Contribution of a time-dependent and hyperpolarization-activated chloride conductance to currents of resting and hypotonically shocked rat hepatocytes

Wen-Zhi Lan, Houria Abbas, Hung D. Lam, Anne-Marie Lemay, and Ceredwyn E. Hill

Gastrointestinal Diseases Research Unit, Hotel Dieu Hospital and Queen's University, Kingston, Ontario, Canada

Submitted 18 May 2004 ; accepted in final form 8 September 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatocellular Cl flux is integral to maintaining cell volume and electroneutrality in the face of the many transport and metabolic activities that describe the multifaceted functions of these cells. Although a significant volume-regulated Cl current (VRAC) has been well described in hepatocytes, the Cl channels underlying the large resting anion conductance have not been identified. We used a combination of electrophysiological and molecular approaches to describe potential candidates for this conductance. Anion currents in rat hepatocytes and WIF-B and HEK293T cells were measured under patch electrode-voltage clamp. With K+-free salts of Cl comprising the major ions externally and internally, hyperpolarizing steps between –40 and –140 mV activated a time-dependent inward current in hepatocytes. Steady-state activation was half-maximal at –63 mV and 28–38% of maximum at –30 to –45 mV, previously reported hepatocellular resting potentials. Gating was dependent on cytosolic Cl, shifting close to 58 mV/10-fold change in Cl concentration. Time-dependent inward Cl currents and a ClC-2-specific RT-PCR product were also observed in WIF-B cells but not HEK293T cells. All cell types exhibited typical VRAC in response to dialysis with hypertonic solutions. DIDS (0.1 mM) inhibited the hepatocellular VRAC but not the inward time-dependent current. Antibodies against the COOH terminus of ClC-2 reacted with a protein between 90 and 100 kDa in liver plasma membranes. The results demonstrate that rat hepatocytes express a time-dependent inward Cl channel that could provide a significant depolarizing influence in the hepatocyte.

volume-regulated chloride current; regulatory volume decrease; ClC-2; liver; chloride ion conductance


PASSIVE FLUX of Cl plays fundamental roles in the hepatocyte, including maintenance of cell volume in the face of nutrient uptake and bile formation and proliferative status (13, 22, 45). It is well established that hepatocytes have a relatively high resting permeability to Cl and that the anion is distributed across the plasma membrane at equilibrium with the membrane potential (1, 4, 11, 41). However, the identification of the channel protein(s) responsible has not been established. With either physiological gradients of Na+ and K+ or impermeant cations, and Cl as the major anion, patch-clamp studies of single rat hepatocytes or the rat hepatoma, HTC, exhibit time-independent currents in response to applied voltages between –120 and +100 mV (8, 22, 23, 32). The current-voltage relationship with Cl as the sole permeant ion demonstrated a weakly outwardly rectifying conductance (23). In the same cells, hypotonic solutions that cause cell swelling and activation of DIDS-sensitive regulatory volume decrease (RVD; see Ref. 13) activate a weakly outwardly rectifying Cl conductance that can also be stimulated by dialysis with cAMP (23), substrates of the canalicular organic anion transporter mrp2 (21), external ATP, or alanine (8, 22). Other than at large positive potentials (above +80 mV) where currents show inactivation, no other time-dependent properties are exhibited (19, 23). Combined with their sensitivity to voltage-dependent block by DIDS and similar compounds, the properties of these currents are identical to volume-regulated anion channels (VRAC) expressed in many other cell types (28).

Transcripts for members of the voltage-gated Cl channels (ClC-2, ClC-3, ClC-4, ClC-5, and ClC-7) have been identified in rat and mouse hepatocytes or liver (17, 30, 36, 38, 40). An NH2-terminal antibody identified multiple bands at ~90 kDa in HTC cell plasma membranes, and immunocytochemistry localized ClC-2 to the plasma membrane in these cells (30) although similar approaches have not been used in primary hepatocytes or intact liver. Conversely, ClC-3 appears to be primarily expressed in intracellular membranes in rat hepatocytes with some localization to the canalicular membrane (20, 34, 36). Heterologous expression of ClC-2 results in a DIDS-insensitive and time-dependent inward current at potentials more negative than –40 mV (29). The pharmacological and electrophysiological properties of ClC-2 support a role for this channel in mediating resting Cl conductance but not for a direct role in volume regulation. A recent report demonstrates that knockout of ClC-2 expression in the mouse removes the hyperpolarization-activated and time-dependent Cl current from salivary epithelial cells, although their ability to generate an RVD in response to hypotonic solutions is not affected (26). Thus there are a number of assays that allow the functional presence of ClC-2 to be isolated from VRAC and identified in native expression systems.

