©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Pyrophosphate Stimulates Wild-type and Mutant Cystic Fibrosis Transmembrane Conductance Regulator Cl Channels (*)

(Received for publication, December 15, 1994; and in revised form, June 14, 1995)

Mark R. Carson Michael C. Winter Sue M. Travis Michael J. Welsh (§)

From the Howard Hughes Medical Institute, Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A unique feature of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel is regulation by ATP through the two cytoplasmic nucleotide-binding domains (NBDs). To better understand this process, we asked how channel activity is affected by inorganic pyrophosphate (PP(i)), a compound that binds to NBDs in other proteins. PP(i) and three nonhydrolyzable PP(i) analogs reversibly stimulated the activity of phosphorylated channels. Kinetic modeling of single channel data demonstrated that PP(i) affected two distinct steps in channel regulation. First, PP(i) increased the rate at which channels opened. Second, once channels were open, PP(i) delayed their closure. PP(i) could only stimulate channels when it was applied in the presence of ATP. PP(i) also increased the photolabeling of CFTR by an ATP analog. These two findings suggest that PP(i) modifies the activity of ATP-dependent CFTR channel gating. Based on these and previous data, we speculate that the effects of PP(i) are mediated by binding of PP(i) to NBD2 where it regulates channel opening by NBD1, and then, because it is not hydrolyzed, it slows the rate of NBD2-mediated channel closing. Because PP(i) stimulated wild-type channels, we tested its effect on CFTR containing the cystic fibrosis mutations: DeltaF508, R117H, and G551S. PP(i) stimulated all three. PP(i) also stimulated endogenous CFTR in the apical membrane of permeabilized T-84 epithelia. These results suggest that PP(i) or an analog might be of value in the development of new approaches to the treatment of cystic fibrosis.


INTRODUCTION

The cystic fibrosis transmembrane conductance regulator (CFTR) (^1)is an epithelial Cl channel with novel structure and regulation (for reviews see Refs. 1 and 2). CFTR is composed of two membrane-spanning domains that contribute to formation of the ion conducting pore and three cytoplasmic domains that regulate channel activity: two nucleotide-binding domains (NBDs) and the R domain. Dysfunction of the CFTR Cl channel causes cystic fibrosis (CF), a common lethal genetic disease(3, 4) . An important goal of CF research is to understand the function of CFTR and to use that knowledge to develop better treatments for the disease.

The presence of two NBDs confers a complex mechanism of regulation on channel activity. Phosphorylation of the R domain by cAMP-dependent protein kinase is necessary but not sufficient for channel activity. Once the R domain has been phosphorylated, ATP is required to open the channel(5) . Functional studies of ATP regulation of variant channels and studies of nucleotide binding to full-length CFTR and to NBD peptides suggested that ATP may interact with both NBDs(6, 7, 8, 9, 10, 11, 12) . The inability of nonhydrolyzable analogs to open the channel suggested that ATP hydrolysis is required for activity(5, 8, 13, 14) . Although NBD1 and NBD2 have some sequence similarity, functional studies of CFTR containing site-directed mutations in the NBDs provided evidence that the two NBDs have distinct functions in controlling the channel(6, 7, 8) . Through work with nucleotide analogs, vanadate, and beryllium, Gadsby and co-workers (13, 15) suggested that hydrolysis of ATP is not only required for channel opening but is also involved in channel closure from the bursting state. This conclusion was supported and expanded upon by our work in which mutations expected to decrease the rate of hydrolysis by NBD2 but not by NBD1 increased the duration of bursts(8) . Thus ATP binding and hydrolysis at the two NBDs appear to regulate both channel opening and closing, perhaps with the rate of ATP hydrolysis at NBD1 regulating opening and the rate of ATP hydrolysis at NBD2 regulating closure.

To further understand how the two NBDs function to regulate channel activity, we examined the effect of inorganic pyrophosphate (PP(i)) on CFTR channel gating. Gunderson and Kopito (14) recently showed that PP(i) could prolong bursts of activity, an effect similar to that observed with the nonhydrolyzable ATP analog AMP-PNP(8, 15) . However, the mechanism of this effect was not examined. We hypothesized that PP(i) might interact with the NBDs of CFTR to alter ATP-dependent channel activity. Previous studies have demonstrated that PP(i) binds with high affinity to the NBDs of other ATP-binding proteins. For example, PP(i) interacts with both intact mitochondrial F(1)-ATPase (16, 17, 18) and a 50-amino-acid F(1)-ATPase peptide(19) . PP(i) also binds to myosin, producing a charge change effect similar to that produced by ATP binding(20) , and produces dissociation of S-1 myosin from actin (21) without being hydrolyzed(22) .

