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Am J Physiol Cell Physiol 275: C1040-C1047, 1998;
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Vol. 275, Issue 4, C1040-C1047, October 1998

Cytosolic pH regulates GCl through control of phosphorylation states of CFTR

M. M. Reddy1, Ron R. Kopito2, and P. M. Quinton1

1 Department of Pediatrics, School of Medicine, University of California, San Diego, La Jolla 92093-0831; and 2 Department of Biology, Stanford University, Palo Alto, California 94305

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Our objective in this study was to determine the effect of changes in luminal and cytoplasmic pH on cystic fibrosis transmembrane regulator (CFTR) Cl- conductance (GCl). We monitored CFTR GCl in the apical membranes of sweat ducts as reflected by Cl- diffusion potentials (VCl) and transepithelial conductance (GCl). We found that luminal pH (5.0-8.5) had little effect on the cAMP/ATP-activated CFTR GCl, showing that CFTR GCl is maintained over a broad range of extracellular pH in which it functions physiologically. However, we found that phosphorylation activation of CFTR GCl is sensitive to intracellular pH. That is, in the presence of cAMP and ATP [adenosine 5'-O-(3-thiotriphosphate)], CFTR could be phosphorylated at physiological pH (6.8) but not at low pH (~5.5). On the other hand, basic pH prevented endogenous phosphatase(s) from dephosphorylating CFTR.After phosphorylation of CFTR with cAMP and ATP, CFTR GCl is normally deactivated within 1 min after cAMP is removed, even in the presence of 5 mM ATP. This deactivation was due to an increase in endogenous phosphatase activity relative to kinase activity, since it was reversed by the reapplication of ATP and cAMP. However, increasing cytoplasmic pH significantly delayed the deactivation of CFTR GCl in a dose-dependent manner, indicating inhibition of dephosphorylation. We conclude that CFTR GCl may be regulated via shifts in cytoplasmic pH that mediate reciprocal control of endogenous kinase and phosphatase activities. Luminal pH probably has little direct effect on these mechanisms. This regulation of CFTR may be important in shifting electrolyte transport in the duct from conductive to nonconductive modes.

sweat duct; phosphatase; kinase; chloride transport; chloride conductance; fluid transport; electrolyte transport; sweat gland; cystic fibrosis

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

A PRIME PHYSIOLOGICAL function of the human sweat duct is to reabsorb NaCl from the isotonic primary sweat secreted by the sweat gland secretory coil. As the primary sweat secreted by the secretory coil enters the lumen of the absorptive duct, Na+ is reabsorbed down a persistent electrochemical gradient via an amiloride-sensitive Na+ channel in the apical membrane (17, 18, 22). At least part of the control of absorption of NaCl from the duct is exerted through regulation of cystic fibrosis transmembrane conductance regulator (CFTR), the Cl- channel through which Cl- passes during this process.

Recently, we demonstrated that the electrochemical driving force for Cl- (Delta µ Cl-) across the apical and basolateral membranes is a function of luminal salt concentration (18, 22), which can change rapidly from essentially isotonic to <15 mM in vivo (18, 22). Accordingly, the Delta µ Cl- may be favorable for absorption at high luminal salt concentration (i.e., >50 mM) or paradoxically unfavorable at low luminal salt concentration (i.e., <50 mM). A necessary consequence of such changes in Delta µ Cl- across the duct epithelium is that Cl- be absorbed by electroconductive Cl- channels when the salt concentration is high enough to favor passive Cl- diffusion across the apical membrane through CFTR Cl- channels and by other transporter(s) or mechanisms when the salt concentration is low and the Delta µ Cl- is unfavorable for passive Cl- diffusion into the cell (22). Because intracellular Cl- activity is above the level required for passive distribution across the cell membranes at physiologically low luminal salt concentration (22), the electroconductive Cl- shunt through CFTR must close to prevent secretion of Cl- into the lumen (22-24). However, little is known about the physiological processes that couple luminal salt concentration to activation/deactivation of CFTR Cl- conductance (GCl).

Because the luminal pH (4.5-7.8) and salt concentration [~10-100 mM (3)] decrease with decreasing sweat secretory rates, one signal that might link changing luminal salt concentration to CFTR GCl activation could be a change in extra- and/or intracellular pH. Acidic pH was also shown to affect CFTR channel function in ex vivo model systems (27). Therefore, we hypothesized that a change in luminal and/or cytosolic pH after reduced luminal Cl- concentration would deactivate CFTR Cl- channels, facilitating another nonconductive process for Cl- absorption.

