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Originally published In Press as doi:10.1074/jbc.M405517200 on July 31, 2004

J. Biol. Chem., Vol. 279, Issue 40, 41658-41663, October 1, 2004
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Novel Regulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Gating by External Chloride*

Angela M. Wright{ddagger}, Xiandi Gong§, Burns Verdon{ddagger}, Paul Linsdell§, Anil Mehta¶, John R. Riordan||, Barry E. Argent{ddagger}, and Mike A. Gray{ddagger}**

From the {ddagger}Institute of Cell and Molecular Biosciences, University of Newcastle Upon Tyne, Framlington Place, Newcastle Upon Tyne, NE2 4HH, United Kingdom, §Department of Physiology and Biophysics, Dalhousie University, Sir Charles Tupper Medical Building, Halifax, Nova Scotia, B3H 1X5, Canada, Division of Maternal and Child Health Sciences, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, United Kingdom, and ||Mayo Foundation, S. C. Johnson Medical Research Center, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259

Received for publication, May 18, 2004 , and in revised form, July 6, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The cystic fibrosis transmembrane conductance regulator (CFTR) is vital for Cl– and transport in many epithelia. As the concentration in epithelial secretions varies and can reach as high as 140 mM, the lumen-facing domains of CFTR are exposed to large reciprocal variations in Cl– and levels. We have investigated whether changes in the extracellular anionic environment affects the activity of CFTR using the patch clamp technique. In fast whole cell current recordings, the replacement of 100 mM external Cl– () with , Br–, , or aspartate– inhibited inward CFTR current (Cl– efflux) by ~50% in a reversible manner. Lowering alone by iso-osmotic replacement with mannitol also reduced Cl– efflux to a similar extent. The maximal inhibition of CFTR current was ~70%. Raising cytosolic calcium shifted the Cl– dose-inhibition curve to the left but did not alter the maximal current inhibition observed. In contrast, a reduction in the internal [Cl–] neither inhibited CFTR nor altered the block caused by reduced . Single channel recordings from outside-out patches showed that lowering markedly reduced channel open probability with little effect on unitary conductance. Together, these results indicate that alterations in alone and not the ratio regulate the gating of CFTR. Physiologically, our data have implications for current models of epithelial secretion and for the control of pH at epithelial cell surfaces.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cystic fibrosis transmembrane conductance regulator (CFTR)1 is a cyclic AMP activated epithelial Cl– channel, the mutation of which causes the potentially fatal inherited disease cystic fibrosis (CF) (1). The CFTR protein is a member of the ABC transporter family and is regulated by phosphorylation (2–4) and by ATP binding and hydrolysis (5–7). Dephosphorylation of CFTR by membrane-bound protein phosphatases is also important in the physiological regulation of channel activity (2, 8, 9).

Although CF is generally considered to result from a defect in Cl– transport, most of the affected epithelia also transport ions. Indeed, recent work suggests that there is a better correlation between defects in CFTR-dependent transport than defects in Cl– transport and the severity of disease (10). is an important component of epithelial secretions and, via its buffering role, controls the pH at the epithelial cell surface. It has been reported that CF-affected epithelia, particularly in the gastrointestinal tract, secrete fluid with a more acidic pH than normal epithelia (11, 12). Whether this is also true for airway surface liquid is still controversial (13, 14), but recent measurements of the pH and concentration of fluid secreted by polarized human submucosal gland cells, Calu-3, indicate that these cells are capable of secreting substantial amounts of (~80 mM) under appropriate stimulation, a process that would be defective in CF (15). An acidic luminal environment affects the physical properties of mucus (16, 17) and promotes bacterial binding to mucins (18, 19), both of which may have important implications for CF lung disease (20). As many CF-affected epithelia normally secrete substantial amounts of , the luminal concentration will vary under different physiological situations. This is particularly the case for the pancreas, a tissue in which CFTR is highly expressed and is severely affected in CF. Although plasma is ~25 mM, the concentration of in pancreatic juice can reach ~140 mM in humans, and because pancreatic juice is isotonic with plasma, these high concentrations are accompanied by a reciprocal fall in juice Cl– concentration (21, 22). Thus under physiological conditions, the apical surface of pancreatic duct cells and therefore the extracellular face of CFTR will be exposed to large variations in external Cl– () and () concentrations. A similar but less extreme situation is also likely to exist in many other -transporting epithelia, such as the small intestine (12), airways (15, 23), liver (24), and reproductive tract (25).