Here we attempted to define the molecular nature of the Cl channel(s) underlying the high resting Cl conductance in short-term cultured rat hepatocytes and hepatocyte couplets and to determine whether this same channel could be involved in the slowly developing swelling-activated current. Results of the molecular expression and electrophysiological studies reported herein suggest that a ClC-2-like time-dependent inward current makes a significant contribution to resting Cl currents. The amplitude of the time-dependent inward current was not affected by conditions inducing cell swelling, whether in the absence or presence of an inhibitor of the volume-activated Cl conductance, suggesting that this current does not contribute to RVD. Similar results were obtained in the polarized and bile-secreting rat hepatoma, WIF-B. Conversely, a third cell type, HEK293T, was capable of generating VRAC-like but not time-dependent inward currents in the absence of ClC-2 expression. The results suggest that rat hepatocytes functionally express at least two Cl channels, one of which is involved in mediating the large resting Cl conductance of these cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials. Male and female Sprague-Dawley rats (200–225 g) were obtained from Charles River (Montreal, QC) and maintained on a 12:12-h light-dark regime with access to water and rat chow ad libitum according to the Canadian Council on Animal Care. WIF-B cells were a gift from Dr. Ann Hubbard (Johns Hopkins University). Unless otherwise noted, chemicals were purchased from Sigma-Aldrich Chemicals (St. Louis, MO) or British Drug Houses (Toronto, ON) and were of the highest grade available. DIDS and FCS were supplied by Toronto Biochemicals (Toronto, ON) and GIBCO/Life Technologies (Grand Island, NY), respectively.

Cell culture. Rat hepatocytes and couplets were isolated using a collagenase (Liberase, 0.35 mg/ml; Roche Biochemicals, Montreal, QC) perfusion technique and plated at 1 x 105 cells/ml on glass coverslips in 35-mm petri dishes (15). Cells were incubated in DMEM containing 0.15% NaHCO3, 10 mM HEPES, 10% (vol/vol) FCS, 2 mM glutamine, 5 µg/ml insulin, 1 µM dexamethasone, and 100 U·100 µg–1·ml–1 penicillin-streptomycin, pH 7.4, in a humidified atmosphere of 95% air-5% CO2 at 37°C. Electrophysiological experiments were carried out between 12 and 48 h of culture.

WIF-B cells were maintained in Coon's F-12 medium with further addition of 0.15% NaHCO3, 10 µM hypoxanthine, 40 nM aminopterin, 1.6 µM thymidine, 200 U·50 µg–1·0.5 µg–1·ml–1 penicillin-streptomycin-amphotericin, and 5% FCS according to the protocols provided by Shanks et al. (35). After passaging every 7 days, single cell suspensions were plated on glass coverslips, as were the hepatocytes, and patch clamped within 48 h.

HEK293T cells were maintained in DMEM medium with further addition of 0.15% NaHCO3, 2 mM glutamine, 100 U·100 µg–1·ml–1 penicillin-streptomycin and 10% FCS. After passaging every 4 days, single cell suspensions were plated on glass coverslips, as were the hepatocytes, and patch clamped within 48 h.

Electrophysiology. Patch-clamp recordings were made in the whole cell configuration using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA) and Clampex 7 software, as described previously (16). Patch pipettes were made from borosilicate glass (no. G85165T-4; Warner Instrument, Hamden, CT) and had resistances between 2 and 4 M{Omega} when filled and immersed in Cl-containing solutions. Whole cell currents were digitized (Digidata 1200B) at 2 or 5 kHz. Cells were bathed in solutions at pH 7.4 containing (in mM) 5 glucose, 5 HEPES, 1 CaCl2, and 1 MgSO4 and, where noted, either 145 N-methyl-D-glucamine (NMDG) chloride, 140 sodium gluconate, and 5 potassium gluconate, 140 NaI, and 5 KCl, 140 NaCl and either 5 CsCl or 5 KCl. Pipettes were filled with solutions at pH 7.2 containing (in mM) 1 MgSO4, 1 EGTA, 5 HEPES and, where noted, either 140 NMDG-Cl, 140 sodium gluconate, 140 CsCl, or 130 sodium gluconate and 10 CsCl, in the absence or presence of 80 raffinose.

To monitor the development of swelling-activated currents, voltage ramps from +150 to –150 mV and 400-ms duration were applied 30 s after attaining the whole cell configuration, and one time each successive minute for 15 min. Time-dependent properties of resting and swelling-activated currents were measured by applying voltage steps from –150 or –140 mV and incremented by 15 or 20 mV. Before construction of current-voltage relationships, currents were corrected for junction potentials present in solutions in which Cl was not equivalent in pipette and bath. Current-voltage relationships are shown for instantaneous current and time-dependent inward current at either 0.2 or 2 s. The latter was calculated as the difference between the instantaneous current at the beginning of the voltage step and that at 0.2 or 2 s. Mean data were pooled after normalization of individual records to whole cell capacitance, determined through the amplifier circuitry in response to a 5-mV step from a holding potential of 0 mV, and expressed as mean picoampere/picofarad ± SE.