We examined the effect of PP(i) on CFTR channel activity using the excised inside-out configuration of the patch-clamp technique. We found that PP(i) strongly potentiated the activity of phosphorylated channels in the presence but not the absence of ATP. We also assessed the effect of PP(i) on photolabeling of CFTR using the photolabile ATP analog 8-N(3)ATP. We then used kinetic modeling of single channel data to investigate the mechanism of how PP(i) stimulated channel activity. Our results led us to test the effect of PP(i) on three CF-associated mutant CFTRs and in a permeabilized epithelial preparation to explore the possibility that PP(i) or an analog might be utilized in the development of new approaches to therapy.


EXPERIMENTAL PROCEDURES

Chemicals and Solutions

Sodium pyrophosphate (PP(i)) was obtained from EM Science (Gibbstown, NJ). Catalytic subunit of cAMP-dependent protein kinase was from Promega Corp. (Madison, WI). Methylenediphosphonic acid (PCP, trisodium salt tetrahydrate) was from Aldrich. Etidronate disodium (1-hydroxyethylidenebisphonic acid, Didronel, 300 mg/6 ml H(2)O) was from Pharma (Minneapolis, MN). ATP (disodium salt), imidodiphosphate (PNP, sodium salt), and all other reagents were from Sigma.

For experiments with excised inside-out membrane patches, the pipette (extracellular) solution contained (in mM): 140 N-methyl-D-glucamine, 100 aspartic acid, 35.5 HCl, 5 CaCl(2), 2 MgCl(2), 10 HEPES, pH 7.3, with 1 N NaOH. The bath (intracellular) solution contained (in mM): 140 N-methyl-D-glucamine, 135.5 HCl, 3 MgCl(2), 10 HEPES, 4 cesium, and 1 EGTA, pH 7.3, with 1 N HCl ([Ca] < 10M). PP(i) and PP(i) analog stock solutions were 200 mM in cesium EGTA-free bath solution, pH 7.3, and were diluted to desired the final concentration in bath solution, except for etidronate disodium, which was diluted from commercial preparation. For Ussing chamber experiments, the mucosal (apical) solution contained (in mM): 135 NaCl, 1.2 MgCl(2), 1.2 CaCl(2), 2.4 K(2)HPO(4), 0.6 KH(2)PO(4), and 10 HEPES, pH 7.3. The submucosal (basolateral) solution contained (in mM): 135 sodium gluconate, 7 mM MgSO(4), 2.4 K(2)HPO(4), 0.6 KH(2)PO(4), 10 HEPES, 10 dextrose, no added calcium, and 1 magnesium ATP, pH 7.3.

Cells and Transfection Procedure

For patch-clamp experiments, two different cell types expressing wild-type and mutant CFTR were used: stably transfected C127 mouse mammary epithelia cells and transiently transfected HeLa cells. Transient transfection of HeLa cells with the vaccinia virus bacteriophage T7 hybrid expression system was as described previously(23, 24) . For experiments with DeltaF508 CFTR, stably transfected C127 cells were incubated at 25 °C for 12-72 h prior to use(25) . For experiments with polarized T-84 intestinal epithelial monolayers, cells were plated at a density of 5 10^5 cells/cm^2 on permeable filter supports (Millicell HA filters, Millipore, Bedford, MA; pore size, 0.4 µm; diameter, 27 mM). Transepithelial resistance was monitored with a EVOM epithelial volt-ohmmeter (World Precision Instruments, Sarasota, FL), and filters with resistances between 4 and 7 kilo-ohms were used.

Patch-Clamp Technique

Methods for excised, inside-out patch-clamp recording are similar to those previously described(5, 24, 26) . An Axopatch 200 amplifier (Axon Instruments, Inc., Foster City, CA) was used for voltage-clamping and current amplification. A microcomputer and the pClamp software package (Axon Instruments, Inc.) were used for data acquisition and analysis. Data were recorded on videotape following pulse code modulation using a PCM-2 A/D VCR adapter (Medical Systems Corporation, Greenvale, NY). Patch pipettes were fabricated as described(24) , with pipette resistances of 5-15 megaohms and with seal resistance routinely greater than 5 gigaohms. Voltages are referenced to the extracellular side of the membrane. Excised macro-patch experiments were performed at a holding potential of -40 or -80 mV; single channel data were recorded at a holding potential of -80 mV. Experiments were conducted at 34-36 °C using a temperature-controlled microscope stage (Brook Industries, Lake Villa, IL).

For excised macro-patch data, replayed records were filtered at 1 kHz using a variable 8-pole Bessel filter (Frequency Devices Inc., Haverhill, MA) and digitized at 2 kHz. Each time course data point represents the average current from 1 s with one data point collected every 5 s. Average currents for an intervention were determined as the average of the last 12 data points (the last minute) during the intervention. To compensate for any channel run-down during an experiment, specific interventions were bracketed when possible with current measurements made with similar concentrations of ATP but without the test compound; the intervention current was then compared with the average of pre- and post-intervention currents. For single channel analysis, replayed data were filtered at 1 kHz using a variable 8-pole Bessel filter, digitized at 5 kHz, and digitally filtered at 500 Hz. Idealized records were created using a half-height transition protocol; transitions less than 1 ms in duration were not included in the analysis. For the purpose of illustration, time course figures are inverted so that an upward deflection represents an inward current, data points during solution perfusion were not included in some figures, and single channel traces were digitally filtered at 200 Hz.