We have previously shown using fast whole cell patch clamp recordings (fWCR) that raising extracellular inhibits CFTR currents in native guinea pig pancreatic duct cells (26). Our data showed that increasing caused an inhibition of both inward and outward CFTR currents. The inhibition of outward current (Cl– influx) was expected as was replaced by the less permeant ion. However, the inhibition of inward current (Cl– efflux) was surprising as the intracellular Cl– concentration should remain constant because of the large Cl– reservoir in the pipette and currents would not have been predicted to alter from simple theories of ion flow through channels. Thus replacing external Cl– with resulted in "trans-inhibition" of Cl– efflux through CFTR. The inhibitory effect of was concentration-dependent, and ~70% of the CFTR conductance was inhibited in the presence of 140 mM . CFTR inhibition was not caused by changes in either extracellular or intracellular pH or in either the pCO2 or content of the -rich solutions. We proposed that this effect of on CFTR was a novel negative feedback mechanism for the control of secretion and thus the luminal surface pH in epithelia (26). That external anions can modulate CFTR function is also supported by the recent report of Shcheynikov et al. (27). These authors found that reducing to below 20 mM caused a remarkable time-dependent increase in the OH–/ permeability of oocytes transfected with CFTR. This switch in selectivity was suggested to arise from a conformational change in the CFTR protein, which may involve an external Cl– binding site on the ion channel (27). This effect of is consistent with our previous proposal (26) that an extracellular "anion" binding site on CFTR is important for modulating channel function.

The main aim of this study was to clarify whether the inhibitory effect of on CFTR that we previously observed was specific to the pancreatic duct. We did this by working with human CFTR heterologously expressed in Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cells. We also wanted to determine whether the trans-inhibition of CFTR is caused solely by the increase in external concentration, by the reciprocal fall in , or by both mechanisms. And finally we wished to investigate whether trans-inhibition was due to an effect on channel gating or on the rate of Cl– permeation through the CFTR pore. To do this, we employed excised outside-out membrane patches and studied the effects of changing on single channel activity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Electrophysiology—CHO and BHK cells, stably transfected with human wild type CFTR, were grown as previously described (2, 28). Cells were cultured on glass coverslips for use in patch clamp experiments between 2 and 5 days after plating. Whole cell patch clamp recordings were carried out at room temperature as described previously (26). An EPC-7 amplifier (List Electronic, Darmstadt, Germany) was used to record currents from CHO or BHK cells. Two configurations of the whole cell technique were used: 1) fWCR in which physical and electrical access to the cell is obtained by rupturing the patch of membrane underneath the patch pipette and 2) the slow whole cell (sWCR), a perforated patch recording technique in which a pore-forming antibiotic is included in the pipette solution. In this case, electrical access is obtained after sufficient integration of the antibiotic into the plasma membrane. In both configurations, whole cell currents were monitored using two different voltage protocols. For monitoring time-dependent changes in currents, the membrane potential (Vm) was held at 0 mV and alternately clamped to ±60 mV for 1 s with a 1-s hold at 0 mV between each pulse. Steady-state current-voltage (I/V) relationships were obtained by holding Vm at 0 mV and clamping to ±100 mV in 20-mV increments for 500 ms with an 800-ms interval between each pulse. Data were filtered at 1 kHz and sampled at 2 kHz with a CED 1401 interface (Cambridge Electronic Design, Cambridge, United Kingdom). Reversal potentials (Erev) and current densities were determined from I/V plots after fitting a third order polynomial using regression analysis. Mean current amplitudes were calculated at Erev ± 60 mV and normalized to cell capacitance (pF) measured using the EPC-7 amplifier. The relative permeability (Px–/PCl–) for each anion (X–) compared with Cl– was determined from changes in Erev (26). Outside out, single channel experiments were conducted on BHK cells at room temperature as described previously (28) at a Vm of –50 mV. Single channel currents were recorded using an Axopatch 1D amplifier (Axon Instruments, Union City, CA), filtered at 50 Hz, and digitized at 1 kHz using pCLAMP8 software (Axon Instruments). Channel activity was quantified by measuring the mean number of channels open (NPo) during recordings lasting between 1–3 min at each Cl– concentration. In all of the experiments, Vm was corrected for liquid junction potentials.