RT-PCR of ClC-2. mRNA was isolated (Oligotex suspension; Qiagen, Valencia, CA) from freshly prepared hepatocytes and from cultured WIF-B and HEK293T cells in midlogarithmic growth and reverse transcribed (Expand RT; Roche Biochemicals) according to the manufacturer's directions. Controls were conducted in the absence of enzyme. For each 20-µl PCR reaction, cDNA generated from 40 ng mRNA was amplified in the presence of 10 pmol forward (5'-GGAAGGGATGGAGCCTCGAG-3') and reverse (5'-CCCTGGACACTAGGAACTT-GT-3') primers for rat ClC-2 (NCBI accession no. NM 017137). PCR products were cloned into an A/U cloning vector (pDrive; Qiagen) and sequenced to confirm their identities.

Western blotting of ClC-2. Rat liver membranes were isolated on step gradients of sucrose after perfusion and homogenization in 1 mM NaHCO3 (36, 37). Membranes were cleaned of associated proteins by incubation (0.5 mg/ml) in 100 mM Na2CO3, pH 11.5, for 30 min at 0°C (9). The suspension was centrifuged at 21,000 g, 1 h, and 4°C. Membrane protein (30 µg) was solubilized in 10% SDS and 62.5 mM Tris, pH 6.8, and diluted to 2% SDS with Laemmli buffer, incubated at 50°C for 15 min, 20°C for 30 min, and finally 15 min at 50°C before SDS-PAGE in 10% acrylamide. Proteins were transferred to polyvinylidene difluoride membrane by semidry transfer. Membranes were blocked with 5% milk powder in Tris-buffered saline plus 0.25% Tween (TBST) overnight, followed by a 4-h incubation with a ClC-2 antibody raised in goats against a peptide corresponding to a 12–20 residue fragment in the final 100 amino acids of the COOH terminus of ClC-2 (C-20, 2 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA) in TBST. After four 15-min washes with TBST, membranes were incubated with alkaline phosphatase-conjugated anti-goat IgG for 60 min and visualized with NBT/BCIP. RT-PCR and Western blotting were performed five times with different preparations, and representative results are shown.

Statistical measurements. Where applicable, data were pooled, and means ± SE are shown with the number of samples, n, stated in the legends for Figs. 16. Statistical differences were tested using the Student's t-test for independent samples with Levene's test for equality of variances. Differences of P < 0.05 were considered significant.



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Fig. 1. Resting and volume-activated Cl currents in rat hepatocytes. A: representative original current traces from 2 different rat hepatocytes dialyzed with 140 CsCl in the absence (control) or presence (raffinose) of 80 mM raffinose and voltage clamped from –140 to +120 mV in 20-mV increments (holding potential was 0 mV, final step was to –100 mV, 10 s elapsed between each clamp step). Under control conditions, voltage steps between –140 and –40 mV slowly activated an inward current. With raffinose in the pipette, an outwardly rectifying current that inactivated at potentials above +80 mV superimposed these currents. B: mean ± SE instantaneous (left) and time-dependent (right) currents under control ({circ}, n = 12 cells) and raffinose-dialyzed ({bullet}, n = 14) conditions and normalized to cell capacitance. Instantaneous currents were measured following the capacitance transient at the onset of the time-dependent activation of inward current at –140 mV and normalized to cell capacitance; raffinose-treated cells are significantly different from control. Time-dependent current was the difference between current at 2 s and the instantaneous current. *Significantly different from control.

 


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Fig. 6. Resting and volume-activated Cl currents in WIF-B and HEK293T cells. A and B: representative current traces from a WIF-B and an HEK293T cell dialyzed with 140 CsCl and voltage clamped from –140 to +120 mV in 20-mV increments in the absence (top) and presence (bottom) of 80 mM raffinose in the dialysate (the holding potential was 0 mV, final step was to –100 mV, 10 s elapsed between each clamp step). Under control conditions, voltage steps between –140 and –40 mV slowly activated an inward current in WIF-B but not in HEK293T cells. With raffinose in the pipette, these currents were superimposed on an outwardly rectifying current that inactivated at potentials above +80 mV. C and D: WIF-B (C) and HEK293T (D) mean ± SE instantaneous (top) and time-dependent (bottom) currents under control ({circ}, n = 4) and raffinose-dialyzed ({bullet}, n = 6) conditions were measured, respectively, after the capacitance transient or as the difference between the instantaneous current and current at 2 s and normalized to cell capacitance.