Burst analysis was performed as described previously(8, 27) , using a t(c) (the time which separates interburst closures from intraburst closures) of 20 ms. This value was derived from analysis of wild-type CFTR closed-time histograms derived from excised inside-out membrane patches containing a single channel studied in the presence of 1 mM ATP plus cAMP-dependent protein kinase and by the method of Sigurdson et al.(28) . Closures longer than 20 ms were considered to define interburst closures, whereas closures shorter than this time were considered gaps within bursts. We used a 20-ms interburst discriminator for studies done in both the absence and presence of PP(i) and analogs; this is justified by the facts that closed-time histograms showed a similar minimum at approximately 20 ms in the presence of PP(i) and that kinetic analyses showed little change in rates within bursts (note that in Fig. 5the beta(1) is 20 times slower than beta(2)). Burst data for PP(i), PNP, and PCP were derived from experiments in which the membrane patch contained one active channel. For experiments with etidronate and DeltaF508 CFTR, burst data were from patches containing four or fewer active channels; bursts in which there were no superimposed openings and that were separated from other bursts by greater than 20 ms were included in the analysis. We have previously shown that no discernible bias is observed by including burst data from patches with more than one channel(8, 27) .


Figure 5: Effect of PP(i) on kinetically modeled rate constants. A, a linear three state model of CFTR channel activity composed of two closed states (C(1) and C(2)), one open state (O), and four rate constants (beta(1), beta(2), alpha(1), and alpha(2)). B-E show values of rate constants before and after the addition of 5 mM PP(i). Rate constants were derived as described under ``Experimental Procedures'' from four experiments in which the membrane patch contained only one active channel, studied in the presence of 0.3 mM ATP and 75 nM cAMP-dependent protein kinase. The asterisks indicate p < 0.05.



Maximum Likelihood Analysis and Kinetic Modeling

Maximum likelihood analysis and kinetic modeling were performed as described (29) . Briefly, single channel data were filtered with an 8-pole Bessel filter (Frequency Devices Inc., Haverhill, MA) at a corner frequency of 1 kHz and digitized at 5 kHz on a microcomputer (Apple Macintosh, Apple Computer, Inc., Cupertine, CA) equipped with a multifunctional data acquisition board (NB-MIO-16) and LabVIEW 2 software (National Instruments, Austin, TX). Data were then digitally filtered at 500 Hz for analysis. Our previous work has shown that a linear three state model (C(1) C(2) O) best describes the kinetics of CFTR channel activity(29) . The Maple 5 symbolic algebra program (Waterloo Maple Software, Waterloo, Canada) was used to derive the open- and closed-time probability density functions for this model by solving the matrix equations in terms of the rate constants. The resulting equations were used in LabVIEW 2 to determine the set of rate constants that yielded the maximum likelihood for the open and closed times observed with different experimental interventions.

[alpha-P]8-NATP Photolabeling

Photolabeling of membrane-associated CFTR was performed as described previously(12) . Monolayer cultures of Spodoptera frugiperda (Sf9) cells were infected with a baculovirus containing the entire coding sequence for human CFTR (gift of R. J. Gregory and A. E. Smith, Genzyme Corp., Framingham, MA). Membranes were prepared by differential centrifugation and resuspended in 20 mM HEPES, pH 7.5, 50 mM NaCl, 3 mM MgSO(4), with 2 µg/ml each of leupeptin, aprotinin, and pepstatin. Membrane-associated CFTR was phosphorylated prior to photolabeling by incubation with 100 nM cAMP-dependent protein kinase and 0.1 mM ATP in buffer containing 10 mM MgCl(2) and 50 mM PIPES, pH 6.8, for 20 min at 30 °C and then diluted in 12 volumes of buffer containing 20 mM Tris, pH 7.5, 0.2 mM EGTA, and 1 µM calyculin A. Membranes were collected by centrifugation and resuspended at a concentration of 2.5 mg protein/ml in the same buffer.

Photolabeling was performed by preincubating membranes (50 µg of membrane protein/sample) on ice with [alpha-P]8-N(3)ATP (30 µM, 6-12 Ci/mmol) and PP(i) (in mM) as indicated in the figures. After 60 s of UV irradiation, CFTR was solubilized and immunoprecipitated as described (12) using antibodies raised against the R domain (M13-1, 0.3 µg/sample) and against the C terminus (M1-4, 10 µg/sample). Immunocomplexes were analyzed by SDS-polyacrylamide gel electrophoresis, and incorporation of [alpha-P]8-N(3)ATP was quantitated with an AMBIS radioanalytic imaging system (AMBIS Systems, Inc., San Diego, CA). Data are expressed as the percentage of radiolabel incorporation relative to control that had no added PP(i). We have previously shown the specificity of the labeling(12) .