Solutions—For fWCR, the standard pipette solution contained as follows: 110 mM CsCl; 5 mM EGTA; 10 mM HEPES; 2 mM MgCl2; and 1 mM Na2ATP (pH 7.2 with CsOH) (240 mosm). For experiments in which intracellular [Cl–] was varied, CsCl was replaced with equimolar N-methyl-D-glucamine-Cl) or iso-osmolar mannitol. To achieve a free Ca2+ concentration in the pipette solution of 10–7 M, 2.12 mM CaCl2 was added to the standard solution. For perforated patch sWCR, 240 µg/ml amphotericin B was added to the standard pipette solution with the Na2ATP omitted. The pipette solution used in outside out patch experiments contained the following: 110 mM CsCl; 10 mM HEPES; 5 mM EGTA; 1 mM MgCl2; 2.12 mM CaCl2; 1 mM MgATP; and 50 nM protein kinase A (Promega, Madison, WI), pH 7.2 with CsOH. The standard bath solution in all of the experiments contained 145 mM NaCl, 4.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 5 mM glucose, pH 7.4 with NaOH. For anion replacement studies, NaCl in the standard bath solution was replaced with equimolar concentrations of sodium , Br–, NO–3, or aspartate–. When using NaHCO3 solutions, CaCl2 was omitted to prevent precipitation. containing solutions were not routinely gassed with CO2, so their pH was higher than that of the standard bath solution (pH 7.4). For the reduced [Cl–] bath solutions, NaCl was removed from the standard bath solution and replaced with sufficient mannitol to maintain osmolarity as measured using an osmometer. 2 mM CaCl2 was omitted to ensure that the solution was comparable with the substitution experiments. During WCR, CFTR was activated using 5 µM forskolin (Tocris, Avonmouth, United Kingdom).

Statistics—Data are presented as mean ± S.E. The significance of difference between groups was determined using Student's paired or unpaired t tests where appropriate. Alternatively for multiple comparisons, the data were analyzed using one-way analysis of variance (ANOVA). A probability of p ≤0.05 was considered statistically significant.

Chemicals—Stock solutions of forskolin (50 mM) were made up in 100% ethanol. Unless otherwise stated, all of the chemicals were from Sigma.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
External HCO–3 Inhibits Human CFTR Currents—Results presented in Fig. 1 demonstrate that extracellular inhibits human CFTR expressed in CHO or BHK cells in a manner similar to that observed for CFTR in native guinea pig pancreatic duct cells (26). The data shows dose-response curves for the inhibitory effect of external on human CFTR currents expressed in CHO cells (open circles) and for guinea pig CFTR (closed circles). The data are expressed as the percent inhibition of the inward current measured at Erev –60 mV using fWCR, and the curves have been fit to a simple one-site Michaelis-Menten equation. Overall, there is no significant difference in the maximal inhibition achieved between species (human, 70 ± 10.3%; guinea pig, 65 ± 4.0%); however, human CFTR had a significantly higher Km value (human, 37.4 ± 14; guinea pig, 5.2 ± 1.4 mM), suggesting that human CFTR is less sensitive to block by external . CFTR expressed in BHK cells (Fig. 1, closed triangle) behaved in a similar fashion to CFTR expressed in CHO cells. In all of the cases, the block by external was voltage-independent over the potential range ±100 mV and fully reversible (data not shown), similar to our previous results from guinea pig duct cells (26).



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  		FIG. 1.
Human and guinea pig CFTR are inhibited by raising external HCO–3 concentration. Concentration-response curves showing the inhibition of the forskolin activated inward CFTR current (Cl– efflux) over a range of external concentrations. Mean percent inhibition was calculated from I/V data obtained at Erev –60 mV using the fast whole cell recording technique. Closed circles, data from guinea pig pancreatic duct cells (n = 9–17). Open circles, data from CHO cells stably transfected with CFTR (n = 5–18). Closed triangle, data from BHK cells stably transfected with CFTR (n = 3). Data for guinea pig adapted from O'Reilly et al. (26).