 

    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Time-dependent, hyperpolarization-activated Cl currents in resting and hypotonically stressed hepatocytes. A complete description of the Cl currents present in resting rat hepatocytes has not yet been reported, even though this anion is a major contributor to the resting conductance of these cells. Figure 1A, left, shows that voltage clamp of short-term cultured rat hepatocytes bathed in NaCl and CsCl and dialyzed with CsCl evoked a slowly activating inward current between –40 and –140 mV. Outward currents elicited by voltages up to +140 mV were much smaller, demonstrating the inwardly rectifying nature of the resting Cl conductance. When the cells were dialyzed with a hypertonic raffinose solution, the well-characterized VRAC in these cells (23) developed over 10–15 min. VRAC was routinely measured in these studies to establish to what extent they contribute to the time-dependent inward currents. Currents resulting from voltage steps imposed at 10 min after attainment of the whole cell configuration are shown in Fig. 1A, right. Large outward and instantaneous currents were activated at all potentials positive to 0 mV. Currents slowly inactivated at potentials above +80 mV. Tail currents at –100 mV from these depolarizing potentials show time-dependent reactivation. The current-voltage relationship of the instantaneous currents under control conditions has a mean conductance of 68 ± 5 pS/pF (Fig. 1B, left). Dialysis with raffinose increased outward instantaneous currents (Fig. 1B, left) but did not significantly affect time-dependent inward currents (Fig. 1B, right). The contribution of the time-dependent currents to resting Cl conductance at the membrane potential of rat hepatocytes and intact liver reported previously (–30 to –45 mV; see Ref. 25) can be calculated from the normalized control currents (Fig. 1B) and the driving force [applied potential (–1.2 mV), where –1.2 mV is the Nernst potential of Cl]. The mean conductance of the time-dependent current is 1.7 and 5 pS/pF at –37.5 and –45 mV, respectively, accounting for 2–7% of total Cl conductance under the conditions used herein. No detectable current is observed at –30 mV. We also measured currents in couplets of hepatocytes that had clearly visible canaliculi. These currents were not significantly different from single cells after normalizing to cell capacitance (data not shown), indicating that both single cultured cells and polarized hepatocytes express the same Cl currents.

To demonstrate that the inward, time-dependent currents represent ion flow through channels, we applied clamp protocols that would permit the illustration of deactivating, or tail, currents at voltages positive to the Cl equilibrium potential. Figure 2A shows representative current traces resulting from successive applied potentials of –140 to +60 mV followed by a depolarizing step to +120 mV. Slowly deactivating currents at +120 mV are largest from –140 mV and are reduced with more depolarized initial steps. The tail current amplitudes from three different cells were corrected for the time-independent current contribution, normalized to currents evoked from –140 or –150 mV, and plotted as functions of the level of the hyperpolarizing step (Fig. 2B). The data were fitted to a Boltzmann distribution. The resulting steady-state activation curve shows that the time-dependent current is half maximally activated at –63.1 ± 3.6 mV and, at the resting potential of hepatocytes (–30 to –45 mV), 28 to 38% of these channels are open.



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Fig. 2. Voltage-dependent activation of the time-dependent inward current. A: representative original current traces from a rat hepatocyte bathed in Na+ and Cs+ salts of Cl dialyzed with 140 CsCl and voltage clamped from –140 to +60 mV in 20-mV increments (holding potential was 0 mV, final step was to +120 mV, 10 s elapsed between each clamp step). Large, slowly deactivating tail currents decreased in amplitude as the size of the initial hyperpolarizing step was reduced. B: apparent open probability (Popen) of the time-dependent current from 3 cells voltage clamped as in A, or from –150 to +60 mV in 15-mV increments before stepping to +120 mV, calculated from the deactivating current amplitude normalized to current amplitude from an initial step of –140 or –150 mV. Data were fitted to a Boltzmann distribution having a half-activation potential of –63.1 ± 3.6 mV and slope factor of –36.9 ± 3.5 mV. The rectangle demarcates the range of resting potentials reported from rat hepatocytes and intact rat liver, as noted in text.

 
Cl selectivity of the time-dependent inward current in resting hepatocytes. To demonstrate the Cl dependence of the time-dependent inward current and characterize the halide selectivity of the resting hepatocyte anion conductance, current-voltage relationships were analyzed from cells in which we altered the Cl concentration in the bath and/or pipette solutions. Figure 3A shows representative traces from four cells dialyzed and bathed with Cl at 140 and 2 mM (A1), 140 and 7 mM (A2), 140 and 147 mM (A3), or 10 and 147 mM (A4). Where Cl was reduced, it was replaced by equimolar concentrations of either gluconate (A1, A3, and A4) or iodide (A2). Currents observed under these conditions reflected a similar selectivity between I and Cl, with a greatly reduced permeability to gluconate. Specifically, with gluconate as major anion in the bath (an outwardly directed Cl concentration gradient), instantaneous currents were inward below +75 mV (Fig. 3A1). However, with I as major bath anion, outward currents were similar to those observed with Cl (Fig. 3A2 vs. A3 and A4). With an inwardly directed Cl gradient, the outward rectification was still present, but inward time-dependent currents were only observable when the membrane potential was clamped to –165 mV or more negative (Fig. 3A4). The ragged records at large negative potentials are likely to represent increased channel activity rather than membrane destabilization, since both subsequent currents at less negative potentials and deactivating tail currents retain, respectively, their rectifying characteristics and time dependence. Mean current-voltage relationships for the instantaneous and time-dependent currents observed under these conditions are shown in Fig. 3B. The curvilinear instantaneous relationship (Fig. 3B, left), indicative of inward and outward rectification, is clearly visible when outward currents are carried by either Cl or I. However, outward currents were greatly reduced with gluconate replacement of most of the bath Cl. The mean reversal potential of the instantaneous current shifted from +11.2 to –17 and +67 mV with partial replacement of, respectively, bath Cl with I and gluconate corresponding to relative permeabilities of I-Cl-gluconate of 1.08:0.59:0.05. The time-dependent current demonstrates a higher selectivity for Cl, since voltage gating was dependent on the cytosolic concentration of the anion (Fig. 3B, right), confirming a recent report showing that decreases in intracellular Cl concentration cause a negative shift in activation of both heterologously expressed ClC-2 and ClC-2-like currents in colonocytes (3). In the hepatocyte, the activation threshold shifted from –50 to –130 mV, with a change in cytosolic Cl from 140 to 10 mM, reflecting a 58-mV change/10-fold increase in cytosolic Cl concentration. The combined results presented in Fig. 3 indicate that, whereas the instantaneous anion currents measured in resting hepatocytes are more permeable to I than Cl, gating of the time-dependent inward current is similar to ClC-2 in being dependent on the intracellular Cl concentration.



