Regulation of a hyperpolarization-activated chloride current in murine respiratory ciliated cells

Abstract

1. The properties of a hyperpolarization-activated Cl⁻ current (I_hyp-act) in murine ciliated respiratory cells have been studied using whole cell patch clamping.

2. The current–voltage relationship was inwardly rectifying which was due to voltage-dependent gating of the channel.

3. Inward current was markedly sensitive to the extracellular Cl⁻ concentration, an effect that was not related to changes in transmembrane Cl⁻ gradient. Decreasing extracellular Cl⁻ concentration to 6 mM caused a 70 % reduction in inward current with the dose–response relationship exhibiting a Hill coefficient of ≈2·0 and an IC₅₀ of 29 mM.

4. External anion replacement gave a selectivity sequence of Br⁻ ≥ I⁻ > Cl⁻ > gluconate = aspartate. The more permeant halides significantly increased current density while the less permeant anions decreased current density, indicating that an extracellular anion is important for channel activity.

5. The conductance was unaffected by exposure to anisotonic pipette solutions or to increases in intracellular cAMP; however, current density was reduced dose dependently by increases in intracellular calcium concentration from 0·1 to 0·5 μM. These results indicate that I_hyp-act is unlikely to be involved in either volume regulation or cAMP/Ca²⁺-stimulated fluid secretion.

6. Decreasing extracellular pH to 5·0 irreversibly inhibited I_hyp-act. However, the current was fully active over the pH range 5·4–9·0 making it unlikely that it is modulated by extracellular pH under physiological conditions.

7. We speculate that I_hyp-act may have a role in basal Cl⁻ absorption, acting as a Cl⁻ sensor to maintain optimal volume and composition of airway surface liquid.

Chloride channels are common in epithelial cells where they have particularly important roles in electrolyte and fluid transport and cell volume control. Chloride flux through these channels is tightly regulated and is typically controlled by increases in second messengers such as Ca²⁺ and cAMP, or by changes in cell volume (reviewed by Valverde et al. 1995). An important class of epithelial anion channel, which displays characteristic voltage-dependent gating, is the ClC family, which includes up to ten distinct conductances (Jentsch & Gunther, 1997). ClC-0, the first member of this family to be characterized, was initially cloned from the Torpedo electric organ (Jentsch et al. 1990). This channel is thought to have a double-barrelled ‘protochannel’ arrangement where the current through these protochannels is dependent on the extracellular Cl⁻ concentration, with the permeant anion acting as part of the gating mechanism (Chen & Miller, 1996). Using probes made from ClC-0, two more Cl⁻ channels, termed ClC-1 and -2, were identified and shown to share significant homology with ClC-0. While ClC-1 was predominantly expressed in muscle (Steinmeyer et al. 1991), ClC-2 was shown to be ubiquitously expressed in both epithelial and non-epithelial cells. When ClC-2 cRNA was injected into Xenopus oocytes the resulting currents were characterized by activation at hyperpolarizing potentials, insensitivity to extracellular DIDS and an anion permeability sequence of Cl⁻ > Br⁻ > I⁻ (Thiemann et al. 1992). Endogenous ClC-like currents have also been detected electrophysiologically in a variety of other epithelial and non-epithelial cell types (Jentsch & Gunther, 1997). However, the properties of these currents vary in what appears to be a tissue specific manner. For example, hyperpolarization-activated Cl⁻ currents have been reported that are both phosphorylation dependent (Fritsch & Edelman, 1996) and phosphorylation independent (Komwatana et al. 1994), are DIDS sensitive (Clark et al. 1998) and insensitive (Komwatana et al. 1994) and that have ion permeability sequences ranging from Cl⁻ > Br⁻ > I⁻ (Fritsch & Edelman, 1996) to I⁻ > Br⁻ > Cl⁻ (Kowdley et al. 1994).

We have previously reported that the dominant basal Cl⁻ current in native murine nasal ciliated cells was a ClC-like, hyperpolarization-activated Cl⁻ current (I_hyp-act) (Tarran et al. 1998) and that this current is regulated by the cystic fibrosis transmembrane conductance regulator (CFTR). The role of Cl⁻ currents in airway ion transport is currently controversial. One school of thought maintains that the airway surface liquid (ASL) [NaCl] is low (∼50 mM) as a result of transcellular Cl⁻ absorption across a relatively tight epithelium (Smith et al. 1996; Zabner et al. 1998). Moreover, these authors proposed that a low ASL [NaCl] was important for maintenance of the antibacterial properties of the ASL (Smith et al. 1996; McCray et al. 1999). It has also been proposed that the ASL [NaCl] is isotonic to plasma and that Na⁺ absorption is transcellular with Cl⁻ and water moving paracellularly and as salt is reabsorbed the ASL volume changes accordingly to maintain isotonicity (Boucher, 1994; Matsui et al. 1998). Here, maintenance of an optimum ASL volume has been proposed to be important for mucus clearance, which in turn allows removal of airborne pathogens.

Since ASL volume and composition are important in defence against disease, it is likely that airway ion channels are regulated by factors in the ASL as part of a feedback mechanism for ASL homeostasis. Hence, one of the aims of this present work was to further characterize the regulation of I_hyp-act in an effort to better comprehend such possible regulatory systems.