The topic assumes added significance, since CFTR Cl- channels are expressed in other membranes that also may experience pH environments ranging from the extremely acidic compartments of intracellular organelles such as endosomes (1, 15, 29) to the highly basic lumen of pancreatic ducts (10, 26). In addition, sweat duct cells, which are extremely rich in CFTR (17, 21), seem to present a unique opportunity to investigate pH effects, since large fluctuations in pH occur in the luminal and cytosolic compartments, depending on the status of physiological stimulation (7). The effect of these in vivo pH environments on the functional properties of CFTR in a native epithelial membrane is unknown. Therefore, we sought to 1) determine possible physiological mechanisms for deactivating the Cl- shunt in the sweat duct and 2) better understand the effect of such extreme pH changes on CFTR GCl.

The basolaterally alpha -toxin-permeabilized sweat duct preparation offers a good opportunity to study pH regulation of apical CFTR GCl, because 1) the apical membrane is rich in CFTR, which comprises most, if not all, GCl, such that changes in membrane GCl can be directly attributed to the activity of CFTR GCl, and 2) the cytoplasmic pH can be directly and freely manipulated through the alpha -toxin pores in the basolateral membrane. We show that, in the native sweat duct, luminal pH changes seem not to directly affect CFTR GCl but cytoplasmic pH changes may modulate CFTR GCl through a reciprocal control of relative kinase phosphorylation and phosphatase dephosphorylation of CFTR.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Tissue Acquisition

Sweat glands were obtained from adult male volunteers who gave informed consent. Full-thickness skin biopsies (3 mm diameter) were taken over the scapula and stored overnight in Ringer solution at room temperature. Individual sweat glands were isolated from the plug in cold Ringer solution by dissection with fine-tipped tweezers visualized at a magnification of ×80. The isolated glands were transferred to a dissection cuvette with Ringer solution cooled with a Peltier block to 4°C, where the segments of reabsorptive duct (>1 mm long) were separated from the secretory coil of the sweat gland. The sweat duct was transferred to a perfusion chamber containing Ringer solution at 35 ± 2°C.

Selective Permeabilization of the Basolateral Membrane

The basolateral membrane of the sweat duct was selectively permeabilized with the pore-forming agent alpha -toxin. alpha -Toxin (1,000 units) derived from Staphylococcus aureus in cytoplasmic Ringer solution containing 140 mM potassium gluconate and 5 mM ATP was applied to the basolateral surface of the microperfused sweat duct for 15-30 min. alpha -Toxin forms pores that pass molecules of 3,500-5,000 mol wt (19, 23), so that the concentration of intracellular molecules such as cAMP and ATP, as well as intracellular pH, could be directly controlled as a function of their concentration in the extracellular bath solution.

Intracellular pH

Qualitative changes in intracellular pH in response to luminal Cl- substitution were measured using a pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM (16). BCECF-AM permeates the cell membrane and is cleaved by endogenous esterases to impermeant BCECF with highly pH-sensitive fluorescence. We monitored the emitted fluorescence intensities from the dye at 505-560 nm after exciting it alternately at its isobestic point (435-440 nm) and then in a highly pH-sensitive region (505 nm). Cytoplasmic pH is indicated by the ratio of the intensity emitted when excited at 435 nm to the intensity emitted when excited at 535 nm. Changes in intracellular pH were monitored while the lumen was perfused at relatively high rates (>50 nl/min). High perfusion rates ensure that the composition of the perfusate changes insignificantly within the duct lumen. In this way, the compositions of the luminal and bath solutions are known and controlled.

Electrical Measurements

Electrical setup. After the lumen of the sweat duct was cannulated with a double-lumen cannula made from theta glass (1.5 mm diameter, Clark Electromedical Instruments, Reading, UK), a constant-current pulse of 50-100 nA for a duration of 0.5 s was applied through one barrel of the cannulating pipette containing NaCl-Ringer solution. The other barrel of the cannulating pipette served as an electrode for measuring transepithelial potential (Vt) with respect to the contraluminal bath and as a cannula for perfusing the lumen of the duct with selected solutions. Vt was monitored continuously using one channel of a dual electrometer (model WPI-700) referenced to the contraluminal bath. Transepithelial conductance (Gt) was measured as described earlier (17, 21, 23) from the amplitude of transepithelial voltage deflections in response to 50- to 100-nA transepithelial constant-current pulses using a cable equation.