 
Effect of Other External Anions on CFTR Currents in CHO Cells—Trans-inhibition of CFTR currents was also observed with Br–, aspartate–, and NO–3 (Table I). The efficacy sequence was = Br– = aspartate– > NO–3 (Table I). The extent of trans-inhibition caused by these anions was not obviously related to their relative permeabilities (Table I) as calculated from reversal potential shifts. For instance, Br– is more permeant through CFTR than (Px–/PCl– = 1.53) yet has an equivalent inhibitory effect on inward currents.


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  		TABLE I
Effect of external anion substitution on inward CFTR current and relative anion selectivity

{Delta}Erev values represent the mean change in reversal potential following substitution of 100 mM NaCl with other anions and were obtained from I/V plots. Relative permeability (Px–/PCl–) and percent inhibition of inward current were determined as stated under "Experimental Procedures." Asp– represents aspartate. Note that the mean shift in Erev following substitution of 100 mM Cl– with is much lower than the 27.8 mV predicted by the Nernst equation for a Cl–-selective current. Some of this difference can be explained by the fact that can permeate CFTR, albeit to a lesser extent than Cl– (26, 27, 32). However, in additional experiments in which the CsCl was replaced by NMDG-Cl in the pipette solution (i.e. no internal permeant cations), the replacement of 100 mM with caused Erev to shift by 19.7 ± 3.2 mV (n = 4), which is much closer to the predicted shift. However, the mean inhibition of inward current caused by the replacement under these conditions was not significantly different to the CsCl pipette experiments (38 ± 5.3% versus 50.2 ± 5.1%, measured at Erev –60 mV). This observation indicates that a component of the forskolin-activated whole-cell current in CHO cells is cation-selective. However, this does not appear to prevent or affect the trans-inhibition of CFTR by changes in the extracellular anionic environment.

 
External Cl– Concentration Underlies the Inhibition of CFTR in CHO Cells—All of the anions tested caused some inhibition of the inward CFTR Cl– current (Table I), suggesting that trans-inhibition of CFTR was not anion-specific. To investigate this further, was reduced without replacing the ionic component and osmolarity maintained at 300 mosm by the addition of mannitol. As shown in Fig. 2, A and B, using fWCR both the outward and inward CFTR currents were inhibited by the replacement of 100 mM with mannitol. In this representative experiment, the inward CFTR Cl– current was reduced by 57.9% at Erev –60 mV in the presence of 51.5 mM compared with the current magnitude in the presence of 155.5 mM . Note that the degree of CFTR inhibition is very similar regardless of whether extracellular Cl– is replaced with mannitol or with (compare Figs. 1 and 2A). Fig. 2B shows I/V plots for the experiment illustrated in Fig. 2A. In these experiments, the average change in Erev following replacement of 100 mM extracellular Cl– with mannitol was 12.8 ± 0.8 mV (n = 6), somewhat less than the predicted value (27.8 mV), which is probably explained by the presence of a cation conductance in the CHO cells (see legend to Table I). Fig. 2C shows the Cl– dose-inhibition curve for inward CFTR currents measured using fast WCR when extracellular NaCl is replaced with mannitol. Note that the plot is curvilinear and appears biphasic. The degree of current inhibition increased markedly to ~50% as was reduced from 155 to 71.5 mM, but further reduction in to 10.5 mM caused rather less inhibition (Fig. 2C). Note that a substantial fraction (~30%) of the CFTR current could not be inhibited, even when was reduced to 10.5 mM.