METHODS

Isolation of ciliated respiratory cells from the nasal epithelium

Mice of either sex from a BALB/c breeding colony at the University of Newcastle upon Tyne were used for these experiments. Ciliated respiratory cells, suitable for patch clamping, were obtained from the murine nasal epithelium using a technique that we have described previously (Tarran et al. 1998). In brief, mice were killed by cervical dislocation and the nasal epithelia removed and incubated overnight at 4°C in Dulbecco's modified Eagle's medium (DMEM) plus 0.05 % Protease XIV (Sigma). After 24 h, the enzyme activity was halted by placing the tissue in DMEM plus 10 % fetal calf serum. The tissue was then kept in this media for up to 48 h. To isolate single cells, small pieces of protease-treated tissue were then further dissected using sharpened entomology pins in a low Ca²⁺ modified Krebs Ringer buffer solution to yield single cells. The respiratory epithelial cells could easily be distinguished from other cell types by their prominent cilia. Our criteria for cell viability were: (i) a clear, bright phase contrast image, and (ii) beating cilia. On the basis of these criteria the majority of isolated respiratory cells were viable.

Electrophysiology

The cell preparations were transferred to a tissue bath (volume 1.5 ml) mounted on a Nikon Diaphot inverted microscope (Nikon, UK), and viewed using phase contrast optics. Pipettes were pulled from borosilicate glass (Clarke Electromedical, UK) and had resistances, after fire polishing, of between 2 and 4 MΩ. Giga-ohm seals (typically 10–30 GΩ) were obtained on the non-ciliated basolateral membrane of the isolated cells, with a success rate of about 70 % provided the cells were used within 30 min of isolation. Recordings were made at room temperature either from single cells or from small groups of cells (≤ 7), using the whole cell configuration of the patch clamp technique. We noticed that cilia beat frequency usually increased when a cell-attached seal was obtained. However, once whole cell recording was established, whether the cilia continued to beat or not was dependent on the composition of the pipette solution. In the presence of a low intracellular Ca²⁺ concentration (< 1 nM) the cells stopped beating; however, if the intracellular Ca²⁺ was fixed at ≥ 0.1 μM then the cilia continued to beat.

Whole cell currents were recorded with an EPC-7 patch clamp amplifier (List Electronic, Darmstadt, Germany). During continuous recording of membrane currents (voltage clamp experiments), the membrane potential (V_m) was held at 0 mV and alternately clamped to -60 mV and +60 mV for 1 s (here called the ±60 mV protocol). To obtain I–V relationships, the membrane potential was held at 0 mV, and then voltage clamped over the range -100 mV to +100 mV in steps of 20 mV (here called the ±100 mV protocol). Each voltage step lasted 500 ms and there was an 800 ms interval at the holding potential (V_h) between steps. In order to investigate the voltage dependence of I_hyp-act, a second voltage step protocol was used. A conditioning voltage step of -80 mV was applied for 1500 ms in order to fully open the channels prior to stepping to ±100 mV in 20 mV steps. For this protocol (here called the -80 mV protocol) each step lasted 500 ms and following a 100 ms rest period at 0 mV, V_m was returned to the original prepulse potential of -80 mV before each successive step. Data were filtered at 1 kHz and sampled at 2 kHz with a Cambridge Electronic Design 1401 interface (CED, Cambridge, UK), and stored on computer hard disk. I–V plots were constructed using the average current measured over a 2 ms period starting 495 or 5 ms into the voltage pulse for the ±100 mV and -80 mV protocols, respectively. The currents were not leak corrected. Series resistance (R_s) was typically 2–3 times the pipette resistance, and R_s compensation (40-50 %) was routinely used. Membrane potentials (V_m) have been corrected for current flow (I) across the uncompensated fraction of R_s using the relationship: V_m=V_p- IR_s, where V_p is the pipette potential. Reversal potentials (V_rev) were obtained from I–V plots by interpolation after fitting a third or fourth order polynomial using least squares regression analysis. The input capacitance (C_i) of the cells was routinely measured using the analog circuitry of the EPC-7 amplifier and compensated prior to the start of recording. C_i values were used to calculate current density which is expressed as picoamps per picofarad. Junction potentials were measured and the appropriate corrections applied to V_m.

Solutions and chemicals

To tease out single ciliated cells, a low Ca²⁺ medium was used which contained (mM): 145 NaCl, 4.5 KCl, 1 MgCl₂, 2.0 EGTA, 10 Hepes, pH 7.4.

In order to isolate anion-selective currents, cation conductances were blocked with N-methyl-D-glucamine⁺ (NMDG⁺). Murine ciliated cells also contain volume-activated chloride currents which were distinguishable from I_hyp-act on the basis of their markedly greater sensitivity to tamoxifen and DIDS (data not shown, see Tarran et al. 1998 for pharmacology of I_hyp-act), their pronounced outward rectification and inactivation at positive membrane potentials. To prevent activation of these volume-sensitive currents, the pipette solution was made 20 mosmol l⁻¹ hypotonic to the bath solution. The pipette solution contained (mM): 120 NMDG-Cl, 2.0 MgCl₂, 2.0 EGTA, 1.0 ATP, 10.0 Hepes, pH 7.2. The calculated free Ca²⁺ concentration was < 1 nM to inhibit Ca²⁺-activated Cl⁻ currents and mannitol was added to raise the osmolarity to 280 mosmol l⁻¹. The standard bath solution contained (mM): 149.5 NMDG-Cl, 2.0 CaCl₂, 1 MgCl₂, 5 glucose, 10 Hepes, pH 7.4. To examine the Ca²⁺ dependency of I_hyp-act, the pipette EGTA was increased to 5.0 mM and the intracellular Ca²⁺ concentration fixed at 0.1, 0.3, 0.5 or 1.0 μM by adding 2.12, 3.44, 3.93 and 4.8 mM CaCl₂, respectively (final free calcium calculated using Eqcal software, Biosoft, UK). To examine the effects of reducing intracellular Cl⁻ on I_hyp-act, 80 mM NMDG-Cl was removed from the pipette solution and the osmolarity fixed at 280 mosmol l⁻¹ by the addition of mannitol. When comparing conditions which necessitated the use of different pipette solutions, several different pipette solutions would be tested on cells isolated from the same mouse to make the experiments ‘paired’.