Apical GCl. Cl- diffusion potential (VCl) and GCl were monitored to indicate the level of activation of GCl. After alpha -toxin permeabilization of the basolateral membrane, the epithelium is simplified to a single (apical) membrane with parallel Na+ and Cl- conductances (19, 20, 23). Application of amiloride further limited the system to that of a predominantly Cl--selective membrane. The composition of Ringer solution in bath and lumen was designed to create single ion permeation of the membrane for Cl- [140 mM potassium gluconate (bath)/150 mM NaCl + 10-5 M amiloride (lumen)]. Under these conditions the Vt and Gt can be regarded essentially as VCl and GCl, respectively.

Solutions

The luminal perfusion Ringer solutions contained (in mM) 150 NaCl, 5 K, 3.5 PO4, 1.2 MgSO4, 1.0 Ca2+, and 0.01 amiloride (pH 7.4). The cytoplasmic/bath solution contained (in mM) 145 K, 140 gluconate, 3.5 PO4, and 1.2 MgSO4 and 260 µM Ca2+ buffered with 2.0 mM EGTA (Sigma Chemical) to 80 nM free Ca2+ (pH 6.8). The effect of luminal or cytoplasmic pH on CFTR GCl was evaluated by directly manipulating bath (cytoplasmic) or luminal pH from 4.5 to 9.0 under activated (0.1 mM cAMP/5 mM ATP) and deactivated conditions (with ATP but without cAMP, with cAMP but without ATP, or without both ATP and cAMP).

Data Analysis

Values are means ± SE (where n is the number of ducts from >= 4 subjects). Statistical significance was determined on the basis of Student's t-test for paired samples. P < 0.05 indicated a statistically significant difference. The data presented as electrophysiological traces are representative of experiments repeated at least three times.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Effect of Luminal pH on CFTR GCl

We investigated the effect of changing luminal pH on the apical CFTR GCl. While maintaining constant pH in the cytoplasmic bath, changing luminal pH between 5.0 to 8.5 had virtually no effect on activated (0.1 mM cAMP and 5 mM ATP) or nonactivated CFTR GCl (Fig. 1).


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  		Fig. 1.   Lack of effect of luminal pH on cystic fibrosis transmembrane conductance regulator (CFTR) Cl- conductance (GCl). Cytoplasmic pH was maintained at 6.8 while luminal pH was manipulated from 8.5 to 5.0. Neither transepithelial GCl nor Cl- diffusion potentials (VCl) are affected by changes in luminal pH. Initial transient changes in transepithelial potential after different luminal pH solution changes reflect junction potentials that were mimicked by similar luminal pH solution changes after sweat duct was removed from perfusion pipette.

Effect of Luminal Cl- Concentration on Cytoplasmic pH

We examined the effect of changing luminal Cl- concentration on the cytoplasmic pH. We substituted luminal Cl- with the impermeable anion gluconate and followed the cytoplasmic pH of nonpermeabilized duct cells. Luminal Cl- substitution consistently acidified cytoplasmic pH (Fig. 2). The changes in cytoplasmic pH occurred within seconds and were fully reversible on reintroduction of Cl- into the lumen. Even though we did not follow the steady-state effect of luminal Cl- substitution on changes in pHi, we found that cytosolic acidification was maintained for >10 min (duration of observation, Fig. 2).


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  		Fig. 2.   Effect of luminal Cl- concentration on cytosolic pH of a sweat duct cell. Fluorescence studies indicated that cytosolic pH is coupled to luminal Cl- concentration. Effect of luminal Cl- on cytosolic pH was tested by replacing Cl- with impermeant anion gluconate. Substitution of Cl- by gluconate significantly lowered cytosolic pH. Physiologically, luminal Cl- concentration can vary from <15 to >100 meq, depending on conditions in vitro. A reduction in luminal Cl- concentration caused a reduction in intracellular Cl- (18, 22). Mechanism by which luminal Cl- effects intracellular pH changes is not known, but these observations show that intracellular pH can change with luminal Cl- in vivo. Data are representative of 4 such measurements from as many ducts. 505 I/439 I, ratio of fluorescence intensity at 505 nm to fluorescence intensity at 439 nm.

Effect of Cytoplasmic pH on CFTR GCl

Changes in cytoplasmic pH had little effect on nonactivated CFTR (i.e., in the absence of cAMP and ATP). However, changes in cytoplasmic pH had significant effects on cAMP/ATP-activated CFTR GCl. The dose-response relationship of the magnitude of activated CFTR GCl vs. cytosolic pH clearly showed that the CFTR GCl activity is a function of cytosolic pH. We found that acidic pH inhibited, while higher pH enhanced, CFTR GCl (Figs. 3 and 4). Although we used a wide range of pH to maximize the effect, CFTR GCl activity is clearly sensitive to cytosolic pH changes within a narrower, more physiological range (Fig. 4). An earlier study (7) revealed that, unlike most cell types, the cytosolic pH of sweat duct cells seems to fluctuate over a wide range (~1 pH unit) depending on the status of stimulation. Therefore, although the experimental range of intracellular pH changes imposed on the tissue in these studies was large, it may not be aphysiological.