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  		FIG. 2.
Trans-inhibition of CFTR is observed when external alone is reduced. Representative fWCR current recordings from CHO cells transfected with CFTR measured between ±100 mV in 20-mV steps. A(i), unstimulated current records; (ii), currents following stimulation with 5 µM forskolin; (iii), stimulated currents after was reduced to 51.5 mM (the removal of 100 mM NaCl and replaced with mannitol). B, corresponding I/V relationship for this experiment. Filled circles represent the base-line current, open circles represent the forskolin-stimulated current in the presence of 151.5 mM bath Cl–, and inverted triangles represent the stimulated current in the presence of 51.5 mM bath Cl–. C, chloride concentration dose-response curve showing the inhibition of the forskolin-activated current over a range of with mannitol replacement. Percent inhibition was calculated from I/V data at Erev –60 mV using the fast whole cell recording technique (n = 4–7).

 
Internal Cl– Concentration Does Not Modulate CFTR in CHO Cells—We next used fWCR to investigate whether a reduction in internal [Cl–] also regulated CFTR. The intracellular [Cl–] in epithelial cells is usually in the 30–60 mM range (29, 30). Fig. 3A shows that we could not detect any significant change in forskolin-stimulated CFTR current density when pipette [Cl–] was either raised above or decreased below the standard value of 114 mM. Thus changes in internal [Cl–] down to 54 mM has no effect on Cl– influx (outward current), in marked contrast to the effect of lowering external [Cl–] by this amount on Cl– efflux (inward current). Lowering cytosolic [Cl–] also had no marked effect on inward current. In the presence of 54, 79, 114, and 154 mM internal Cl–, the mean current densities measured at Erev –60 mV were: –202.1 ± 76.5 (n = 5); –269.9 ± 95.4 (n = 3); –180.33 ± 30.8 (n = 10); and –275.0 ± 99 (n = 8) pA/pF, respectively (p = 0.97, Fig. 3A). Because the Km of CFTR for internal Cl– is ~38 mM (31), one might expect some reduction in inward CFTR conductance at the lowest internal [Cl–] used in our experiments. The reason why we do not see a change is unclear, but we do not think it is because cytosolic Cl– was not being fixed adequately in our experiments since the zero current Erev measurement for the 54 mM internal Cl– data set (–11.7 ± 2.2 mV) is significantly different to the experiments employing 79 (–4.27 ± 2.3 mV, p < 0.05), 114 (–1.24 ± 0.75 mV, p < 0.001), and 154 mM internal Cl– (–0.7 ± 0.5 mV, p < 0.001).



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  		FIG. 3.
Internal Cl– concentration does not modulate CFTR in CHO cells. The concentration of CsCl in the pipette solution was varied and, where appropriate, replaced by iso-osmolar mannitol. A, mean-stimulated current density (pA/pF), measured at Erev ± 60 mV, using the fast whole cell recording technique at different internal [Cl–] but constant external [Cl–] of 155.5 mM. B, mean percent inhibition of inward CFTR current density following exposure to 51.5 mM external Cl– at different internal [Cl–]. Percent inhibition was determined from I/V relationships at Erev –60 mV and were not found to be significantly different from the control value (114 mM internal Cl–).

 
Importantly, the degree of trans-inhibition of CFTR currents following reduction in was not affected by these changes in internal [Cl–] (Fig. 3B). The mean percent inhibition of the inward current in the presence of 54, 79, 114, and 154 mM internal Cl– following the replacement of 100 mM external [Cl–] with mannitol was 49.8 ± 9.8 (n = 3), 71.5 (n = 2), 63.2 ± 7.0 (n = 7), and 54.9 ± 9.2% (n = 8), respectively (p = 0.96). Taken together, these observations suggest that trans-inhibition of CFTR is mediated solely by the concentration of external Cl– and that neither the internal [Cl–] nor the [Cl–] gradient across the cell membrane are important factors.