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Fig. 3. Cl selectivity of instantaneous and slowly activating inward currents in resting rat hepatocytes. A: representative current records of cells dialyzed with 140 mM CsCl (1–3) or 10 mM CsCl and 130 mM sodium gluconate (4) and bathed in 5 mM K+ and 140 mM Na+ salts of gluconate (1), 5 mM KCl and 140 mM NaI (2), or 5 mM K+ and 140 mM Na+ salts of Cl (3 and 4). Cells were voltage clamped from –150 to +135 mV (1–3) or –195 to +135 mV (4) in 15-mV increments [holding potential was 0 mV, final step was to –100 mV (1), +100 mV (2 and 3), or +60 mV (4), 10 s elapsed between each clamp step]. B: mean ± SE instantaneous (left) and time-dependent (right) current normalized to cell capacitance from rat hepatocytes dialyzed with and bathed in Cl-containing solutions of 10 and 147 mM ({blacktriangleup}, n = 3), 140 and 147 mM ({circ}, n = 14), 140 and 7 mM ({blacksquare}, n = 3), and 140 and 2 mM ({bullet}, n = 7). Voltages were corrected for liquid junction potentials. Instantaneous and time-dependent currents were obtained as described in the legend to Fig. 1, except that the current amplitude at 0.2 s rather than 2 s was used. Reversal potentials of instantaneous currents indicate a relative permeability sequence of I > Cl > gluconate. Gating of time-dependent currents is dependent on cytosolic Cl concentration.

 
DIDS inhibits outward volume-activated currents but not hyperpolarization-activated time-dependent inward currents in rat hepatocytes. To provide further support for the hypothesis that two different Cl channels are involved in resting and recovery from swelling, we measured currents in raffinose-dialyzed cells in the absence or presence of external DIDS. Whereas ClC-2 channels are insensitive to DIDS, the stilbene inhibits VRAC, providing an independent assessment of their functional expression (2). Figure 4A shows current records from two different cells dialyzed with raffinose to activate VRAC. These cells were dialyzed and bathed in Cl salts of the impermeant cation NMDG. Compared with permeant cation solutions, the resulting whole cell currents were not visibly different from those depicted in Fig. 1A, left. When 0.1 mM DIDS was added to the external medium, the peak amplitude of the outward current was greatly diminished, and the rate of inactivation was increased. In both examples, the tail currents resulting from stepping the membrane potential to –100 mV for the last 500 ms were similar in amplitude, suggesting that the recovery from inactivation and number of VRAC channels open at hyperpolarizing potentials were not affected by DIDS. We measured current amplitude at 2 s in the absence or presence of DIDS to determine its effect on the current-voltage relationships for the VRAC and the hyperpolarization-activated, time-dependent current. DIDS inhibited the outward current but did not affect either the inward raffinose-activated current or the time-dependent inward current (Fig. 4B).



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Fig. 4. DIDS inhibition of outward volume-activated Cl currents but not slowly activating inward currents. A: representative current records from 2 different rat hepatocytes bathed in 145 mM N-methyl-D-glucamine (NMDG) chloride in the absence (left) or presence (right) of 0.1 mM DIDS and dialyzed with 140 NMDG-Cl and 80 mM raffinose. Cells were voltage clamped from –140 to +120 mV in 20-mV increments (holding potential was 0 mV, final step was to –100 mV, 10 s elapsed between each clamp step). Under control conditions, raffinose activated currents similar to those in Fig. 1A. With DIDS in the bath, the peak amplitude of the outward currents was smaller, and these currents inactivated more rapidly. Note that tail currents upon return to –100 mV were not significantly different, confirming that DIDS inhibition of these channels is voltage dependent. B: mean ± SE current at 2 s in the absence ({bullet}, n = 9) and in the presence ({blacktriangleup}, n = 4) of DIDS normalized to cell capacitance (left); mean time-dependent current was the difference between current at 2 s and the peak current normalized to cell capacitance, as described in the legend for Fig. 1 (right). *Significantly different from control.