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  		Fig. 3.   Reversible effect of pH on CFTR GCl activity. CFTR GCl activity in apical membranes of native sweat ducts is reversibly activated and deactivated by basic and acidic cytoplasmic pH, respectively. Cytosolic pH of basolaterally permeabilized duct was directly manipulated by perfusing cytoplasmic bath with Ringer solution buffered to different pH values. Acidic pH deactivates and basic pH activates CFTR GCl in presence of 0.1 mM cAMP and 5 mM ATP in cytoplasmic bath. Relative activation (or deactivation) of CFTR GCl is indicated by magnitude of VCl and transapical GCl. GCl is inversely related to voltage deflections in response to transepithelial constant-current pulses. Lumen was continuously perfused with 150 mM NaCl + 10-5 M amiloride so that apical membrane conductance reflects changes in CFTR GCl only, not in Na+ conductance.


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  		Fig. 4.   Dose-response relationship for effect of cytosolic pH on CFTR GCl. pH response data are taken from experiments similar to that described in Fig. 3. Basolateral membranes of sweat duct were permeabilized. CFTR GCl was activated by adding 0.1 mM cAMP and 5 mM ATP to cytoplasmic bath, then pH was incrementally increased from 5.0 to 8.0 in cytoplasmic bath while CFTR GCl was activated. CFTR GCl increases with increasing cytosolic pH.

Effect of Intracellular pH on Phosphorylated CFTR

Activation of CFTR GCl in the sweat duct requires protein kinase A (PKA) phosphorylation and a physiological concentration of ATP (19, 23). However, once CFTR is irreversibly phosphorylated, ATP alone can activate CFTR GCl. Therefore, activation of CFTR GCl by ATP without cAMP is a clear indication that CFTR has been irreversibly phosphorylated. CFTR can be irreversibly phosphorylated using different pharmacological tools (2, 23, 24). In these experiments we achieved irreversible phosphorylation of CFTR by 1) using phosphatase-resistant adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S) as substrate during cAMP-activated PKA phosphorylation of CFTR (2, 23, 24), 2) inhibiting endogenous phosphatases pharmacologically (24) with a phosphatase-inhibiting cocktail consisting of okadaic acid (10-6 M) and fluoride (5 mM), or 3) elevating cytoplasmic pH to 8.5 for 5-10 min (see DISCUSSION). Once CFTR is irreversibly phosphorylated, we were able to examine the effects of pH on ATP activation of CFTR GCl independent of effects of phosphorylation on the channel. We found that the ATP activation of irreversibly phosphorylated CFTR GCl was dependent on cytoplasmic pH (Figs. 5 and 6). Under these conditions, lowering pH inhibited and elevating pH activated CFTR GCl in the constant presence of ATP (Fig. 5). This activation of CFTR GCl was not affected by the presence of staurosporine, a nonspecific kinase inhibitor (Fig. 5).


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  		Fig. 5.   Irreversible phosphorylation of CFTR at basic cytoplasmic pH. CFTR was phosphorylated by first exposing apical membranes to 0.1 mM cAMP and 5 mM ATP at a basic pH of 8.5 for ~10 min. cAMP was then washed out. Normally, when CFTR is phosphorylated at pH 6.8 in presence of cAMP and ATP, CFTR GCl is deactivated immediately after cAMP washout, as shown in Fig. 10. However, when CFTR was phosphorylated at pH 8.5, as shown in this experiment, CFTR GCl remained activated as long as ATP was present in medium. Furthermore, CFTR remained phosphorylated (as indicated by ATP activation), even when pH of medium was reduced to control value of 6.8 as in Fig. 6, showing that CFTR was not dephosphorylated. To determine whether ATP activation of CFTR GCl involved any other kinase phosphorylation, we studied effect of nonspecific kinase inhibitor staurosporine on ATP activation of CFTR GCl previously phosphorylated in presence of cAMP at basic pH. Putatively inhibiting all kinases had little effect on ATP activation, indicating that CFTR became irreversibly phosphorylated at basic pH.