External Cl– Inhibits CFTR Activity in Cell-free Membrane Patches from BHK Cells—To gain insight into the mechanism of the effect, we performed similar experiments using cell-free outside out membrane patches. Because of the density of channels in the transfected cells, only multi-CFTR-containing patches were obtained; therefore, kinetic analysis was limited to a comparison of the channel activity (NPo) before and after changes in external Cl– concentration. Nonetheless, Fig. 4A shows that a reduction in from 155.5 to 35.5 mM led to a marked decrease in the probability of channel opening without any obvious change in the size of the single channel currents. In three experiments, NPo decreased by 82.9 ± 12% and Student's paired t tests showed a highly significant change in this parameter (Fig. 4B, p = 0.012). The reverse experiment was also conducted in which the bath solution was changed from the lower to higher Cl– concentration, and results are summarized in Fig. 4C. From four such experiments, there was a significant change in NPo (p = 0.013) and an overall 62.3 ± 11.1% increase in channel activity, a value not significantly different to that obtained for the high to low experiments. Therefore, these results suggest that the marked inhibition in whole cell inward current upon bath anion-replacement depicted in Figs. 1 and 2 is primarily due to a reduction in open state probability of CFTR channels.



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  		FIG. 4.
External Cl– modulates CFTR gating. Cell-free, outside out patch clamp data obtained from BHK cells stably transfected with CFTR at a membrane potential of –50 mV. A, lefthand trace shows single channel records obtained in the presence of 155.5 mM . Right-hand trace shows current records after reducing to 35.5 mM in the same experiment. Expanded sections at higher time base are also shown to illustrate the marked change in CFTR channel activity. Solid lines to the left of the traces represent the current level when all of the channels are closed. B, plot of NP in the presence of high and low . In separate experiments, patches were also excised into bath solutions containing 35.5 mM and then exposed to the 155.5 mM bath solution (C). NPo data were obtained from current records lasting between 1 and 3 min under each condition (see "Experimental Procedures").

 
Recording Configuration Affects the Regulation of CFTR by External Cl– in CHO Cells—All of the experiments described so far were performed under situations in which the cytosolic solution was controlled by the composition of the pipette solution. The question we next asked was whether CFTR inhibition by changes in also occurred under more physiological conditions where the cytoplasmic compartment remains intact. To address this possibility, we turned to the sWCR-perforated patch recording technique (see "Experimental Procedures"). Using this approach, CFTR current density following stimulation with 5 µM forskolin was approximately twice as large as that found for fWCR (sWCR, 440.6 ± 57.7 and –387.9 ± 46.4 pA/pF (n = 14); fWCR, 238.2 ± 39.3 and –186.3 ± 25.1 pA/pF (n = 19) at Erev +60 mV and Erev –60 mV, respectively (p < 0.01 at both voltages)), indicating that cytoplasmic integrity is important for full CFTR current activation.

Fig. 5 shows the dose-inhibition curve for obtained using sWCR (closed triangles) with being replaced iso-osmotically with mannitol. For comparison, fWCR data (closed circles), previously shown in Fig. 2C, are also included. In contrast to the data obtained in fWCR, the dose-inhibition curve for sWCR clearly shows a different profile. In sWCR, the percent reduction of inward CFTR current following a decrease in was significantly less over the range 116.5–71.5 mM Cl–. Moreover, the concentration of causing a 50% inhibition of the forskolin-stimulated CFTR current at Erev –60 mV in sWCR was 31.8 mM compared with a value of 77.5 mM in fWCR, giving a leftward shift of the dose-inhibition curve of ~47 mM. Nevertheless, the maximum degree of CFTR current inhibition was similar in fast and slow WCR (Fig. 5). The same difference in the Cl– dose-inhibition curves for fast and slow WCR was observed when extracellular Cl– was replaced with . In sWCR, the response curve was shifted to the left by ~39 mM (data not shown).



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  		FIG. 5.
Inhibition of inward CFTR current by is dependent on recording configuration and intracellular Ca2+ concentration from CHO cells stably transfected with CFTR. Data shows the Cl– concentration-response curve for the fWCR data shown in Fig. 2C, filled circles. Results from similar experiments using the sWCR configuration are also shown (filled triangles) (n = 3–7). Slow WCRs were made with amphotericin B in the pipette solution (see "Experimental Procedures"). The mean percent inhibition was determined from I/V data at Erev –60 mV. Asterisk indicates data for which there is a significant difference between the fast and slow WCR (p ≤ 0.05). In separate fWCR experiments, the free intracellular [Ca2+] in the pipette solution was fixed to 10–7 M (open circles). There was no significant difference between this data set and that obtained for sWCR.

