 1.58 NIH image analysis software package. The volume was calculated, assuming that each cell was a perfect sphere, and normalized to the zero time point before the solution change. Results are expressed as means ±s.e.m.

Electrophysiology

Swelling-activated and hyperpolarization-activated Cl⁻ currents were measured using the whole-cell recording mode of the patch-clamp technique as described previously (Valverde et al. 1996). The intracellular (pipette) solution contained (mm): 140 NMDGCl, 1.2 MgCl₂, 1.0 EGTA, 2 ATP, 10 Hepes; pH 7.4, 285 ± 5 mosmol kg⁻¹ (n= 4). Occasionally, ATP was omitted from the intracellular solution when hyperpolarization-activated Cl⁻ currents were observed (Fritsch & Edelman, 1996). The isosmotic bathing solution contained (mm): 140 NMDGCl, 0.5 MgCl₂, 1.3 CaCl₂, 10 Hepes (pH 7.4), 20 D-mannitol; 308 ± 4 mosmol kg⁻¹ (n= 3). The standard 20 % hyposmotic bathing solution contained (mm): 105 NMDGCl, 0.5 MgCl₂, 1.3 CaCl₂, 10 Hepes; pH 7.4, 220 ± 4 mosmol kg⁻¹ (n= 12).

The osmolalities of the solutions were adjusted using D-mannitol and measured by a Wescor 5100c vapour pressure osmometer.

RESULTS

I_Cl(swell) and ClC-2 currents in T₈₄ cells

Whole-cell currents recorded in T₈₄ cells under isosmotic conditions in which Cl⁻ was the main charge carrier were negligible (Fig. 1A). Upon exposure to a 20 % hyposmotic bathing solution a Cl⁻ current was activated (Fig. 1B). This current, termed I_Cl(swell), showed typical outward rectification and time-dependent decay at depolarizing voltages (Worrell et al. 1989; Solc & Wine, 1991; Valverde et al. 1993). Addition of 300 μm Cd²⁺ to the bath had no affect on I_Cl(swell) (Fig. 1C), which continued to increase in magnitude under the osmotic stress (n= 15). However, this current, as reported previously (Valverde et al. 1993; Zhang et al. 1994), was potently blocked by 10 μm tamoxifen (Fig. 1D). Similar results to those reported for tamoxifen were obtained with DDFSK, a specific blocker of I_Cl(swell) (Valverde et al. 1992), which does not inhibit ClC-2 current (results not shown; see also Fritsch & Edelman, 1997).

Figure 1. Cd²⁺ and tamoxifen sensitivity of I_Cl(swell) in T₈₄ cells.

A, Cl⁻ currents recorded under isosmotic (Iso) conditions. B, swelling-activated outwardly rectifying Cl⁻ currents appeared after exposure to a 20 % hyposmotic (Hypo) bathing solution. Currents were measured after 3 min in hyposmotic solution. C, addition of 300 μm CdCl₂ to the bath had no effect upon I_Cl(swell) (measured after 3 min in CdCl₂). However, addition of 10 μm tamoxifen (D) immediately blocked this current (measured 1 min after addition of tamoxifen). Current traces were generated by holding the potential at 0 mV and pulsing from -80 to +80 mV in 40 mV steps, each step lasting 500 ms.

Strong hyperpolarization activated a different type of Cl⁻ current in eight of nine T₈₄ cells (Fig. 2). This current demonstrated inward rectification and time-dependent activation at hyperpolarizing voltages (Fig. 2B), similar to that described for the ClC-2 channels expressed in Xenopus oocytes (Thiemann et al. 1992). The magnitude of the inwardly rectifying current, measured at -120 mV with ATP-free intracellular solution, before and after strong hyperpolarization was 6 ± 2.1 and 27 ± 7 pA pF⁻¹, respectively (n= 8). In the presence of intracellular ATP the inwardly rectifying current was increased from 4.1 ± 2 pA pF⁻¹ before strong hyperpolarization to 16.2 ± 4 pA pF⁻¹ (n= 3) after strong hyperpolarization. The difference in the magnitude is similar to that reported by Fritsch & Edelman (1996) and implies an ATP-mediated decrease in its magnitude. Also, as previously described in T₈₄ cells (Fritsch & Edelman, 1996), this current was rapidly and reversibly blocked by the addition of 300 μM Cd²⁺ to the bathing solution (Fig. 2C). The percentage inhibition of ClC-2-like currents by 300 μm Cd²⁺ was 88 ± 6 % (n= 4). The addition of 10 μm tamoxifen was without effect (n= 6; Fig. 2D). In order to check that the development of ClC-2-like currents was not due to spontaneous cell swelling under isosmotic conditions (Worrel et al. 1989), a series of experiments were carried out in the presence of a 45 mosmol kg⁻¹ difference between intracellular (280 mosmol kg⁻¹) and extracellular (325 mosmol kg⁻¹) solutions (Fig. 3). Under these conditions, ClC-2-like currents, which were absent in the first family of currents recorded immediately after breaking into the whole-cell configuration (Fig. 3A), were subsequently activated (Fig. 3B and C). The magnitude of the inward current, measured at -120 mV, increased from 3 ± 1.2 to 10 ± 2 pA pF⁻¹ (n= 3).