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  		Fig. 6.   Effect of pH on ATP activation of CFTR GCl. Sensitivity of ATP interaction with CFTR to changes in cytosolic pH is shown. CFTR was first irreversibly phosphorylated by applying 10-5 M cAMP + 5 mM ATP in cytoplasmic bath at pH 8.5. Once irreversibly phosphorylated, CFTR was activated by ATP alone (without cAMP), indicating that no additional protein kinase A phosphorylation was needed to activate CFTR GCl. ATP activation of irreversibly phosphorylated CFTR GCl in continued presence of ATP is dependent on cytoplasmic pH. Acidic pH inhibits ATP activation of CFTR after irreversible phosphorylation. These results show that, irrespective of effects of pH on kinases and phosphatases, acidic pH has a significant inhibitory effect on nucleotide activation of CFTR.

Effect of Lowering Cytoplasmic pH on Phosphorylation/Dephosphorylation of CFTR

We investigated the effect of lowering cytoplasmic pH on endogenous PKA phosphorylation and endogenous phosphatase dephosphorylation of CFTR.

Effect of Lowering pH on PKA Phosphorylation

To test whether PKA phosphorylation of CFTR was affected by acidic pH, we incubated the apical membranes in a phosphatase-inhibiting cocktail containing 0.01 mM cAMP and 5 mM ATPgamma S at pH 5.5 or 6.8. Under these conditions, CFTR GCl was activated only when membranes were incubated at pH 6.8 (Figs. 7 and 8). cAMP and ATPgamma S were washed out, and the cytoplasmic solution bathing the apical membrane was changed to pH 6.8. At pH 6.8, ATP alone activated CFTR GCl only in preparations previously incubated in irreversibly phosphorylating cocktail at pH 6.8, but not at pH 5.5 (Fig. 9). These results show that CFTR GCl was phosphorylated at pH 6.8, but not at pH 5.5, even though we used phosphatase-resistant ATPgamma S as substrate (Figs. 7 and 8).


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  		Fig. 7.   Lack of phosphorylation activation of CFTR GCl at acidic pH. Effect of pH (5.5 vs. 6.8) on CFTR GCl activation by 0.01 mM cAMP + 5 mM adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S) is shown. Again, phosphatase-resistant ATPgamma S was used as substrate in protein kinase A phosphorylation of CFTR to prevent subsequent dephosphorylation by endogenous phosphatases. CFTR GCl could be activated only after phosphorylation was attempted at pH 6.8. These results show that lack of activation of CFTR GCl at pH 5.5 is not simply due to an activation of endogenous phosphatases at this pH, because phosphatases could not have dephosphorylated irreversibly phosphorylated CFTR. Similarly, activation of CFTR GCl at pH 6.8 cannot be attributed simply to deactivation of endogenous phosphatases, because irreversibly phosphorylated CFTR cannot be dephosphorylated, even if endogenous phosphatases were activated. These results show that acidic cytoplasmic pH inhibits protein kinase A phosphorylation of CFTR.


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  		Fig. 8.   Summary of results of experiments in Fig. 7. There was no significant difference (P < 0.05) between conductance in ducts before activation with cAMP and ATPgamma S and after attempted activation at pH 5.5 in presence of cAMP and ATPgamma S. However, activation with cAMP and ATPgamma S at pH 6.8 resulted in a large increase in GCl. K Glu, potassium gluconate. *P < 0.05 vs. Control.


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  		Fig. 9.   Effect of pH on CFTR phosphorylation. CFTR in perfused, basolaterally permeabilized ducts were treated with 0.01 mM cAMP and 5 mM ATPgamma S at acidic pH 5.5 or pH 6.8 for ~5 min. These solutions were washed out. Cytosolic solutions were then changed to pH 6.8 + 5 mM ATP (standard perfusion solutions contained 140 mM potassium gluconate in cytoplasmic bath/150 mM NaCl + amiloride in lumen), and GCl was measured. GCl = 7.7 ± 1.5 mS/cm2 in ducts phosphorylated at pH 5.5 (A) and 89.3 ± 29.6 mS/cm2 in ducts that had been phosphorylated at pH 6.8 (B); GCl = 6.5 ± 1.2 mS/cm2 in control ducts at pH 6.8 that had not been exposed to phosphorylation activation cocktail (C), which is very similar to GCl measured in ducts phosphorylated at pH 5.5. These results showed that CFTR was not phosphorylated during incubation at acidic pH and that lack of activation of CFTR GCl at acidic pH is not simply due to inhibition of ATP interaction with CFTR. *P < 0.05 vs. no ATP.

Effect of Elevating Cytoplasmic pH on CFTR

Alkalinizing cytoplasmic pH resulted in a pH-dependent increase in the magnitude of the cAMP/ATP-activated CFTR GCl revealed as significantly larger increases in VCl and GCl (Fig. 10). The following experiments were designed to test the effect of high pH on phosphorylation/dephosphorylation of CFTR.