To test whether I_hyp-act was dependent on extracellular Cl⁻ concentration, the NMDG-Cl concentration in the standard bath solution was reduced to 120, 70, 49.5, 30, 19 or 0 mM, whilst maintaining the osmolarity with mannitol.

To investigate anion selectivity, 149.5 mM NMDG-Cl in the bath solution was replaced with 149.5 mM NMDG-I, -Br, -gluconate or -aspartate. The effects of acid pH were tested by lowering the pH of the standard bath solution to 5.5 or 5.0 with extra HCl. For these acid solutions we ensured that the pH remained constant by repeatedly measuring the pH of these solutions during the experiments. To create an alkaline bath solution, NMDG-Cl was prepared using Trizma HCl rather than HCl, and Hepes was omitted from the solution. The pH was then adjusted to 9.0 with Trizma base. All bath solutions had an osmolarity of 300 mosmol l⁻¹ and the osmolarities of all solutions were checked prior to use with a freezing point depression osmometer (Roebling, model no. 10B).

Intracellular adenosine 3′,5′-cyclic monophosphate (cAMP) was raised by acutely stimulating the cells with a ‘cAMP cocktail’ consisting of forskolin (1 μM), dibutyryl cAMP (0.1 mM) and 3-isobutyl-1-methylxanthine (0.1 mM) for ∼5 min. 1,2-Epoxy-3-(p-nitro-phenoxy)-propane (ENPP; 1.0 mM) was made up daily as a stock solution in dimethyl sulphoxide and then diluted 1000-fold to give the final concentration used.

Statistics

Significance of difference between means was determined using ANOVA followed by Dunn's multiple comparison test, the Mann-Whitney U test, or the Wilcoxon signed rank test as appropriate. Significance of difference between the number of cells responding to a particular manoeuvre was assessed using the χ² test. The level of significance was set at P≤ 0.05. All values are expressed as means ±s.e.m., with number of observations (n) given in parentheses.

The dose-response curves to Ca²⁺ and Cl⁻ were fitted using the following allosteric sigmoid function:

(1)

where i_max and i_min are the maximal and minimal currents, x and x₅₀ are the concentration and half-maximal concentration, respectively, of Ca²⁺ or Cl⁻, and h is the Hill coefficient.

RESULTS

Biophysical properties

We have recently shown that in the absence of agonist, ciliated respiratory cells isolated from the nasal epithelium of mice possess a number of distinct chloride currents (Tarran et al. 1998). In approximately 30 % of cells isolated from wild-type animals a hyperpolarization-activated Cl⁻ current (I_hyp-act) was present which was characterized by slight inactivation at positive potentials and marked activation at negative potentials (Fig. 1A). We have now gone on to further characterize the properties of this current. When the cell is held at 0 mV the steady-state current-voltage (I–V) plot is inwardly rectifying (Fig. 1C, squares). To examine whether this rectification was due to an intrinsic voltage dependence of the channels, the membrane potential was first held at -80 mV for 1500 ms in order to fully activate all channels and then stepped to between -100 and +100 mV in 20 mV steps. Comparing Fig. 1A and B it can be seen that when the cell is held at -80 mV (Fig. 1B) the instantaneous currents are markedly increased, an effect that is particularly noticeable at depolarizing potentials. In the five cells tested, instantaneous current densities (measured at V_rev± 60 mV) were increased from 81.5 ± 13.7 and -139.0 ± 17.5 pA pF⁻¹ at a holding potential of 0 mV to 142.2 ± 13.9 and -151.0 ± 13.7 pA pF⁻¹ at a holding potential of -80 mV (P < 0.01 for outward currents; P = 0.06 for inward currents). A consistent finding using the -80 mV protocol was the absence of any time dependence to the whole cell currents at hyperpolarizing potentials, and a wave-like appearance of the currents at large depolarizing voltages (Fig. 1B), perhaps suggesting a transient activation of another current at these potentials. Looking at the instantaneous I–V relationship for cells held at -80 mV (Fig. 1C, circles), we can see that the plot has become almost linear over the range -100 to +100 mV. These data suggest that the inward rectification displayed in the steady-state I–V plots is due to voltage-dependent gating of the channels rather than to an intrinsic asymmetry in chloride movement through the channel pore.

Figure 1. Characteristics of I_hyp-act in ciliated nasal epithelial cells.

A, typical current trace recorded using the ±100 mV protocol (V_h, 0 mV) (see Methods). B, typical current trace recorded using the -80 mV protocol (V_h, -80 mV) (see Methods). C, current-voltage plots from the data in A (▪) and B (•).

Intracellular regulation – ATP, cAMP, Ca²⁺ and osmolarity

To investigate the regulation of I_hyp-act, we examined the effects of three common intracellular regulators of Cl⁻ channels, ATP, cAMP and Ca²⁺, and also changed the intracellular osmolarity to shrink or swell the cells by the addition or removal of mannitol.

ATP was routinely added to the pipette solution to help maintain cell viability. Omitting ATP from the pipette had no significant effect on the size or frequency of I_hyp-act (no ATP, 47.4 ± 16.5 and -80.2 ± 21.8 pA pF⁻¹, observed in 4/11 cells; +ATP, 61.8 ± 8.1 and -104.0 ± 13.6 pA pF⁻¹, observed in 28/93 cells; P > 0.05 for density and frequency, respectively).