 
ClC-2 is expressed in the polarized, hepatocyte-derived WIF-B cells but not HEK293T cells. To provide corroborative independent evidence that the time-dependent inward Cl currents in hepatocytes result from ClC-2 expression, we looked for evidence for differential expression of the hyperpolarization-activated Cl channel, ClC-2, in a cell line that is closely related to primary rat hepatocytes (WIF-B) and in cells that express a VRAC-like Cl current but not ClC-2 (HEK293T; see Refs. 24 and 2931). We used rat hepatocyte mRNA as a positive control, since it is already established that ClC-2 mRNA is present in these cells (30). We measured, using RT-PCR, {beta}-actin and ClC-2 expression in mRNA from freshly isolated rat hepatocytes and cultured HEK293T and WIF-B cells. Figure 5A shows that, although all three preparations express actin, detectable ClC-2 transcript was observed in the hepatocyte and WIF-B but not in HEK293T cells. Cloning and sequencing of the fragments confirmed the identity of ClC-2 (data not shown). The 239-bp fragments from rat hepatocytes and WIF-B cells were identical to the sequence from rat brain (GenBank X64139; see Ref. 40). These results demonstrate that ClC-2 message is expressed in rat liver cells and the derived polarized cell line, WIF-B. Conversely, HEK293T cells do not express detectable ClC-2 transcript. To detect ClC-2 protein in liver plasma membranes, which has not been reported in the literature, we used a ClC-2-specific antibody raised, according to the supplier, to a region in the COOH terminus corresponding to a 12- to 20-residue peptide within the last 100 amino acids. From sequence comparisons of ClC-2 with ClC-1, and the information supplied by the supplier, the peptide against which the antibody ClC-2 was raised can be narrowed to a region distal to the underlined CBS2 domain (see Fig. 5B). Western blotting showed that the COOH-terminal antibody recognized three protein bands, two between 85 and 100 kDa and close to the predicted molecular mass of ClC-2 (99 kDa) and a larger one >130 kDa, possibly reflecting an incompletely solubilized dimer (Fig. 5C). No bands were detected when the antibody was preabsorbed with antigen (data not shown). In preliminary studies, in which the initial 10% SDS solubilization step was omitted, only the high-molecular-mass band was observed in rat hepatocyte and WIF-B cells, whereas, as expected from the RT-PCR results, no bands were detected in HEK293 membranes (data not shown).



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Fig. 5. Expression of ClC-2 in rat hepatocytes and WIF-B and HEK293T cells. A: RT-PCR products of mRNA isolated from rat hepatocytes and HEK293T and WIF-B cells amplified using primers specific for {beta}-actin or ClC-2. Forty nanograms mRNA in the presence or absence (–) of RT were used in each reaction; M, 100-bp markers. WIF-B cells expressed the largest amount of ClC-2 message/unit cDNA, whereas no detectable signal was present in HEK293T cells. B: alignment of the COOH termini of rat ClC-2 (Swiss Prot P35525 [GenBank] ) and human ClC-1 (Swiss Prot P35523 [GenBank] ) to show the common cystathionine {beta}-synthase (CBS2) domain (underlined) and the unique 40-residue terminus of ClC-2 (bold font). The Santa Cruz antibody (Ab) ClC-2 likely recognizes a 12- to 20-residue domain within the final 40 amino acids. Dashes (-) indicate identical residue; periods (.) indicate the end of ClC-2 sequence. C: Western blotting of 30 µg solubilized protein from rat liver plasma membranes using the COOH terminal Ab ClC-2 detected an intense band between 90 and 100 kDa, plus 2 additional bands at ~ 85 and >120 kDa. Preabsorbing the antibody with the antigen resulted in no reaction products (data not shown). M, markers; L, liver plasma membranes.