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  		Fig. 10.   Inhibition of phosphatases at high pH. CFTR GCl activity returns to baseline after washout of cAMP. Rate of deactivation of CFTR GCl after cAMP washout indicates rate of spontaneous dephosphorylation by endogenous phosphatases (24). Dephosphorylation deactivation of CFTR GCl was progressively delayed or even completely prevented at higher basic pH values.

Role of Dephosphorylation in Activating CFTR GCl by Basic Intracellular pH

Progressively elevating cytosolic pH not only increased the magnitude of activation of CFTR GCl, but results also suggested that the deactivation of CFTR GCl after cAMP washout became progressively slower at higher pH values (Fig. 10). Prolonged exposure of CFTR to cytoplasmic alkaline pH (~10 min at pH >7.5) resulted in an irreversible phosphorylation of CFTR, because CFTR GCl did not fall as long as ATP was present. In fact, phosphorylation of CFTR was so stable that it could be repeatedly activated and deactivated by addition and deletion of ATP alone, even after pH is lowered to 6.8 (Figs. 5 and 6).

Parenthetically, we also found that the magnitude of cAMP/ATP-activated CFTR GCl responses at pH 6.8 was spontaneously very low in some ducts (VCl ~15 mV compared with that of most preparations, which show a change of about +50 mV; Fig. 11). Previously, we discarded such ducts, assuming that they were damaged and physiologically not viable. However, we now find that increasing the cytoplasmic pH (>7.5 pH) of what we interpret as undamaged ducts often activates CFTR GCl to normal levels (Fig. 11).


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  		Fig. 11.   Effect of basic pH on a sweat duct apical membrane with spontaneously low-cAMP-activated CFTR GCl. Spontaneous CFTR GCl in apical membranes of ducts from certain individuals were sometimes uncharacteristically low. Attempt to activate one such duct with 0.1 mM cAMP and 5 mM ATP at pH 6.8 is shown. Only a very small change in VCl and GCl was observed. However, elevating cytosolic pH from 6.8 to 8.5 dramatically increased CFTR GCl, as shown by increase in VCl and GCl to values comparable to average responses from most ducts. This result shows that, for reasons yet to be explained, relative activities of kinases and phosphatases in native tissue seem to vary considerably among different sweat ducts.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

CFTR GCl Is Insensitive to Changes in Luminal pH

In the sweat duct, where CFTR is richly expressed, the luminal pH undergoes extreme changes from alkaline (pH 7.8) to highly acidic values (pH <5.0), depending on the sweat secretory rates (3). Therefore, we sought to determine the role of luminal pH in regulating CFTR GCl. We tested the effect of lowering luminal pH from 8.5 to 5.0 on CFTR GCl. We were surprised that such extreme changes in luminal pH had little effect on the magnitude of cAMP- and ATP-activated CFTR GCl (Fig. 1). These results are in contrast to previous reports that extracytosolic acidic pH significantly reduced the open probability of CFTR Cl- channels in lipid bilayer studies (27). Even though we cannot explain the apparent differences in the effect of extracytosolic pH on CFTR, we note that CFTR is continuously exposed to acidic pH of the endosomes (1, 15, 29) and highly basic pH of the pancreatic duct lumen (10, 26). In addition, our findings show that CFTR can function effectively as a Cl- channel over a wide range of extracellular pH environments. We also point out that this function occurs in a native tissue physiologically.

It has been reported that CFTR Cl- channels in planar lipid bilayers are significantly affected by pH changes in the cytoplasmic side (27). Cytosolic pH is also known to regulate a variety of epithelial transport mechanisms, including Na+-K+ pump activity (9, 12, 28), basolateral K+ conductance (4, 8, 13, 28), and other epithelial Cl- channels (6). Therefore, we asked whether changes in intracellular pH could in fact be a factor that determines when CFTR GCl should open and close as a function of luminal Cl- concentration.

To be relevant, we expect that 1) cytosolic pH should be a function of luminal Cl- concentration and 2) such a change in cytosolic pH activates and deactivates CFTR GCl when luminal Cl- concentration changes (22). Preliminary studies support this possibility, in that cytosolic pH is coupled to luminal Cl- concentration. As shown in Fig. 2, a decrease in luminal Cl- concentration significantly and reversibly decreased the intracellular pH. Even though we did not quantify the magnitude of these changes in intracellular pH, the change is qualitatively consistent with acidifying and deactivating CFTR GCl when luminal Cl- concentration falls, as found ex vivo (27). Therefore, we sought to determine whether and how cytosolic pH might regulate endogenous CFTR GCl in vivo.