I_hyp-act did not require cAMP for its activation, since it was present in 28/93 unstimulated cells from 15 animals, nor was it affected by acute exposure (5 min, n = 6) to a cAMP-elevating cocktail (see Methods; data not shown).

Under standard conditions (2.0 mM EGTA in the pipette solution, [Ca²⁺]_i∼10 nM), I_hyp-act was present immediately after establishing a whole cell recording in 28/93 cells. To investigate the effects of changing [Ca²⁺]_i on I_hyp-act, the pipette EGTA was increased to 5 mM and [Ca²⁺]_i fixed at 0.1, 0.3, 0.5 and 1.0 μM to construct a dose-response curve. Figure 2A–C shows representative current traces of I_hyp-act at 0.1, 0.3 and 1.0 μM Ca²⁺₁ and Fig. 2D shows current densities fitted to the Hill equation (eqn (1); see Methods) over the range 10.0 nM to 1.0 μM Ca²⁺₁. These data show that Ca²⁺ inhibits I_hyp-act and that the dose-response curve for this effect is quite steep, with half-maximal inhibitory (IC₅₀) values of 0.26 ± 0.14 and 0.23 ± 0.07 μM Ca²⁺ for outward and inward current, respectively. The Hill coefficients were 1.49 ± 0.94 (outward current) and 1.78 ± 0.75 (inward current). The frequency at which I_hyp-act was observed was not significantly altered over the range 0.1-0.5 μM Ca²⁺₁. However, if [Ca²⁺]_i was increased to 1.0 μM, I_hyp-act was never observed immediately upon establishing whole cell recording, although it subsequently activated in 4/11 cells after an average time of 2.5 min, albeit with a much diminished current density (Fig. 2).

Figure 2. Effect of increasing intracellular Ca²⁺ concentration on I_hyp-act current density.

A-C, representative current traces, recorded using the ±100 mV protocol, where intracellular Ca²⁺ was fixed at 0.1 (A), 0.3 (B) and 1.0 μM (C) (see Methods). D, dose-response curve for the effect of intracellular Ca²⁺ on I_hyp-act. Data are expressed as current densities (pA pF⁻¹) measured at V_rev± 60 mV for each calcium concentration. The lines are best fits to eqn (1). Data are shown as means ±s.e.m. with the number of cells given in parentheses.

The pipette was routinely made 20 mosmol l⁻¹ hypotonic to the bath solution to prevent activation of outwardly rectifying, swelling-induced Cl⁻ currents. Further reducing the pipette osmolarity to 250 mosmol l⁻¹ or increasing it to 360 mosmol l⁻¹ had no effect on the magnitude or frequency of I_hyp-act (each n = 4; data not shown).

Cl⁻ dependency

Some members of the ClC family of Cl⁻ channels have been shown to be dependent on Cl⁻ for their activation (ClC-0, Richard & Miller, 1990; Pusch et al. 1995; ClC-1, Fahlke et al. 1995) and since I_hyp-act has similar properties to some ClC channels (reviewed by Valverde et al. 1995), we decided to investigate the effects of changing the extracellular and intracellular Cl⁻ concentrations on its activity. Figure 3A shows that I_hyp-act was markedly dependent on the extracellular Cl⁻ concentration. In this experiment, the membrane potential was initially held at 0 mV and then alternately pulsed to -60 and +60 mV. V_m was first changed to offset junction potentials (Fig. 3A, *), and then the bath solution was switched from 155.5 mM to 6 mM Cl⁻. As predicted this manoeuvre caused a substantial decrease in outward current, due to the reduction in the extracellular Cl⁻ concentration, but unexpectedly also caused a rapid, large inhibition (70 %) in inward current (Fig. 3A). The reduction in inward current was fully reversible (Fig. 3A) and was not due to a change in intracellular Cl⁻ concentration, since the pipette Cl⁻ concentration remained constant at 124 mM. Figure 3B–D shows typical current traces for cells exposed to 155.5, 76 and 6 mM extracellular Cl⁻. Note that although there is a clear reduction in the size of the whole cell currents on reducing extracellular Cl⁻, there is no appreciable change in the kinetics of the currents indicating that the effect of Cl⁻ is unlikely to involve a substantial change in gating of the channels. That the reduction in outward and inward current was dependent on the extracellular Cl⁻ concentration is clearly shown by the I–V plots in Fig. 3E where one cell was exposed to a range of Cl⁻ concentrations between 155.5 mM and 6 mM Cl⁻. These data therefore suggest that reducing extracellular Cl⁻ concentration inhibits I_hyp-act directly.

Figure 3. Inhibition of I_hyp-act following a reduction in extracellular Cl⁻ concentration.

A, continuous whole cell recording showing the effect of reducing the extracellular Cl⁻ concentration while keeping pipette Cl⁻ concentration at 124 mM. Starting with 155.5 mM Cl⁻ in the bath, V_m was changed (*) to offset the junction potential that developed, then 6 mM Cl⁻ solution was perfused into the bath. Once I_hyp-act was inhibited the ±60 mV protocol was interrupted by the ±100 mV protocol to establish an I–V relationship. The 126 mM Cl⁻ solution was then perfused into the bath and V_m was again readjusted for the new junction potential. Each cell was exposed to several different Cl⁻ concentrations in order to construct a dose-response curve for the inhibitory effect on I_hyp-act. B–D, representative current traces, recorded with the ±100 mV protocol, where the extracellular Cl⁻ concentration was 155.5 (B), 76 (C) and 6 mM (D). E,I–V relations obtained from a cell exposed to the following extracellular Cl⁻ concentrations: 154 (▪), 126 (•), 76 (▴), 55.5 (▾), 36 (⋆), 25 (♦), 6 mM (|). Note that inward and outward currents are inhibited to about the same extent.