Measurement of Apical Membrane ClCurrent

Apical membrane Cl current was measured as described previously(30) . T-84 monolayers with resistances between 4 and 7 kilo-ohms were mounted in modified Ussing chambers (Jim's Instruments, Iowa City, IA), and the basolateral membrane was permeabilized by the addition of approximately 100 µg/ml Staphylococcus aureus alpha-toxin to the serosal solution. S. aureus alpha-toxin produces pores in the basolateral membrane that are large enough to allow passage of ions and small molecules such as nucleotides without permitting exchange of larger proteins and cellular constituents(30, 31) . Permeabilization was confirmed by both a transient current upon addition of toxin that returned to baseline, as well as by stimulation of short circuit current by addition of 10 µM cAMP, which is cell impermeant in the absence of permeabilization. Data are expressed as current observed 5 min after the intervention.

Results are the means ± S.E. of n observations. Statistical significance was assessed using a paired, unpaired, or one-population Student's t test as appropriate.


RESULTS AND DISCUSSION

Pyrophosphate Stimulates CFTR ClCurrent in Excised Patches of Membrane

To assess the effect of PP(i) on macroscopic CFTR Cl channel activity, we added PP(i) to excised, inside-out membrane patches containing many CFTR channels that had been phosphorylated by cAMP-dependent protein kinase. Fig. 1A shows an example in which addition of 5 mM PP(i) in the presence of 0.3 mM ATP produced a reversible increase in Cl current. Addition of PP(i) in the absence of ATP did not stimulate current, suggesting that PP(i) alone cannot open CFTR Cl channels or substitute for ATP in supporting activity. Fig. 1B shows that as the concentration of PP(i) increased, the stimulation increased with an apparent EC of approximately 500 µM. The stimulatory effect of PP(i) was only observed when added to the cytosolic aspect of the membrane patch; external addition of 5 mM PP(i) did not alter currents from cells studied in the whole-cell configuration (n = 4, data not shown). Because PP(i) reversibly stimulated in the absence of cAMP-dependent protein kinase, the effect of PP(i) is probably not mediated through a membrane-associated phosphatase.


Figure 1: A, effect of PP(i) on CFTR Cl current. Data show the time course of current in an excised membrane patch from a HeLa cell transiently expressing wild-type CFTR. Prior to starting the time course, CFTR Cl channels had been phosphorylated with 75 nM cAMP-dependent protein kinase and 0.3 mM ATP (not shown). ATP (0.3 mM) and PP(i) (5 mM) were present during times indicated by bars. Data were not collected while solutions were changed. B, effect of PP(i) concentration on CFTR Cl currents. All values were determined in the presence of 0.3 mM ATP and are expressed as the percentages of current supported by 0.3 mM ATP. Data points are mean ± S.E. of 3-15 observations at each point; some errorbars are smaller than data symbols.



To determine how PP(i) stimulated CFTR currents, we studied membrane patches that contained only a single active channel. Examination of the traces in Fig. 2A suggests four things. First, PP(i) did not increase Cl current by changing the amplitude of current flowing through a single channel. In eight experiments, current amplitude was 0.90 ± 0.05 pA before and 0.91 ± 0.02 pA after addition of 5 mM PP(i) (p = 0.812). Second, it is apparent that the increase in total current is due to an increase in the probability that single channels are in the open state (P(o)). Third, it appears that the increased P(o) is at least in part caused by an increase in the duration of bursts of activity. (Note that a burst is defined as the time in which the channel is open with only brief flickers to the closed state. A closure of greater than 20 ms separates bursts). Fourth, PP(i) appeared to decrease the duration of the long closed states between bursts of activity.


Figure 2: Effect of PP(i) on the single channel characteristics of wild-type CFTR. A, traces from an excised inside-out membrane patch from a C127 cell containing a single, active channel. Dotted lines show closed state, and downward deflections correspond to openings. ATP and PP(i) concentrations (in mM) are indicated; cAMP-dependent protein kinase concentration is 75 nM. B and C, effect of 5 mM PP(i) on channel open probability (P(o)) and burst duration. Asterisks indicate p < 0.001; each of eight excised patches was studied an average of 3.0 ± 0.5 and 2.7 ± 0.8 min for control and PP(i), respectively.



Some of the changes that are apparent from visual inspection of tracings of single channels are quantitated in Fig. 2, B and C. PP(i) increased P(o) from 0.39 ± 0.02 to 0.81 ± 0.03 (n = 8, p < 0.001) and increased mean burst duration from 175 ± 6 ms to 1568 ± 219 ms (n = 8, p < 0.001). We considered that PP(i) might increase P(o) through a ``foot in the door'' blocking mechanism, in which blockers can actually increase channel opening duration (and sometimes opening rate) due to increased residence times within the pore(32, 33) . However, such a mechanism cannot explain the kinetic effects of PP(i) because it did not decrease macroscopic or single channel current, whereas a decrease would have been expected if PP(i) were binding to a site within the pore of CFTR.