 
Internal Ca2+ Concentration Modulates Trans-inhibition in CHO Cells—The pipette solutions used for the fWCR experiments contained 5 mM EGTA, which will buffer the intracellular free [Ca2+] at ~10–9 M. However, EGTA is unable to cross the amphotericin-permeabilized membrane during sWCR; thus the internal free [Ca2+] in these experiments is unknown but is expected to be within the physiological range. To determine whether internal free [Ca2+] was modulating the inhibitory response, cytosolic Ca2+ was fixed at a more physiological resting value of 10–7 M during fWCR experiments (see "Experimental Procedures").

Fig. 5 (open circles) shows that increasing the intracellular [Ca2+] from 10–9 to 10–7 M in fWCR changes the shape of the dose-inhibition response to depletion. When intracellular Ca2+ is raised, the inhibition curve moves to the left, closely resembling that obtained in sWCR (Fig. 5, triangles). In fWCR, with 10–7 M Ca2+ in the pipette, the mean percent inhibition of the inward CFTR current at Erev –60 mV was 9.3 ± 7.7%, 23.5 ± 3.5%, and 30.7 ± 4.9% at 116.5, 91.5, and 71.5 mM , respectively (n = 3–7). Two of these values (116.5 and 71.5 mM Cl–) are significantly different from the values obtained in fWCR with 10–9 M Ca2+ in the pipette solution (p = 0.005 and 0.01, respectively, at Erev –60 mV), and the other value (91.5 mM Cl–) just fails to reach significance (p = 0.06). These results suggest that differences in the cytoplasmic Ca2+ concentration are a major factor contributing to the apparent difference between trans-inhibition observed using fast and slow WCR.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The external face of CFTR is exposed to wide variations in luminal pH, [], and [Cl–] that occur normally at the epithelial cell surface. We have asked whether such changes in the composition of surface/secreted fluids can influence the activity of CFTR. Here we show that human CFTR expressed in CHO or BHK cells is inhibited by a reduction in external Cl– through a mechanism that can be modulated by internal [Ca2+]. How such a signal is transmitted across the luminal membrane remains to be explained. Our data show that the replacement of extracellular Cl– with another anion (as will occur physiologically when secretion occurs) causes a trans-inhibition of Cl– efflux through CFTR. Trans-inhibition of CFTR currents was also observed in fWCR following isoosmotic replacement of extracellular NaCl with mannitol. This finding suggests that a reduction in external Cl– per se is the factor that causes inhibition. Similar changes in internal Cl– concentration had no effect on CFTR activity, illustrating the strict sidedness of the effect.

In fWCR, a substantial proportion of the CFTR current (~45%) was inhibited when extracellular Cl– was reduced from a supra-physiological value of 155 mM to the normal plasma value of ~110 mM. Further reduction in extracellular Cl– below 110 mM (as will occur in the luminal secretions produced by -transporting epithelia) caused little additional inhibition of the CFTR channels. A substantial proportion of the CFTR current (~30%) remained, even when external Cl– was reduced to 10.5 mM, which is approximately the minimum value observed in pancreatic juice (21). Under the more physiological conditions of the sWCR configuration where the cytoplasmic compartment remains essentially intact, we found that the Cl– dose-inhibition curve was shifted to the left by ~45 mM. Thus needed to be reduced to ~32 mM to cause a 50% inhibition of CFTR current in sWCR compared with ~78 mM in fWCR. This means that, in sWCR, the inhibitory effect of Cl– removal is more apparent over the normal range of Cl– concentrations found in the fluids secreted by epithelia. Nevertheless, the maximal levels of CFTR current inhibition were similar in fast and slow WCR. Increasing the intracellular [Ca2+] to physiologically relevant resting levels (100 nM) during fWCR shifted the Cl– dose-inhibition curve to the left mimicking the curve obtained using sWCR. The mechanism by which an increase in cytosolic Ca2+ blunts the response of CFTR to reduced is unclear. CFTR activity has not been shown to be directly dependent on Ca2+ (32) and thus the effect is likely to be indirect. Calcium could modulate the interaction of CFTR with regulatory protein kinases, such as protein kinase C, which is known to enhance CFTR activity through the facilitation of CFTR phosphorylation by PKA (4, 32). Alternatively, Ca2+ may alter the interaction of CFTR with regulatory protein phosphatases (2, 8, 9, 32).