Figure 4 summarizes the effect of changing extracellular Cl⁻ on I_hyp-act, and plots the percentage current remaining against the extracellular Cl⁻ concentration (with the current present at 155.5 mM Cl⁻ being taken as 100 %). Outward current is positive while inward current is negative. When fitted to the Hill equation (eqn (1); see Methods), the plot for outward current shows that 50 % of the current is inhibited by reducing extracellular Cl⁻ to 47.8 ± 7.8 mM, whereas for inward current the IC₅₀ was significantly lower at 29.1 ± 4.7 mM (P = 0.008). For outward current the inhibition plot had a Hill coefficient close to unity (1.27 ± 0.32; r²= 0.97), suggesting that Cl⁻ ions do not interact with each other in activating outward current, whereas the plot for inward current has a significantly different Hill coefficient of 2.4 ± 0.05 (r²= 0.99; P = 0.03), suggesting that positive cooperativity between Cl⁻ ions is involved in activating this portion of the current.

Figure 4. Dose-response curve for the effect of extracellular Cl⁻ concentration on I_hyp-act.

The data are plotted as the percentage of I_hyp-act current remaining, with the current present at 155.5 mM Cl⁻ being taken as 100 %. Intracellular Cl⁻ concentration was 124 mM. Positive values denote outward current and negative values denote inward current. Each cell was exposed to several different extracellular Cl⁻ concentrations, and the data are plotted as means ±s.e.m. The lines are best fits to eqn (1). The number of observations is given in parentheses.

Richard & Miller (1990) suggested that the flux of Cl⁻ through the ClC-0 channel, caused by asymmetric concentration gradients, was responsible for closing down the channel. To investigate whether this was the case for I_hyp-act, cells were held at the equilibrium potential for Cl⁻ (12 or 76 mV) when intracellular Cl⁻ was 124 mM and extracellular Cl⁻ was 76 or 6 mM, respectively, in order to abolish the driving force for Cl⁻. This manoeuvre had no effect and the current still decreased as previously described (n = 4; see Fig. 5A and B). The percentage decreases for outward currents were: 76 mM Cl⁻, 37.0 ± 8.2 % (n = 4); 6 mM Cl⁻, 82.1 ± 4.4 % (n = 4); and the percentage decreases for inward currents were: 76 mM Cl⁻, 33.2 ± 10.2 % (n = 4); 6 mM Cl⁻, 61.9 ± 3.6 % (n = 4).

Figure 5. Effect of changing holding potential on the inhibition of I_hyp-act by a low extracellular Cl⁻ solution.

Control I–V plots (▪) were obtained with 155.5 mM extracellular Cl⁻ at a holding potential of 0 mV. A, extracellular [Cl⁻] was 76 mM. Holding potentials: 0 mV (▴) and 12 mV (= chloride equilibrium potential (E_Cl), ▾). B, extracellular [Cl⁻] was 6 mM. Holding potentials: 0 mV (▴) and 76 mV (=E_Cl, ▾).

We next investigated the effect of reducing intracellular Cl⁻ to 44 mM. This manoeuvre significantly reduced inward current from -104.0 ± 13.6 pA pF⁻¹ (n = 28) to -51.1 ± 12.6 pA pF⁻¹ (n = 9; P = 0.02) as would be predicted from the change in intracellular Cl⁻ concentration, but it had no effect on outward current (61.8 ± 9.5, n = 28 versus 68.5 ± 16.6, n = 9; P = 0.86). These data are summarized in Fig. 6A and suggest that the ‘Cl⁻ sensor’ which controls current flow is only accessible from the extracellular side of the channel. This idea is further supported by the observation that if extracellular Cl⁻ concentration was reduced to either 55.5 or 6 mM (while keeping the intracellular Cl⁻ concentration constant at 44 mM), the percentage decrease in both inward and outward current were similar to those observed with a high Cl⁻ pipette solution (Fig. 6B). In the same series of experiments extracellular Cl⁻ was also decreased to 44 mM so that there would be no Cl⁻ gradient across the cell membrane and the current was still inhibited (Fig. 6B). Taken together, these results show that the inhibitory effect of low extracellular Cl⁻ on I_hyp-act is not influenced by the Cl⁻ concentration inside the cell.

Figure 6. Effect of changes in intracellular Cl⁻ concentration on I_hyp-act current density and inhibition by extracellular Cl⁻.

A, effect of reducing the intracellular Cl⁻ concentration from 124 mM (□) to 44 mM () on the size of I_hyp-act. The extracellular [Cl⁻] was 155.5 mM. Data are expressed as current densities (pA pF⁻¹) measured at V_rev± 60 mV. The number of observations is given in parentheses. B, percentage of current remaining after extracellular Cl⁻ was removed when intracellular [Cl⁻] was set at either 44 (continuous lines) or 124 mM (dashed lines). The I_hyp-act current density recorded with 155.5 mM Cl⁻ in the bath solution was taken as 100 %. The data for 124 mM Cl⁻ are taken from Fig. 5. Note that the same percentage decrease in I_hyp-act occurs upon removal of extracellular Cl⁻ regardless of the intracellular Cl⁻ concentration. Positive values denote outward current and negative values inward current. The lines are best fits to eqn (1). The number of cells is given in parentheses.