Cytosolic pH Modulates CFTR GCl

We determined that cytosolic pH has little effect on deactivated CFTR (no cAMP/ATP). However, intracellular pH had a significant effect on CFTR GCl activated by cAMP/ATP. Lowering intracellular pH inhibited, while elevating intracellular pH enhanced, CFTR GCl (Figs. 3 and 4). These observations suggested that cytosolic pH might not be directly affecting, but rather might be modulating, previously activated CFTR GCl. Because CFTR GCl is regulated by ATP and phosphorylation/dephosphorylation (2, 11, 13, 19, 23, 24), we sought to determine which of these components of activation is specifically affected by changes in cytosolic pH.

Does Intracellular pH Affect ATP Regulation of CFTR GCl?

To test whether ATP regulation of CFTR GCl is dependent on intracellular pH, we irreversibly phosphorylated CFTR, as described in METHODS (Figs. 5-9; see below). We found that, even after irreversible phosphorylation, ATP regulation of CFTR GCl was dependent on intracellular pH (Fig. 6). These results suggested that at least some of the effects of intracellular pH on CFTR GCl may be independent of phosphorylation/dephosphorylation events and involve an effect on nucleotide interaction with CFTR molecule. However, we do not know whether such regulation is caused by a direct effect of pH on CFTR molecular conformation and/or the protonation status of ATP.

Reciprocal Regulation of CFTR Phosphorylation/Dephosphorylation by Intracellular pH

We previously showed that pharmacological inhibition of endogenous phosphatases did not enhance cAMP-activated baseline CFTR GCl in sweat duct (24). These results suggested that the phosphatase that is responsible for dephosphorylating CFTR may be subject to coordinated inhibition synchronous with the activation of PKA to optimize CFTR phosphorylation (24). The following results show that intracellular pH can exert a reciprocal effect on the activities of endogenous kinases and phosphatases that regulate CFTR GCl.

Intracellular pH-Dependent PKA Phosphorylation

We found that a basic cytosolic pH was favorable, whereas an acidic pH was inhibitory, for PKA phosphorylation of CFTR. We know that CFTR can be irreversibly phosphorylated in the presence of ATPgamma S and cAMP when intracellular pH is maintained at 6.8. The irreversibly phosphorylated CFTR can be induced to express GCl activity by application of ATP alone (without renewed PKA phosphorylation; Figs. 5, 6, and 9) (23-25). To determine whether intracellular pH affects PKA phosphorylation of CFTR, we attempted to irreversibly phosphorylate CFTR (with ATPgamma S/cAMP) at acidic (5.5) and near-physiological (6.8) pH (Figs. 7 and 8). We subsequently tested for the status of CFTR phosphorylation at each pH by measuring the magnitude of CFTR GCl activated by ATP alone at physiological pH (6.8). The rationale of these maneuvers is as follows. If PKA phosphorylation of CFTR is independent of pH, we should be able to phosphorylate CFTR at both pH values. Once stably phosphorylated, ATP alone should activate CFTR GCl at near-physiological pH (6.8). However, if any given medium pH blocks phosphorylation of CFTR, ATP alone will not be able to activate CFTR when returned to normal cytoplasmic pH (6.8). As shown in Fig. 9, ATP alone activated CFTR GCl only in ducts previously incubated in ATPgamma S phosphorylation cocktail at pH 6.8, not at pH 5.5. These results showed that acidic pH inhibits PKA phosphorylation of CFTR. Our results are also consistent with the previous findings that PKA enzyme catalytic efficiency is optimal around neutral pH and is inhibited in acidic pH (5, 30). However, these results do not rule out the possibility that a pH-dependent conformational change in CFTR or the ionization state of ATP (or its analogs) might render it a less effective substrate for PKA phosphorylation.