Pyrophosphate Increases 8-N(3)ATP Photolabeling

Because PP(i) stimulated CFTR channel activity only in the presence of ATP, because an interaction between ATP and the NBDs is required for CFTR activity, and because PP(i) binds to the glycine-rich loop of nucleotide-binding domains in a number of proteins(16, 17, 18, 19, 20, 21) , we asked whether PP(i) might alter the binding of ATP to the NBDs of CFTR. This question was also raised by the findings of Gunderson and Kopito (14) , who said that as the ATP concentration decreased the effect of PP(i) increased, suggesting competition between PP(i) and ATP at an ATP-binding site. To answer this question, we examined the effect of PP(i) on 8-N(3)ATP photolabeling of membrane-associated CFTR. Our previous studies showed that CFTR was labeled in a specific and saturable way by 8-N(3)ATP, that 8-N(3)ATP supported channel function, and that ATP competed with 8-N(3)ATP(12) . These data plus other studies using peptides from the NBDs indicate that the NBDs are the site of ATP and 8-N(3)ATP interaction with CFTR(10, 11, 34) .

Fig. 3shows that PP(i) produced a concentration-dependent increase in 8-N(3)ATP photolabeling of CFTR, suggesting that PP(i) alters the interaction of nucleotide with the protein. If PP(i) and ATP both interact (in fact compete) at an NBD, why would PP(i) increase photolabeling by 8-N(3)ATP? At first inspection, the opposite effect would be predicted. However, the observation can be explained if PP(i) binds to one active site (one NBD) and thereby affects the properties of another active site in the molecule (the other NBD). Note that 8-N(3)ATP photolabeling does not measure equilibrium binding because the photolabeling reaction was performed over a 60-s period and is irreversible. Increased labeling of CFTR by PP(i) could be due to an increase in the rate of nucleotide binding to an NBD, a decrease in the rate of nucleotide release from an NBD, or exposure of more ATP-binding sites. With which NBD does PP(i) interact? Neither our present labeling data nor our previous study of 8-N(3)ATP photolabeling of CFTR variants allow us to answer this question. We previously showed that mutations predicted to disrupt hydrolysis at the NBDs did not alter 8-N(3)ATP photolabeling(8) . However, the data suggested that those mutations may have disrupted interactions between the two NBDs.


Figure 3: Effect of PP(i) on 8-N(3)ATP photolabeling of membrane-associated CFTR. Membranes of Sf9 cells infected with CFTR baculovirus were photolabeled with [alphaP]8-N(3)ATP in the presence of the indicated amount of PP(i). Incorporation is expressed as the percentage of photolabeling observed without added PP(i). Each value is average ± S.E. of 10-14 samples from four separate experiments. The asterisk indicates p < 0.05.



Nonhydrolyzable PP(i)Analogs Stimulate CFTR Channel Activity

It seemed possible that PP(i) might stimulate channel activity simply by binding to CFTR. In some systems PP(i) can mimic the functional effects of ATP binding without being hydrolyzed(22) . Alternatively, channel stimulation might require hydrolysis of PP(i)(35, 36) . To choose between these alternatives, we studied the effect of PP(i) analogs that do not support hydrolysis(35) . Examples of the effects of the nonhydrolyzable PP(i) analogs PNP, PCP, and etidronate disodium (a drug used clinically for treatment of hypercalcemia(37) ) are shown in Fig. 4A. Like PP(i), PCP, PNP, and etidronate can all support prolonged bursts of activity. However, the increase in burst duration generated by these compounds was smaller than that observed with PP(i), suggesting that these compounds were less potent than PP(i).


Figure 4: Effect of nonhydrolyzable PP(i) analogs on CFTR single channel characteristics. A, traces are from two separate excised membrane patch experiments from C127 cells that contained only a single active channel. To the right of the traces are the chemical structures of the PP(i) analogs. Traces shown were chosen to illustrate the effects of PCP, PNP, and etidronate and may not be representative of average P(o) or burst duration. B and C, effect of PCP (n = 6 and 5), PNP (n = 7 and 6), and etidronate (n = 5 and 3) on P(o) and burst duration, respectively. The asterisks indicate p < 0.05; P(o) and burst duration were calculated from greater than 3-min recording in each experiment.



The effects of these PP(i) analogs on P(o) and average burst duration are shown in Fig. 4, B and C. Although all three nonhydrolyzable analogs altered channel activity, the effects of PCP did not achieve statistical significance, suggesting that small differences between compounds, such as the electronegativity and/or the angle of the bridging group, can produce a large difference in the ability to alter channel gating. This is similar to the finding that AMP-PNP, a nonhydrolyzable ATP analog with a structure very similar to ATP(38) , competes for photolabeling at one-twentieth the potency of ATP(12) . Although these data do not rule out the possibility that hydrolysis of PP(i) may occur, they suggest that binding of PP(i) is sufficient for stimulation of the channel activity and prolongation of burst duration.