Anion selectivity

To examine the anionic selectivity of I_hyp-act, and to verify that its inhibition was indeed caused by a reduction in the extracellular Cl⁻ concentration rather than by a reduction in NMDG⁺, 149.5 mM NMDG-Cl in the bath solution was replaced with an equal amount of NMDG-I, -Br⁻, -gluconate or -aspartate. I⁻ and Br⁻ were only marginally more permeant than Cl⁻ through the conductance and gave permeability ratio (P_anion/P_Cl) values of 1.15 ± 0.04 and 1.07 ± 0.02, respectively (n = 6, and see legend to Fig. 7). Interestingly, addition of either I⁻ or Br⁻ to the bath solution caused a significant increase in both inward and outward current of about 25 %. The increase in outward current would be expected for a more permeant anion (see Fig. 7). However, the pipette solution still contained 124 mM Cl⁻ in these experiments, so it is likely that we are seeing the reverse of the low extracellular Cl⁻ effect described in the previous section, i.e. a facilitation of inward current by a more permeant extracellular anion. Conversely, replacement of Cl⁻ by aspartate or gluconate caused a decrease in both inward and outward current of about 40 % (Fig. 7). This is consistent with the idea that a reduction in extracellular anions inhibits the conductance. Gluconate and aspartate were both less permeant than Cl⁻ and gave P_anion/P_Cl values of 0.48 ± 0.03 and 0.52 ± 0.04, respectively (n = 6, and see legend to Fig. 7).

Figure 7. Effect of changing the permeant anion on the magnitude of whole cell current.

The reversal potentials for each anion were: I⁻, -5.4 ± 0.5 mV; Br⁻, -4.5 ± 0.5 mV; Cl⁻, -3.6 ± 0.8 mV; gluconate, 12.0 ± 2.0 mV; aspartate, 15.9 ± 1.1 mV (n = 6; P < 0.0001). Data are expressed as means ±s.e.m. and P values relate to the size of whole cell currents relative to the currents measured with chloride-rich solutions.

pH sensitivity

Other ion channels including those sensitive to Ca²⁺ have been reported to be pH sensitive (Uchida et al. 1995; Kajita & Brown, 1997) while the ClC-2 analogue isolated from rabbit stomach is active over the pH range 7.4-3.0 (Malinowska et al. 1995). More recently ClC-2 in dissociated rat sympathetic neurons (Clark et al. 1998) was found to be enhanced by extracellular acidification and reduced by extracellular alkalinization, a finding which was also observed for ClC-2 transfected into Xenopus oocytes (Jordt & Jentsch, 1997) and into a human cystic fibrosis (CF) airway cell line (Schwiebert et al. 1998). To see whether I_hyp-act was affected in this manner the pH of the standard, high Cl⁻ bath solution was increased to 9.0. This manoeuvre had no effect on the current (control, 93.7 ± 50.3 and -108.9 ± 48.5 pA pF⁻¹; pH 9.0, 85.3 ± 49.4 and -97.9 ± 58.6 pA pF⁻¹; n = 6; P = 0.56). Reducing the pH to 5.5 also had no effect on the current (control, 25.2 ± 4.9 and -40.4 ± 6.0 pA pF⁻¹; pH 5.5, 25.5 ± 5.1 and -35.1 ± 4.0; n = 6; P = 0.56). However, further reducing the extracellular pH to 5.0 rapidly inhibited approximately 97 % of I_hyp-act in 4/4 cells as can be seen in Fig. 8A (control, 47.8 ± 23.8 and -71.2 ± 25.8 pA pF⁻¹; pH 5.0, 1.75 ± 1.3 and -1.75 ± 1.9 pA pF⁻¹; n = 4; P = 0.04). The effect was irreversible, and the current did not return after a 5 min wash in the control solution. In a further four cells, the extracellular pH was reduced to 5.0 in the presence of the acid protease inhibitor ENPP and the inhibitory effect was abolished, suggesting that ciliated respiratory cells possess an endogenous acid protease, the action of which is inhibited by ENPP (control, 63.9 ± 17.6 and -76.9 ± 22.7 pA pF⁻¹; pH 5.0 + ENPP, 65.6 ± 20.7 and -65.7 ± 15.5 pA pF⁻¹; n = 4; P = 0.03). The results of experiments in which extracellular pH was changed in the presence and absence of ENPP are summarized in Fig. 8B.

Figure 8. Effect of extracellular pH on I_hyp-act current density.

A, inhibition of whole cell current following a reduction in extracellular pH from 7.4 to 5.0. B, summary showing the I_hyp-act currents at pH 9.0, 5.5 and 5.0 expressed as a percentage of the control value at pH 7.4 (ns, not significant). Nasal cells were also exposed to a bath solution at pH 5.0 containing the acid protease inhibitor ENPP at 1 mM.

DISCUSSION

We have previously demonstrated that the hyperpolarization-activated inwardly rectifying Cl⁻ current (I_hyp-act) is observed at a markedly reduced frequency in ciliated cells isolated from the nasal epithelium of murine CF null animals (Tarran et al. 1998). In this paper, we have shown that I_hyp-act is voltage sensitive and dependent on the presence of extracellular Cl⁻ for its activation yet independent of intracellular ATP, cAMP and changes in osmolarity. The conductance also appears to be inhibited by increases in intracellular Ca²⁺ and irreversibly inhibited by extracellular acidification.