To gain some insight into the mechanism of inhibition of CFTR by , outside out single channel patch experiments were employed. The results presented in Fig. 4 show that reducing to 35.5 mM markedly decreased channel open probability with little change in unitary current amplitude. Conversely NPo was increased when the was changed from low to high levels, indicating that primarily affects CFTR gating rather than permeation. Overall, the percent change in channel activity from these single channel experiments (~70%) is consistent with the whole cell results for the same change in external Cl–. How brings about this specific effect is unknown, but modulation of ATP binding and/or hydrolysis at the nucleotide binding domains of CFTR is one possibility. We found that including 5 mM pyrophosphate in the pipette solution during fWCR experiments to lock open CFTR (33) did not affect the ability of low to inhibit CFTR (data not shown). This finding suggests that the gating effect we observe is not because of a change in open-time duration and thus probably occurs by stabilizing the closed state of the channel (32). Gating of the CFTR channel is also known to be affected by phosphorylation/dephosphorylation of the R domain (2–4, 8, 9), and a Cl–-dependent conformational change in CFTR could well affect the phosphorylation status of CFTR, which is known to alter channel activity (32). That changes in can induce a structural change in CFTR is supported by the recent data of Shcheynikov et al. (27). Indeed, the phosphorylation of the R domain itself has been shown to cause a pronounced conformational change in CFTR monitored by fluorescence spectroscopy (34). Clearly, establishing whether changes in alter the structure of CFTR is an important area for future work.

The mechanism by which the CFTR protein senses remains to be determined. Our observation that the inhibitory effect of anion substitution and low on CFTR currents was voltage-independent argues against a Cl– sensor being located within the selectivity filter of the channel pore. This is in marked contrast to the Cl–-dependent gating of the ClC channel pore (35, 36). There are a number of positively charged amino acid residues on the extracellular loops of CFTR, e.g. Arg-104, Lys-114, and Arg-117 in extracellular loop (EL) 1; Lys-329 in EL3; Lys-892, His-897, and Arg-899 in EL4; and Arg-1128 in EL6. It is possible that one or more of these residues form a Cl– sensor. The disease-causing mutation R117H is known to reduce the open state probability of CFTR by ~30% (37). However, Gong and Linsdell (38) have identified Arg-334, which is probably located at the outer mouth of the CFTR pore as an important arginine residue involved in high affinity Cl– binding. Because this residue is very important in coordinating ion-ion interactions, it may well have a role to play in the Cl–-sensing mechanism.

Although the exact mechanism of epithelial secretion is still not fully understood, it is generally accepted that CFTR has a fundamental role in this process (10, 21–27, 29, 39). Physiologically, we propose that, during agonist-stimulated anion secretion, CFTR activity is reduced by the reciprocal changes in luminal Cl– and concentrations. The reduction in CFTR activity as falls would help to maintain the electrical driving force for anion exit by preventing excessive depolarization of the apical membrane. This effect, together with the recently described increase in permeability of CFTR at low (27) would help to drive net secretion. Thus changes in luminal [Cl–] has two major effects on CFTR. It modulates both gating and anion permeability and therefore represents an important novel signal for regulating anion and fluid secretion in epithelial cells.


   FOOTNOTES
 
* This work was supported by a Wellcome Trust project grant (to M. A. G. and B. E. A.), a Canadian Institutes of Health research grant (to P. L.), and by a Wellcome Trust and CF Trust project grant (to A. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** To whom correspondence should be addressed. Tel.: 44-191-222-7592; Fax: 44-191-222-6706; E-mail: m.a.gray{at}ncl.ac.uk.

1 The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; CF, cystic fibrosis; , external Cl– concentration; , external concentration; CHO, ovary; Chinese hamster BHK, baby hamster kidney; fWCR, fast whole cell patch clamp recording; sWCR, slow whole cell patch clamp recording; I/V, current/voltage; Erev, reversal potential; NPo, number of channels multiplied by channel open probability; EL, extracellular loop of the CFTR protein. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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