 
Resting and volume-activated Cl currents in WIF-B and HEK293T cells. To confirm that functional ClC-2-like currents are present in WIF-B but not HEK293T cells, and to provide additional evidence that ClC-2 is not responsible for generating VRAC-like currents, we carried out an electrophysiological characterization of Cl currents in WIF-B and HEK293T cells. Figure 6, A and B, shows representative currents from WIF-B and HEK293T cells recorded in the absence (traces on top) or presence (traces on bottom) of 80 mM raffinose in the patch pipette. In the presence or absence of raffinose, a hyperpolarization-activated and time-dependent current is clearly visible in the WIF-B but not the HEK293T cells. Both cells responded to dialysis with raffinose by activating large, outwardly rectifying, and inactivating current reminiscent of the VRAC seen in the rat hepatocyte (Fig. 1B and Ref. 23). Current-voltage relationships of the mean instantaneous currents normalized to cell capacitance demonstrate the activation of VRAC in both cell types in the presence of raffinose (Fig. 6, C and D, top). Conversely, the current-voltage relationships of the time-dependent inward current show that this is functionally expressed in the WIF-B but not the HEK293T cells (Fig. 6, C and D, bottom).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study shows that resting short-term cultured rat hepatocytes express a hyperpolarization-activated and time-dependent Cl current. Both functional and molecular expression data presented here support the hypothesis that rat hepatocytes express a ClC-2-like channel that is present under resting conditions and is not affected by conditions leading to activation of VRAC. Although ClC-2 transcript has been reported in hepatocytes earlier (30), ours is the first report of native protein expression and functional activity of a Cl-selective current having molecular and electrophysiological properties conforming to ClC-2. Based on the conductance and Cl selectivity of the two components of Cl current observed here, our results also demonstrate that the time-dependent component comprises a significant fraction of resting Cl conductance in these cells. The voltage-sensitive properties of the inward current further endow it with the ability to provide significantly larger Cl efflux with small (5–10 mV) hyperpolarizations of the membrane potential, effectively countering further hyperpolarization. Final identification of this current as ClC-2 awaits successful gene knockout in the rat and functional analysis of Cl currents in hepatocytes from those animals.

Resting Cl currents in hepatocytes. Rodent hepatocytes have a high resting permeability to Cl. In the intact rat liver, Cl accounts for ~60% of total ionic conductance (4). Electrophysiological measurements in single hepatocytes and hepatocyte couplets revealed Cl conductances of between 2.3 and 8 nS (13, 23). In our preparation of rat hepatocytes under patch-clamp, Cl conductance was biphasic, with inward conductance of ~2.4 nS and outward at 1.8 nS, both of which are similar to the earlier observations. However, in contrast to the weakly outwardly rectifying whole cell currents seen earlier (23), the resting currents in our study demonstrated both inward and outward rectification, bridged by a shallow linear conductance encompassing Cl equilibrium. In the earlier study, the hepatocytes were voltage clamped between ±100 mV, and current-voltage relationships were constructed between ±60 mV. This voltage range would have obscured the observation of both the inward rectification and the time dependence of the inward Cl currents in hepatocytes. Alternatively, the hepatocyte isolation technique used (4°C storage up to 24 h before culture on collagen-coated coverslips for 2–6 h at 37°C) may have adversely affected the expression of the time-dependent and inwardly rectifying Cl conductance.

Evidence that ClC-2 may be a significant contributor to resting hepatocyte currents. Here, we present functional, pharmacological, and molecular evidence supporting our hypothesis that ClC-2 underlies the time- and voltage-dependent Cl current in the rat hepatocyte. Kinetically, ClC-2 is the only established candidate for the electrophysiological properties described herein. Steady-state activation analysis shows that the hepatocellular current activates at potentials negative to Cl equilibrium with a relatively slow time course and is not inhibited by the stilbene DIDS, all distinctive properties of ClC-2 (2, 3, 40). At the molecular level, we demonstrated both ClC-2 transcript and protein expression in the hepatocyte. We also compared molecular and functional expression in two other cell types, one known to be a nonexpressor (HEK293T; see Refs. 24 and 2931) and one having a rat hepatocyte origin and capable of forming a polarized epithelium (WIF-B). We found ClC-2 transcript in the WIF-B cells but not in HEK293T cells, the latter confirming earlier reports (24, 29, 30). As expected from the expression results, time-dependent and hyperpolarization-activated currents characteristic of ClC-2 were present in WIF-B but not HEK293T cells.

In contrast to heterologously expressed ClC-2 (12), the hyperpolarization-activated Cl currents of hepatocytes do not increase in response to exposure to hypotonic conditions. Volume sensitivity is localized to a specific region in the NH2 terminus of ClC-2, and deletion results in a constitutively open channel (12). Although the full-length sequence of the rat hepatocyte ClC-2 is identical to the original clone from brain (30), we speculate that an additional regulatory component, possibly linked to external acidification (see Ref. 31), is absent in the heterologous expression systems. This may explain why only a small fraction of recorded heart cells express ClC-2-like currents (5). In the present study, all hepatocytes expressed time-dependent inwardly rectifying Cl currents. It remains to be determined how these currents in hepatocytes may be regulated in addition to voltage gating.

We also assessed the halide selectivity of the instantaneous and time-dependent currents in resting rat hepatocytes. The instantaneous currents were significantly more permeable to I than Cl, and these became more clearly outward rectifiers with I as the major charge carrier of outward current. These properties are very similar to the weakly outwardly rectifying anion currents, or VRAC, measured earlier in hepatocytes exposed to hypotonic media (23). We suggest that the resting currents may result from spontaneous opening of a small fraction of VRAC channels. In contrast, ClC-2 is significantly less permeable to I than Cl (7) and therefore would not contribute to the instantaneous currents. Furthermore, voltage gating of ClC-2 currents is sensitive to the intracellular Cl concentration such that channel opening occurs at more negative potentials with lower intracellular Cl concentrations (27). The hepatocellular hyperpolarization-activated Cl current described herein has a similar sensitivity to intracellular Cl.