Kinetic Effects of PP(i)on CFTR Channel Activity

We have previously shown that the activity of single phosphorylated CFTR channels can be described by a linear three state model (29) and have used this model to describe how ATP, ADP, and inorganic phosphate alter the rate constants that describe transitions between states(27, 29) . To provide more insight into the mechanism by which PP(i) stimulated CFTR, we performed kinetic modeling on the data from four patches that contained only a single active channel. As shown in Fig. 5A, C(1) represents the long closed state between bursts of openings and C(2) O represents the bursting state, in which several channel openings (O) are separated by brief, flickery closures (C(2)) before the channel returns to the longer closed state (C(1)). The rate constants (beta(1), beta(2), alpha(1), and alpha(2)) describe the rate of transition between each state.

Fig. 5(B-E) describes the average values of the rate constants in the presence and the absence of 5 mM PP(i). PP(i) produced large changes in both beta(1) and alpha(1) and a smaller decrease in alpha(2) and did not alter beta(2). These results suggest that PP(i) affects more than one step in channel gating. The increase in burst duration shown in Fig. 2C is caused principally by a 6-fold decrease in alpha(1). alpha(1) is a major determinant of burst duration because it defines the rate at which the channel leaves the bursting mode (i.e. leaves C(2), the closed state within a burst). The duration of bursts can also be affected by beta(2) and alpha(2), the transition rates within a burst, but PP(i) did not alter beta(2) and decreased alpha(2) by 37%. However, because alpha(2) is one-tenth the magnitude of beta(2), the decrease in alpha(2) produced an overall change in P(o) within a burst of less than 5% (n = 4, not significantly different). Thus the decrease in alpha(2) had a minimal effect on net channel activity. In addition to increasing burst duration, PP(i) also increased beta(1), the rate of transition from the long closed state (C(1)) to the bursting state (C(2) O), suggesting that PP(i) facilitated opening by ATP. The decrease in the duration of long closed times between bursts can also be appreciated by examining the traces shown in Fig. 2and Fig. 4.

A Model to Explain Nucleotide-dependent Regulation of CFTR

These data show that PP(i) increased channel activity by affecting two distinct steps: increasing the rate of channel opening to a burst (beta(1)) and decreasing the rate of channel closure from a burst (alpha(2)). To consider how interaction of PP(i) with the NBDs may produce these changes, we discuss the effects on these two rate constants separately.

How does PP(i) increase the rate of channel opening (increase beta(1))? There are four pertinent considerations. First, PP(i) binding alone cannot open the channel. The stimulatory effect of PP(i) required the presence of ATP, consistent with previous data suggesting that ATP binding and hydrolysis are required to open the channel(5, 8, 13, 15) . Second, hydrolysis of PP(i) was not required for stimulation. Third, PP(i) stimulated the only transition that is regulated by ATP concentration (i.e. beta(1)). Fourth, PP(i) increased photolabeling with 8-N(3)ATP. We conclude that PP(i) must bind to a site in CFTR other than the site at which ATP directly opens the channel. When PP(i) binds it facilitates binding (and probably hydrolysis) of ATP at a separate site, increasing the rate at which the channel opens. Given the precedent for PP(i) interaction at the NBDs of other proteins, the presence of two NBDs in CFTR, and the previous suggestion that PP(i) competes with ATP(14) , we propose that PP(i) regulates channel activity by interacting with an NBD.

How does PP(i) prolong burst duration (decrease alpha(1))? Our previous work indicated that mutations expected to reduce the rate of hydrolysis at NBD2 increased the duration of bursts(8) . Nonhydrolyzable analogs of ATP also prolonged the duration of bursts(8, 13, 14) . These data suggest that the rate of hydrolysis of bound nucleotide at NBD2 is the primary determinant of burst duration. While ATP is bound, the burst continues; upon hydrolysis, the burst is terminated. The most straightforward interpretation of the data is that PP(i) prolongs the duration of bursts by binding to NBD2. Because PP(i) is not hydrolyzed, the open state is not terminated by hydrolysis and the burst duration is prolonged, and this is reflected in the model as a decrease in alpha(1).

Obviously, the linear three state model shown in Fig. 5A is a minimal model, and each state or transition in the model may represent more than one physical event or conformational change. We previously proposed a more complex mechanistic model of how events at NBD1 and NBD2 give rise to the opening and closing of phosphorylated channels (see Fig. 8 and (8) ) and proposed that there are interactions between the two NBDs such that binding of ATP to NBD2 may regulate events at NBD1, including a step leading to channel opening.

In keeping with that model, we speculate that PP(i) interacts primarily with NBD2, and in so doing it alters two functions of NBD2. First, binding of PP(i) to NBD2 could mimic the effect of ATP binding. It would effect NBD1, enhancing binding and/or hydrolysis of ATP. Hydrolysis of ATP at NBD1 may then open the channel to the bursting state. Second, because PP(i) is not hydrolyzed, it prolongs the duration of the bursting state preventing channel closure. Although this seems to be the most straightforward explanation and is consistent with previous observations, we acknowledge the possibility that PP(i) may interact at a site other than NBD2 or that PP(i) interaction at more than one site may produce the different effects observed. Consistent with the effects reported here, we previously speculated that AMP-PNP, like PP(i), interacted with NBD2 to increase burst duration(8) . However, AMP-PNP was much less potent (see Fig. 7and Ref 8). Based on our data with PP(i), we predict that AMP-PNP would also increase channel opening (i.e. increase beta(1)), but because of its low potency it was not possible to test this prediction.