Voltage dependency of I_hyp-act

We performed a simple kinetic analysis of I_hyp-act in order to compare its properties with those of current passing via exogenously expressed ClC-2. Similar to the experiments of Thiemann et al. (1992) we found that the steady-state current-voltage relation (I–V) for I_hyp-act was inwardly rectified but the instantaneous I–V, determined following a conditioning voltage step to -80 mV, was linear. These results therefore indicate that the apparent inward rectification is due to voltage-dependent gating of the channels rather than to an asymmetric movement of chloride ions through the channel pore. Inevitably, this means that we have been underestimating I_hyp-act outward currents, since the majority of I–V plots were constructed from current measurements using a holding potential of 0 mV, when channels would not be fully activated. However, our I–V data do indicate that the channel would be open at physiological membrane potentials in intact epithelia, since the membrane potential has been reported to be between -25 and -40 mV for the apical and basolateral membranes, respectively (Boucher, 1994).

Regulation by intracellular ATP, cAMP, Ca²⁺ and osmolarity

We have shown that the size of the I_hyp-act conductance was not modulated by a short (5 min) exposure to cAMP. This suggests that I_hyp-act is not acutely regulated by PKA. These results are different from those of Fritsch & Edelman (1996) who showed that cAMP exposure inhibited the hyperpolarization-activated Cl⁻ conductance from T84 cells within minutes of adding the stimulants. In addition, the conductance was not dependent on the presence of intracellular ATP for its activity nor was it modulated by changes in intracellular osmolarity. This latter finding contrasts with the work on ClC-2 (Grunder et al. 1992), which was shown to be enhanced by cell swelling.

Increasing intracellular [Ca²⁺]_i over the range 0.1-0.5 μM had a marked inhibitory effect on I_hyp-act. Since the resting level of Ca²⁺ in ciliated cells is thought to be around 0.1 μM (Boucher, 1994), I_hyp-act is likely to be open under basal conditions. Only the ClC-3 channel, which was cloned from rat kidney and expressed in a somatic cell line (Kawasaki et al. 1995), has previously been shown to be inactivated by Ca²⁺, and this is the first time that such a phenomenon has been directly demonstrated for a Cl⁻ channel in a native epithelial cell. However, Vayro & Simmons (1996) showed that a putative chloride conductance in rat renal brush border membrane vesicles was inactivated by a rise in intravesicular calcium probably via stimulation of protein phosphatase 2B (PP2B). If a similar mechanism were to exist in nasal cells this would indicate that I_hyp-act requires phosphorylation for activity and would further suggest that the conductance was spontaneously active because of endogenous phosphorylation. This suggestion is not consistent with the observation that I_hyp-act was active in the absence of pipette ATP and clearly further work is required to understand the mechanism of inactivation by calcium.

Taken together our results suggest that a rise in intracellular Ca²⁺ in nasal cells may signal the change from one channel type to another, since as I_hyp-act is inhibited a Ca²⁺-activated Cl⁻ conductance should become active. It is tempting to speculate that this putative reciprocal effect of these second messengers on I_hyp-act and the Ca²⁺-activated Cl⁻ conductance could be part of a mechanism that switches the nasal cells from an absorptive to a secretory mode.

Cl⁻ dependency of I_hyp-act

We have demonstrated that I_hyp-act is regulated by [Cl⁻]_o. Changing [Cl⁻]_o over the range 155.5-6 mM caused a dose-dependent inhibition of both inward and outward currents. The maximal inhibition, observed with 6 mM Cl⁻ was about 70 %. Inhibition of the inward currents was not caused by a decrease in the intracellular Cl⁻ concentration, since this parameter remained constant. Despite causing a large reduction in current density, decreasing the extracellular Cl⁻ concentration only elicited a small shift in reversal potential; the change in V_rev when extracellular Cl⁻ was decreased from 155.5 to 6 mM was 16.3 mV, a value well short of that predicted by the Nernst equation (82 mV). It is possible that this smaller-than-expected change in V_rev is due to the presence of a basal leakage current. At high [Cl⁻]_o, when I_hyp-act predominates, the reversal potential is governed by I_hyp-act. However, at low [Cl⁻]_o, when I_hyp-act is diminished, the leakage current (which is likely to be unaffected by [Cl⁻]_o) plays a larger role in determining the reversal potential.

A Cl⁻ dependency has previously been reported for the ClC-0 channel by Richard & Miller (1990). They suggested that Cl⁻-dependent inactivation of ClC-0 was linked to changes in the amount of Cl⁻ fluxing through the channel and that the energy derived from the Cl⁻ concentration gradient across the membrane powered the conformational changes which gated the channel. To test whether I_hyp-act behaved in the same way, we inhibited the conductance using a low Cl⁻ bath solution and then held the membrane potential at the equilibrium potential for Cl⁻ in order to abolish Cl⁻ flux through the channels. However, this manoeuvre failed to reactivate the conductance, suggesting that I_hyp-act is not dependent on flux ratios. Furthermore, when we reduced [Cl⁻]_i to 44 mM and then reduced [Cl⁻]_o from 155.5 to 44 mM, inward current still decreased in a similar fashion even though the concentration gradient for Cl⁻ across the membrane had been abolished. As yet, we have no idea as to the actual mechanism of the I_hyp-act inactivation, or where the energy to close the channel is derived from. It is unlikely that the energy required to gate I_hyp-act is derived from phosphorylation, which has previously been demonstrated to gate CFTR (Schultz et al. 1995), since I_hyp-act is active in the absence of intracellular ATP.

Reducing intracellular Cl⁻ alone decreased inward current but had no effect on outward current. This is similar to results obtained for the ClC-0 channel by Pusch et al. (1995) but contrasts with the results of Dinudom et al. (1993) who described a hyperpolarization-activated Cl⁻ conductance in submandibular duct cells which was inhibited by low [Cl⁻]_i.