Is ClC-2 responsible for VRAC in hepatocytes? ClC-2 has been implicated in volume regulation in both native (6, 30) and heterologous (5, 10, 12, 46) expression systems. This, however, is still an unsettled issue, since there is substantial evidence that ClC-2 does not underlie VRAC (31). Swelling-activated currents in heterologous expression systems have different pharmacological and electrophysiological properties from those present in native cells, leading to the possibility that these currents do not represent native VRAC. For example, DIDS does not block the swelling-activated outward current in Sf9 cells heterologously expressing ClC-2 (46). The VRAC present in HTC cells presents a different group of characteristics than those reported from many cells. Outward swelling-activated currents can be completely blocked by DIDS, and an antibody against ClC-2 completely blocked the development of both inward and outward Cl currents activated by hypotonic solutions (30). This suggests that these cells do not express classical outwardly rectifying VRAC or that the antibody used is capable of recognizing the DIDS-sensitive current. Strong evidence against ClC-2 being VRAC is based on its pharmacological and biophysical properties, which are very different from VRAC in hepatocytes and many other cell types, as already discussed.

Like many other cell types, hepatocytes respond to hypotonic or isotonic volume increases by an RVD involving increased K+ and Cl efflux and consequent water flow. This response is inhibited by both K+ and Cl channel blockers. DIDS, at 100–150 µM, significantly inhibits hypotonically induced Cl efflux, RVD, and VRAC in hepatocytes (23) and a mouse liver cell line (45). Thus the differential sensitivity to hypotonic solutions, electrophysiological and kinetic properties, and pharmacological profile of the two Cl currents we have identified in rat hepatocytes can be used to assess the role of these channels in mediating VRAC. The hypotonicity-activated current in the hepatocyte exhibits outward rectification and inactivation at extremely positive potentials, inhibition by 100 µM DIDS, and absence from resting cells, all properties that have been reported previously in hepatocytes and other cells (23, 39). Conversely, the hyperpolarization-activated current has, in each of these respects, opposite properties in that it is an inward rectifier that is insensitive to DIDS and conditions in which VRAC is activated in the same cells. Furthermore, we show here that HEK293T cells are capable of activating a VRAC in response to a hypotonic challenge yet do not express ClC-2. These results suggest that two distinct proteins are responsible for the hyperpolarization-activated and hypotonicity-sensitive Cl currents in the hepatocyte.

Last, a physiological role for a hyperpolarization-activated Cl channel in the hepatocyte can be addressed. These cells have a large resting Cl conductance, the molecular identity of which has not been determined (4, 11). Our results suggest that the time-dependent inward Cl currents would contribute up to 8% of total Cl conductance over the range of resting potentials measured in these cells (–30 to –45 mV). Unlike classical secretory epithelia that accumulate Cl above their electrochemical equilibrium to enable a large Cl-mediated secretory response (2), the exocrine activity of hepatocytes does not depend to the same extent on "active" transcellular Cl flux (33). Outward Cl flux mediated by the hyperpolarization-activated channels would be more likely to fulfill a minute-to-minute response to small changes in membrane potential resulting from electrogenic nutrient and organic acid uptake and disposition within the hepatocyte and biliary lumen. For example, exposure to hypoosmotic solutions or uptake of model organic anions, such as bromosulfophthalein, DIDS, and indocyanin green, cause transient membrane hyperpolarization and K+ efflux from isolated hepatocytes and intact liver (14, 18, 4244). The transient nature of these responses may, in part, result from the increased efflux of Cl mediated by the recruitment of open time-dependent, hyperppolarization-activated channels at more hyperpolarized potentials. Thus these channels would be involved in maintenance of membrane potential under physiological conditions.

In conclusion, we have identified a new time- and voltage-dependent Cl current in hepatocytes that appears to be mediated by a channel distinct from the VRAC. This channel is capable of providing a significant fraction of the total hepatocellular Cl conductance. We speculate that this channel is a suitable candidate both for setting the membrane potential of these cells and in countering membrane hyperpolarization resulting from anion uptake.


    ACKNOWLEDGMENTS
 
This work was supported by a grant from the Canadian Institutes of Health Research (CIHR) to C. E. Hill. H. D. Lam and W.-Z. Lan were supported by Natural Sciences and Engineering Research Council and the CIHR Training Grant in Digestive Sciences, and C. E. Hill by the Hotel Dieu Hospital (Jeanne Mance Foundation and GI Diseases Research Unit).


    FOOTNOTES
 

Address for reprint requests and other correspondence: C. E. Hill, Hotel Dieu Hospital, 166 Brock St., Kingston, ON Canada K7L 5G2 (E-mail: hillc{at}post.queensu.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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