Figure 7: Effect of PP(i) on apical membrane Cl currents in permeabilized T-84 epithelia. A, apical membrane Cl current was recorded after basolateral membrane was permeabilized with S. aureus alpha-toxin (100 µg/ml). Basolateral solutions contained 1 mM ATP. The bars indicate the presence of cAMP (10 µM) and PP(i) (in mM). The breaks in the traces omit recordings during change in basolateral solutions because electrical contact was disrupted. B, effect of PP(i) on apical membrane Cl current in the presence (solid bars; n = 10) or the absence (hatched bars; n = 4) of cAMP (10 µM). The asterisk indicates p < 0.05.



Why would PP(i) show preferential binding to NBD2? Perhaps the binding affinity or steric constraints of NBD2 are more favorable for PP(i) binding. The two NBDs do have significantly different amino acid sequences, and there is substantial precedent for nucleotide-binding sites that show preference between different nucleotide analogs and PP(i)(17) .

PP(i)Stimulates CFTR Containing CF-associated Mutations

The ability of PP(i) to potently stimulate CFTR activity suggested that PP(i) or a more stable nonhydrolyzable PP(i) analog might be a useful pharmacological agent to stimulate poorly functional CFTR channels that are associated with disease. To support a possible pharmacological approach, three criteria must be met. First, PP(i) must stimulate CFTR channels that contain CF-associated mutations. Second, PP(i) or an analog must be able to stimulate endogenous CFTR in the apical membrane of epithelia. And third, because stimulation occurs from the cytoplasmic aspect, PP(i) or an analog must be able to gain access to the interior of the cell. To address the first two issues, we performed the following studies.

We examined the effect of PP(i) on CFTR containing the CF-associated mutations DeltaF508, R117H, and G551S. We studied these mutations because they occur in different regions of the protein and have different mechanisms of dysfunction(39) . CFTR-DeltaF508, the most common CF-causing mutation(40, 41) , is defectively processed and fails to traffic to the plasma membrane. In addition to the processing defect, the function of CFTR-DeltaF508 is decreased as indicated by a reduced P(o)(25, 42) . G551S, a mutation in NBD1, is correctly processed but has altered ATP-dependent channel regulation resulting in a reduced P(o)(6) . R117H, which contains a mutation in the membrane-spanning domain, is also correctly processed but has altered ion-conducting properties producing an overall decrease in function(43) .

Fig. 6shows current tracings from a membrane patch containing two active CFTR-DeltaF508 channels. In the presence of 75 nM cAMP-dependent protein kinase and 0.3 mM ATP, channel openings appear qualitatively similar to those of wild-type CFTR, except that both P(o) and burst duration are less than those of wild type. The addition of 5 mM PP(i) stimulated DeltaF508 activity 3-fold (n = 5, Fig. 6, A and D). P(o) increased 3-fold (from 0.07 ± 0.01 to 0.21 ± 0.022), and average burst duration increased 8-fold (from 125 ± 19 ms to 1023 ± 330 ms, n = 3). A similar stimulation of channel activity was observed when 1 mM PP(i) was added to CFTR-R117H and CFTR-G551S (Fig. 6, B, C, and D). This amount of stimulation is similar to that observed with wild-type CFTR (see Fig. 1B).


Figure 6: Effect of PP(i) on CFTR Cl channels containing CF-associated mutations. A, traces from an excised membrane patch from a C127 cell expressing two DeltaF508 CFTR Cl channels. The cell had been incubated at 25 °C to increase protein levels at the plasma membrane(25) . Traces are plotted as described in the legend to Fig. 2. B and C, time course experiments from membrane patches containing multiple channels from transiently transfected HeLa cells expressing R117H (B) or G551S (C) CFTR. Note that PP(i) concentrations for these experiments are 1 mM. Data are plotted as described in the legend to Fig. 1A. D, effect of PP(i) on DeltaF508 CFTR (5 mM PP(i)) (n = 5), R117H (1 mM PP(i)) (n = 9), and G551S (1 mM PP(i)) (n = 6). Data are expressed as the percentages of current relative to current in presence of 0.3 mM ATP and 75 nM cAMP-dependent protein kinase. The asterisks indicate p < 0.05 compared with value in the absence of PP(i).