Anion substitution

In the majority of experiments which examined the Cl⁻ dependency of I_hyp-act, we removed both Cl⁻and NMDG⁺ from the bath solution and replaced these ions with mannitol. This left open the possibility that reductions in either Cl⁻ or NMDG⁺, or perhaps both, were capable of inhibiting the current. Replacing Cl⁻ in the bath with either gluconate or aspartate also caused a decrease in both inward and outward currents, although the net reduction in the currents was not as great as when mannitol was used. Because our data, based on reversal potential shifts (Fig. 7), indicated that both these anions have a significant permeability through I_hyp-act, it is possible then that they could still access the ‘Cl⁻ sensor’ sufficiently to keep the conductance partially open. Alternatively, a reduction in the extracellular NMDG⁺ concentration might inhibit I_hyp-act via a hypothetical ‘cation sensor’ which may be present on the extracellular face of the conductance. While we cannot rule out this latter possibility, NMDG⁺ is a relatively large cation and is unlikely to access the pore of the channel. This type of dependence has been observed for a 30 pS chloride channel in hippocampal neurons (see Franciolini & Petris, 1992 for discussion). However, replacement of NMDG⁺ with TEA⁺, which is a smaller cation, produced no change in the size of the conductance (results not shown).

Conversely, substitution of Cl⁻ in the bath for I⁻ or Br⁻ caused a slight, but statistically significant increase in both inward and outward current. This observation is consistent with the idea that more permeant anions facilitate both inward and outward current flow through the conductance. For I_hyp-act, the halide permeability sequence was I⁻≥ Br⁻ > Cl⁻. Because the relative permeability sequence is inversely related to hydration energy (Hille, 1984) it is likely that a weak field strength binding site is important in determining selectivity. For the two currents that I_hyp-act most closely resembles (ClC-2, Thiemann et al. 1992, and the hyperpolarization-activated Cl⁻ conductance from mouse mandibular glands, Komwatana et al. 1994), the permeability sequences were Cl⁻≥ Br⁻ > I⁻ and Br⁻ > Cl⁻ > I⁻, respectively.

Effects of pH

Decreasing extracellular pH to 5.0 irreversibly inhibited I_hyp-act. However, I_hyp-act is active over the pH range 5.4-9.0 which suggests that this current is unlikely to be modulated by pH under physiological conditions (i.e. ∼pH 7; Boucher, 1994). The addition of ENPP, an acid protease inhibitor, blocked the inhibitory effect of the pH 5.0 solution. Hence, it is likely that an acid protease is activated at pH 5.0 which digests the protein that forms the channel. Recently, an endogenously expressed epithelial serine protease was shown to activate the amiloride-sensitive sodium channel in A6 kidney cells (Vallet et al. 1997). The protease was found to reside on the extracellular side of the apical membrane of these cells but interestingly was also shown to be present in whole, homogenized lung. At the moment, any relationship between this serine protease and the inhibition of I_hyp-act by extracellular acidification remains speculative. Our pH results are similar to those reported for the endogenous hyperpolarization-activated chloride current found in defolliculated Xenopus oocytes (Kowdley et al. 1994). However, recent studies on ClC-2 expressed either in Xenopus oocytes (Jordt & Jentsch, 1997) or in a stably transfected CF airway cell line IB3-1 (Schwiebert et al. 1997), both showed that extracellular acidification activated ClC-2 currents. For rat ClC-2 studied in oocytes, extracellular acidification activated currents by changing the voltage dependence of gating and not by affecting the open pore (Jordt & Jentsch, 1997). Interestingly, mutations in either the amino-terminal inactivation domain or the D7-D8 loop abolished the pH dependency and produced constitutively open channels. Even single point mutations in the D7-D8 region could produce pH-insensitive open pores and it is tempting to speculate that species differences in this region could account for these pH differences.

Physiological role of I_hyp-act

The subcellular location of the channels that underlie I_hyp-act cannot be determined from whole cell recording experiments. However, the fact that I_hyp-act is regulated by CFTR (Tarran et al. 1998), may indicate that the conductance is located in the apical membrane of the nasal cells. I_hyp-act is not activated by any of the classical intracellular messengers that increase airway secretion and so seems unlikely to play a direct role in this process. The channel through which I_hyp-act passes is likely to be open under basal conditions and is inactivated by Ca²⁺, as is the apical Na⁺ channel (Garty & Palmer, 1997) with which it could work in parallel. CF airway epithelia are characterized by an increase in the apical Na⁺ conductance and a decrease in the apical Cl⁻ conductance (Boucher, 1994). These changes result in both an increase in absorption and a decrease in secretion, respectively, which together act to deplete the periciliary liquid layer. Under basal conditions, airway surface epithelia are predominantly Na⁺ absorbing, with Cl⁻ acting as the counter ion (whether Cl⁻ moves paracellularly or transcellularly is still uncertain). However, a decrease in the apical Cl⁻ conductance has been predicted to increase the rate of Na⁺ absorption by increasing the apical membrane driving force for Na⁺ entry (Novotny & Jacobson, 1996). Moreover, by way of its Cl⁻ sensitivity, I_hyp-act could also act as part of a mechanism for regulating the Cl⁻ concentration of the airway surface liquid (ASL). Whether ASL is basally hypotonic or isotonic is also currently under debate and both conditions have been measured in vitro (hypotonicity, Smith et al. 1996; isotonicity, Matsui et al. 1998). Knowing the ionic composition of the ASL will be beneficial in assigning a physiological role to I_hyp-act. Loss of CFTR might, by reducing I_hyp-act, cause disregulation of Cl⁻ absorption and hence, further perturbation of the ASL, which in turn may contribute to making the CF lung prone to infection.