Figure 2. Cd²⁺ and tamoxifen sensitivity of ClC-2 currents in T₈₄ cells.

A, current traces generated by holding the cell at 0 mV and then pulsing from -100 to +40 mV in 20 mV steps (10 s duration) every 20 s. B, hyperpolarization-activated inwardly rectifying Cl⁻ currents activated after holding the cell for 30 s at -120 mV. C, addition of 300 μm CdCl₂ to the bathing solution caused an immediate block of this current, which could be reversed upon removal. Current measured within 3 min in CdCl₂. D, addition of 10 μm tamoxifen to the bath had no effect upon these currents (measured within 3 min in tamoxifen). Intracellular solution as described in Methods but without ATP.

Figure 3. Voltage-dependent activation of ClC-2 currents under mild hyperosmotic gradient.

A, currents recorded immediately after breaking into the whole-cell configuration. B and C, development of ClC-2 currents after repetitive hyperpolarization to -120 mV (every 35 s). The osmolality of the extracellular solution was raised to 325 mosmol kg⁻¹ by the addition of mannitol.

It has been reported that when ClC-2 is expressed in Xenopus oocytes the magnitude of the current can be increased following cell swelling (Gründer et al. 1992). To investigate whether hyposmotic swelling of T₈₄ cells also resulted in a increase in the magnitude of ClC-2 currents, we carried out experiments under conditions in which I_Cl(swell) could not be activated. For that purpose, ATP, an intracellular nucleotide required for the activation of I_Cl(swell) (Gill et al. 1992; Díaz et al. 1993), was removed from the pipette solution and 100 μm DDFSK was added to the extracellular solution. Under these conditions, immediately after breaking into whole-cell mode, negligibly low currents were recorded (Fig. 4A). ClC-2 currents were activated by the repetitive application (20 s interval) of 5 s voltage pulses to -120 mV (Fig. 4B). Maximal activation of ClC-2 current was obtained within 6 min (Fig. 4C). Exposure of the cell to a hyposmotic solution triggered an incresase in the inward current without affecting the outward current (Fig. 4D), which is consistent with an increase in the activity of ClC-2 channels. Similar small increases in the magnitude of ClC-2 currents following cell swelling have been described in T₈₄ cells (Fritsch & Edelman, 1997) and in pancreatic acinar cells (Carew & Thorn, 1996).

Figure 4. Osmosensitivity of ClC-2 currents in T₈₄ cells.

A, Cl⁻ currents recorded immediately after breaking into the whole-cell configuration in a T₈₄ cell. B, ClC-2 currents were activated by the repetitive application of voltage pulses to -120 mV every 20 s. C, maximal activation of ClC-2 current was obtained within 6 min. D, ClC-2 current measured at -80 mV increased upon exposure to hyposmotic conditions. E, ClC-2 currents in D shown on an expanded time scale covering the first 600 ms of the current. Experiments were carried out with ATP-free intracellular solution and bathing solution containing 100 μm DDFSK. Currents were measured in response to 5 s pulses to -80 and +40 mV.

However, in the absence of strong hyperpolarization (< 30 s at -120 mV or repetitive pulses of 5 s to -120 mV), ClC-2 currents could not be modulated by cell swelling. This is exemplified in Fig. 1D where it is shown that, with I_Cl(swell) inhibited by tamoxifen, the hyposmotic shock could not activate ClC-2 currents using short pulses from -80 to +80 mV (n= 15). For comparison with a situation in which ClC-2 current was activated by hyperpolarization and subsequently modulated by cell swelling, note the current pattern recorded at the same time and voltages in the cell shown in Fig. 4E.