Pyrophosphate Stimulates Apical Membrane ClCurrents in Permeabilized Epithelia

To learn whether PP(i) can stimulate endogenous CFTR in the apical membrane of epithelia, we measured the effect of PP(i) on permeabilized T-84 intestinal epithelial monolayers. T-84 intestinal epithelial cells grow as a polarized epithelium on permeable supports and express CFTR in their apical membrane(44, 45) . Because PP(i) is hydrophilic and membrane-impermeant, we permeabilized the basolateral membrane with S. aureus alpha-toxin to provide access of small molecules, such as cAMP and PP(i), to the cell interior(30) . Permeabilizing the basolateral membrane also allowed us to measure current flow across the apical membrane in the absence of the basolateral membrane barrier.

Fig. 7A shows examples of the results. After permeabilization, the baseline Cl current was small, suggesting that in the absence of cAMP, the apical membrane was relatively impermeable to Cl. As we previously reported, addition of cAMP to the basolateral bathing solution stimulated Cl current(30) . Subsequent addition of either 1 or 5 mM PP(i) to the basolateral bathing solution produced a further increase in current. Fig. 7B (solid bars) shows the average values of current 5 min after the addition of the bathing solution with or without PP(i).

Although PP(i) can function as a nonspecific phosphatase inhibitor, and CFTR channel activity is regulated in part by phosphorylation(46) , we think it unlikely that PP(i) stimulated the apical membrane Cl current by acting as a nonspecific phosphatase inhibitor because upon removal of cAMP in the continued presence of PP(i), current decreased to baseline levels within 3 min. This rate is similar to that observed in the absence of PP(i) (Fig. 7A). In other experiments, the addition of 1 or 5 mM PP(i) to permeabilized monolayers in the absence of cAMP did not increase current above baseline levels (Fig. 7B, hatched bars; n = 4). These results suggest that PP(i) only stimulates apical Cl current in the presence of cAMP, suggesting that PP(i) has a direct effect on active channels and not an indirect effect via alteration of the phosphorylation state of CFTR. The conclusion that PP(i) is not acting primarily as a phosphatase inhibitor is consistent with the results shown in Fig. 1and is also supported by the finding that PP(i) stimulated CFTRDeltaR-S660A in excised membrane patch experiments (n = 6, p = 0.004, data not shown). CFTR-S660A is a mutant in which most of the R domain has been deleted and as a result it does not require and is not stimulated by cAMP-dependent protein kinase(47) .

Could PP(i) or a related compound be of value to treat CF? That possibility is suggested by the ability of PP(i) to stimulate CFTR containing several different CF-associated mutations and the ability of PP(i) to potentiate the activity of endogenous CFTR located in the apical membrane of an epithelium. The compounds studied, however, are hydrophilic and are not expected to partition through the lipid membrane to gain access to the cytoplasmically located NBDs when applied extracellularly. Thus delivery of PP(i) or a stable analog may depend upon modification of the compound to allow partitioning through the lipid membrane or use of a delivery vehicle such as liposomes. Certainly, intracellular pyrophosphates or analogs that lack specificity for CFTR might have many undesirable consequences. However, a lipophilic bisphosphonate has been administered systemically to alter cholesterol biosynthesis(48) .

In addition, such a strategy must take into account the specific type of CF mutation, because different mutations have different molecular mechanisms of dysfunction(39) . Poorly functional channels appropriately localized to the plasma membrane, such as R117H and G551S, might be most amenable to regulation by a PP(i) analog. Conversely, because little DeltaF508 mutant is present at the apical membrane in cells grown at 37 °C, an approach based on simulating channel activity might also require a second strategy to increase the amount of DeltaF508 CFTR at the plasma membrane. In summary, although the idea of using PP(i) and PP(i)-like compounds to stimulate defective CFTR remains preliminary, these findings suggest that further investigation of PP(i) and PP(i) analogs may be useful not only to understand the regulation of CFTR but also in the evaluation of future therapeutic applications.


FOOTNOTES

*
This work was supported in part by the Howard Hughes Medical Institute, the National Heart, Lung, and Blood Institute, and the Cystic Fibrosis Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, University of Iowa College of Medicine, 500 EMRB Iowa City, IA 52242. Tel.: 319-335-7619; Fax: 319-335-7623.

(^1)
The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator(s); PP(i), inorganic pyrophosphate; P(o), single channel open-state probability; NBD, nucleotide-binding domain; CF, cystic fibrosis; PNP, imidodiphosphate; AMP-PNP, adenyl-5`-yl imidodiphosphate; PCP, methylenediphosphonic acid; etidronate, 1-hydroxyethylidenebisphonic acid; 8-N(3)ATP, 8-azidoadenosine 5`-triphosphate; PIPES, 1,4-piperazinediethanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Dr. John Marshall and Dr. Seng Cheng of Genzyme Corp. for the gift of M13-1 antibody, Dr. D. Michael Shasby for the gift of S. aureus alpha-toxin, the University of Iowa DNA Core facility for oligonucleotide synthesis and DNA sequencing, S. P. Weber, P. H. Karp, S. R. Struble, J. A. Cieslak, E. C. Kasik, T. A. Mayhew, and E. N. Calease for excellent assistance, and our laboratory colleagues for advice and critical comments.


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