Intracellular pH-Dependent Phosphatase Dephosphorylation

Previously, we showed that the rate of deactivation of CFTR GCl after cAMP washout is a function of endogenous phosphatase dephosphorylation (24). Inhibition of endogenous phosphatases by fluoride and okadaic acid markedly delayed or completely abolished the spontaneous deactivation of CFTR after cAMP washout (24). We found that increasing intracellular pH can also abolish the spontaneous deactivation of CFTR GCl that normally follows cAMP washout (Fig. 10). These results indicated that the endogenous phosphatase responsible for dephosphorylation deactivation of CFTR GCl is more active at acidic pH and inhibited at alkaline pH (Fig. 10). Furthermore, incubating permeabilized ducts in Ringer solution at pH >= 8.5 for 5 min resulted in irreversible inhibition of dephosphorylation by endogenous phosphatases so that CFTR did not spontaneously deactivate. Under these conditions, ATP alone activated CFTR GCl without requiring further phosphorylation. This was substantiated by the fact that ATP activated the GCl, even in the presence of a nonspecific kinase inhibitor, staurosporine (Figs. 5 and 6). CFTR GCl remains irreversibly phosphorylated, even after the cytoplasmic pH is returned to 6.8. Prolonged exposure of apical membranes to alkaline cytoplasmic pH irreversibly inhibits dephosphorylation of CFTR by denaturing the endogenous phosphatases or by inducing an irreversible conformational change in CFTR so that phosphatases cannot deactivate the channel.

Physiological Significance of pH Regulation of CFTR

Recently, we showed that the transapical electrochemical gradient is unfavorable for passive Cl- transport from lumen to cell when the luminal Cl- concentration is low physiologically (22). Under these conditions, we predicted that 1) luminal Cl- would have to be transported against the electrochemical gradient by a putative nonconductive transport mechanism and 2) CFTR GCl would have to be deactivated to prevent the backleak of Cl- from cytoplasm into the lumen down this electrochemical gradient (18, 22-24). We do not know how deactivation of CFTR GCl in the apical membrane is coupled to luminal Cl- concentration, but a close correlation between changes in luminal salt concentration and sweat pH (3) suggested that a luminal or a cytosolic pH change may act in controlling CFTR GCl in the sweat duct. We surmise that at least two mechanisms must exist to transport Cl- out of the sweat duct: 1) an electroconductive mechanism involving CFTR when the luminal salt concentration is high and the transcellular Cl- gradient favors passive diffusion and 2) a nonconductive Cl- transport, which continues Cl- uptake when the luminal salt concentration falls and the electrochemical gradient for passive Cl- uptake disappears (18, 22). For the second mechanism to operate effectively, CFTR GCl must deactivate and close when the electrodiffusion gradient is lost. Otherwise Cl- would flux back into the lumen as soon as it is taken up into the cell (18, 22).

This system begs the question, How could a reduction in the luminal Cl- concentration trigger deactivation of CFTR GCl? Early data suggested that intracellular pH may follow changes in luminal Cl- concentration (Fig. 2). High luminal Cl- concentration raises cytoplasmic pH, resulting in an increase in PKA phosphorylation with a concomitant decrease in the phosphatase dephosphorylation of CFTR. These reciprocal effects should result in increased phosphorylation (activation) of CFTR, which maximally conducts Cl- down an electrochemical gradient (22). On the other hand, low luminal Cl- concentration decreases cytosolic pH, which supports an increase in phosphatase dephosphorylation with a concomitant decrease in PKA phosphorylation of CFTR to close GCl. Deactivation of CFTR GCl at low luminal Cl- concentration should facilitate carrier transport of Cl- against the electrical gradient as described earlier (18, 22). We recognize that acidifying the lumen with a proton pump to drive the anion exchange is not expected to acidify the cytoplasm, but we also are aware that low cytoplasmic concentrations are expected to acidify the cell by virtue of anion exchanger in the basal membrane. A coordinated interaction between luminal Cl-, cytosolic pH, PKA, endogenous phosphatase, and CFTR will be essential for efficient reabsorption of salt from the primary sweat.

Conclusions

We conclude that cytosolic pH could be a factor regulating CFTR GCl activity by exerting reciprocal control over phosphorylation/dephosphorylation of CFTR. Acidic pH inhibits PKA phosphorylation and activates phosphatase dephosphorylation. In contrast, basic pH activates PKA phosphorylation and inhibits phosphatase dephosphorylation of CFTR. Inhibiting CFTR GCl at acidic pH may prevent backleak of Cl- during a carrier-mediated Cl- absorption when the luminal salt concentration is physiologically low. Finally, CFTR GCl is independent of extracytosolic luminal pH in the range of 5.0-8.5.

    ACKNOWLEDGEMENTS

We are grateful to Kirk Taylor for expert technical assistance and the numerous subjects who volunteered for skin biopsy.

    FOOTNOTES

This study was supported by grants from Cystic Fibrosis Research and the National Cystic Fibrosis Foundation.

Address for reprint requests: P. M. Quinton, UCSD School of Medicine, Dept. of Pediatrics---0831, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0831.

Received 17 November 1997; accepted in final form 14 July 1998.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Cell Physiol 275(4):C1040-C1047
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