Therefore, it appears that T₈₄ cells may be armed with two anionic pathways sensitive to cell swelling. In order to investigate the relative contribution of I_Cl(swell) and ClC-2 currents to the total swelling-activated anionic current, a protocol was designed which allowed the activation of ClC-2 under isosmotic conditions prior to exposure to hyposmotic conditions (Fig. 5A-C). A 4 s pulse to -120 mV followed by a 2 s ramp to +40 mV was applied repetitively every 10 s. This protocol activated ClC-2-type currents in all cells tested (n= 6). These currrents showed typical inwardly rectifying current-voltage curves (see ramp) and reached maximal activation within 6 min (Fig. 5C). Upon exposure to hyposmotic conditions the current at -120 mV increased markedly (Fig. 5D), consistent with the activation of either I_Cl(swell) or ClC-2. However, the current-voltage relationship changed from inwardly rectifying to outwardly rectifying (see ramp), suggesting a massive activation of I_Cl(swell). This was further confirmed by the use of tamoxifen, an inhibior of I_Cl(swell), which almost completely inhibited the hyposmotically induced current increase (Fig. 5E). This inhibition was also accompanied by the return of the current-voltage curve to an inwardly rectifying pattern (see ramp). Under these conditions, i.e. in the presence of hyposmotic solution and tamoxifen, there was a small increase in current, compared with the isosmotic condition, which could represent the increase in ClC-2 in response to cell swelling.

Figure 5. Relative contribution of I_Cl(swell) and ClC-2 currents to the total swelling-activated Cl⁻ current in T₈₄ cells.

ClC-2 currents were negligibly low immediately after breaking into the whole-cell configuration (A) but were activated by repetitive application of the voltage pulse shown (B and C). D, addition of a hyposmotic bathing solution. E, superfusion with a hyposmotic solution containing 5 μm tamoxifen.

Altogether, the electrophysiological experiments suggested that the increase in ClC-2 current as a consequence of cell swelling only takes place following the activation of ClC-2 current by hyperpolarization. However, they did not exclude the possibility that, in non-dialysed T₈₄ cells, ClC-2 current might be primarily activated by hyposmotic shocks, and hence play a role in RVD, without the need for prior activation at non-physiological voltages. In order to address this question the ability of T₈₄ cells to perform RVD was studied.

Cell volume regulation of T₈₄ cells

Phase-contrast images of a single T₈₄ cell are shown in Fig. 6. Images were taken under isotonic conditions (Fig. 6A) and after 1 min (Fig. 6B) and 16 min (Fig. 6C) in a 30 % hyposmotic solution. Superfusion of the cell with the hyposmotic solution resulted in a clear increase in cell size (Fig. 6B). After the initial swelling, the cell returned to its size in isosmotic medium (Fig. 6C). The passive initial osmometric behaviour of T₈₄ cells was tested by plotting the induced changes in V_T/V₀ (where V₀ is the volume of the cell measured at time 0 and V_T is the volume of the cell measured at time T) against the reciprocal of the relative osmotic pressure of the medium, π₀/π_T. The peak osmotic responses of T₈₄ cells to changes in the external osmolality from +20 to -30 % are shown in Fig. 6D. Most of the data points fell very close to the theoretical line for a perfect osmometer (dashed line). In fact, extrapolation of the best linear fit for the data yielded a Y-intercept value of 0.18, suggesting that only 18 % of the total cell volume was osmotically inactive.

Figure 6. Osmotic behaviour of T₈₄ cells in response to anisosmotic challenges.

Phase-contrast images of an individual T₈₄ cell in isosmotic solution (A), 1 min after exposure to a 30 % hyposmotic bathing solution (B) and after 16 min in hyposmotic solution (C). Scale bar, 6.5 μm. D, relationship between peak changes in the relative volume (V_T/V₀) of the cell and the reciprocal of the relative external osmotic pressure (π₀/π_T) is shown for the various osmotic solutions used (20 % hyperosmotic, n= 3; 10 % hyposmotic, n= 5; 30 % hyposmotic, n= 20). The dashed line represents the predicted behaviour of a perfect osmometer. E, relative changes in cell volume of individual T₈₄ cells were measured before and after replacement (at 0 min) of the isosmotic bathing solution with a 30 % hyposmotic bathing solution (n= 24). Values are means ±s.e.m.

As seen in Fig. 6A-C, after the initial swelling T₈₄ cells underwent RVD to regain their initial size despite the osmotic gradient. Figure 6E shows the calculated changes in cell volume over time following the exposure to 30 % hyposmotic solution. T₈₄ cells achieved full recovery over a period of 16 min.

Identification of the Cl⁻ current involved in RVD in T₈₄ cells

In order to determine whether either or both of these currents, I_Cl(swell) and ClC-2, play a role in the RVD process, we monitored cell volume changes in response to osmotic stress in T₈₄ cells and the effect upon RVD of specific blockers for each type of Cl⁻ current. Tamoxifen, at a concentration of 10 μM (Fig. 7A), DDFSK (100 μM; Fig. 7B) and DIDS (100 μM; Fig. 7C) were added to the bathing solution 3 min before exposure to a 30 % hyposmotic shock. All compounds, blockers of I_Cl(swell), were effective inhibitors of RVD in T₈₄ cells. In contrast, the presence of 300 μM Cd²⁺ in the bathing solution, which completely blocked ClC-2 current (Fig. 2), did not prevent a full RVD (Fig. 7D).

Figure 7. Effects of tamoxifen, DDFSK, DIDS and Cd²⁺ on RVD after a 30 % hyposmotic shock.

The indicated Cl⁻ channel blocker was added 3 min prior to the addition (at 0 min) of the hyposmotic bathing solution which also contained the appropriate blocker. A, 10 μm tamoxifen (n= 6); B, 100 μM DDFSK (n= 8); C, 100 μm DIDS (n= 4); and D, 300 μM CdCl₂ (n= 9). Values are means ±s.e.m. P < 0.05 at 6 and 16 min for tamoxifen, DDFSK and DIDS with respect to the control values plotted in Fig. 6E. All values in the presence of tamoxifen, DDFSK and DIDS were significantly different from the corresponding controls. For Cd²⁺, P= 0.3 and 0.35 at 6 and 16 min, respectively.

It has been postulated that different levels or types of stimuli which induce cell swelling may activate different RVD mechanisms. Of particular interest is the suggestion that ClC-2 Cl⁻ channels may be activated by small increases in cell volume, as opposed to the activation of I_Cl(swell) which would occur at higher osmotic shocks (Strange, 1994). Therefore, we investigated whether, in response to a smaller hyposmotic challenge (10 %), RVD in T₈₄ cells would become Cd²⁺ sensitive, suggesting the participation of ClC-2 channels in the regulatory process under low osmotic stress. However, Cd²⁺ did not affect the RVD mechanism even after a small 10 % hyposmotic shock (Fig. 8).

Figure 8. Effect of Cd²⁺ on RVD after a 10 % hyposmotic shock.

A, relative changes in cell volume measured before and after the replacement (at 0 min) of the isosmotic bathing solution with a 10 % hyposmotic solution (n= 5). B, cells were pretreated with 300 μm CdCl₂ before replacement with a hyposmotic solution also containing CdCl₂ (n= 3). Values are means ±s.e.m.

DISCUSSION

Osmosensitive Cl⁻ currents in T₈₄ cells

This study has addressed the identity of the anionic conductance mediating the RVD response in T₈₄ cells. One of the most common mechanisms triggered during cell RVD following osmotic stress is the parallel exit of K⁺ and Cl⁻ ions following the activation of Cl⁻ and/or K⁺ channels (Hoffmann et al. 1993). Typically, it is the activation of a Cl⁻ conductance which initiates the RVD response in epithelial cells. However, there are also reports of K⁺ channel activation as the primary regulatory mechanism triggered by cell swelling (Hazama & Okada, 1988). The latter mechanism implies that the Cl⁻ channel involved in the regulatory response may already be active under resting conditions in order to support the co-ordinated exit of K⁺ and Cl⁻.

T₈₄ cells possess both Cl⁻ currents which are active at rest (Valverde et al. 1994) and those which are activated upon exposure to hyposmotic challenge (Worrell et al. 1989; Valverde et al. 1993). Among the latter is the well-characterized outwardly rectifying swelling-activated Cl⁻ current, I_Cl(swell), which has been described in many cell types (for a review see Strange et al. 1996; Sheppard & Valverde, 1997). Despite the fact that this current is primarily activated by cell swelling, its close association with RVD has only been recently demonstrated for CHO cells (Valverde et al. 1996).

Among those currents which do not require cell swelling to be activated is ClC-2 (Thiemann et al. 1992). Interestingly, the observed increase in ClC-2 activity at physiological voltages following hyposmotic shock in Xenopus oocytes (Gründer et al. 1992) makes ClC-2 a good candidate as the anion exit pathway in a system which would rely on the activation of a K⁺ conductance for RVD. However, in all previous studies (Gründer et al. 1992; Carew & Thorn, 1996; Fritsch & Edelman, 1997) the osmosensitivity of ClC-2 was shown on cells which had already seen strong hyperpolarization, and consequently ClC-2 activation prior to their exposure to hyposmotic conditions. Therefore, one of our first aims was to test whether ClC-2 current could be activated by cell swelling at more physiological voltages. For that purpose we used a voltage protocol which does not activate ClC-2 (Fig. 1). Under these conditions and with I_Cl(swell) blocked by tamoxifen no ClC-2 currents were observed under hyposmotic conditions (Fig. 1D). However, once ClC-2 currents were activated by strong hyperpolarization, this current appeared to gain some osmosensitivity (Figs 4 and 5). These results suggest, first, that ClC-2 is a voltage-activated channel that can be modulated by cell swelling. Second, that under conditions in which the cells are dialysed with solutions containing ATP and pulsed to voltages close to the membrane potentials monitored in epithelial cells in response to hyposmotic shock (-80 mV for human 407 enterocytes (Hazama & Okada, 1988) and -60 mV for Necturus enterocytes (Giraldez et al. 1988)), the main Cl⁻ current activated by hyposmotic shock in T₈₄ cells is I_Cl(swell).

A further step in the investigation of whether ClC-2 current and/or I_Cl(swell) are relevant to RVD in T₈₄ cells was to test the sensitivity of both types of current to two Cl⁻ channel inhibitors, Cd²⁺ (Fritsch & Edelman, 1996) and tamoxifen (Valverde et al. 1993). Whole-cell ClC-2 currents were selectively blocked by Cd²⁺ and unaffected by tamoxifen and DDFSK (results not shown; see also Fritsch & Edelman, 1997). In contrast, I_Cl(swell) was blocked by tamoxifen and DDFSK (results not shown) but was insensitive to Cd²⁺. Once the pharmacological profile of these two currents was established this information was used to design experiments that could unveil the nature of the Cl⁻ channel(s) necessary for RVD in T₈₄ cells.

Identification of the Cl⁻ currents relevant to RVD in T₈₄ cells

T₈₄ cells decrease their volume when exposed to hyposmotic shock (Fig. 6), a process believed to be the result of the co-ordinated efflux of Cl⁻ and K⁺ through specific channels. The results reported in this study suggest that I_Cl(swell) would be the major conductance responsible for cell volume regulation in T₈₄ cells. On the other hand, a contribution of ClC-2 to RVD could not be clearly deduced from the patch-clamp data as it could be argued that under the conditions in which the cell was dialysed the modulation of the channel might have changed, i.e. masking its native swelling-dependent activation.

The participation of I_Cl(swell) and ClC-2 current in RVD in T₈₄ cells was investigated using their specific inhibitors in cell volume monitoring experiments. Cd²⁺ was used as a specific blocker of the ClC-2 current and tamoxifen and DDFSK for I_Cl(swell). The results showed that only cells pretreated with tamoxifen and DDFSK were unable to regulate their volume, whereas pretreatment with Cd²⁺ had no effect on RVD in these cells, suggesting that I_Cl(swell), but not ClC-2 current, is required to effect RVD in T₈₄ cells. DIDS, another effective blocker of I_Cl(swell) (Díaz et al. 1993), is unable to block the ClC-2 current (Thiemann et al. 1992; Fritsch & Edelman, 1996). In T₈₄ cells DIDS appeared to be a very effective inhibitor of RVD, which further supports the notion that I_Cl(swell), but not ClC-2 current, is needed for RVD.

There is growing evidence that cells may respond with different regulatory mechanisms to different osmotic shocks (Strange, 1994). In relation to this current view, it has been hypothesized that ClC-2 may be relevant to RVD in response to small volume increases. In order to check this hypothesis, RVD was monitored in response to a smaller 10 % hyposmotic challenge in the presence and absence of Cd²⁺. As was observed following the larger osmotic shock, the ClC-2 blocker Cd²⁺ did not interfere with the capacity of the cells to undergo RVD (Fig. 8).

ClC-2: physiological role revisited

The ClC-2 channel, a member of the ClC family of voltage-gated Cl⁻ channels (Jentsch et al. 1995), was first cloned from a rat cDNA library (Thiemann et al. 1992). When expressed in Xenopus oocytes this channel is electrophysiologically characterized by a hyperpolarization-dependent slow activation. Northern blot analysis has also indicated a wide organ distribution (Thiemann et al. 1992). Subsequent studies showed that ClC-2 channel activity can be increased as a consequence of cell swelling (Gründer et al. 1992). More recently, the human ClC-2 (hClC-2) has been cloned from T₈₄ cells and its wide distribution among human tissues also reported (Cid et al. 1995). Currents with similar characteristics, i.e. hyperpolarization-activated, slow activation at negative voltages, have been described in neurons (Chesnoy-Marchais, 1990) and parotid acinar cells (Arreola et al. 1996). Its wide distribution and swelling sensitivity has led to the proposal that ClC-2 may have a ‘house-keeping’ function contributing to cell volume regulation following hyposmotic stress. However, the results obtained in the present study suggest that cell volume regulation in T₈₄ cells is independent of ClC-2 channel activity.