Fluoride stimulates cystic fibrosis transmembrane conductance regulator Clminus channel activity

Herbert A. Berger, Sue M. Travis, and Michael J. Welsh

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

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

While studying the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR), we found that addition of F- to the cytosolic surface of excised, inside-out membrane patches reversibly increased Cl- current in a dose-dependent manner. Stimulation required prior phosphorylation and the presence of ATP. F- increased current even in the presence of deferoxamine, which chelates Al3+, suggesting that stimulation was not due to AlF<SUP>−</SUP><SUB>4</SUB>. F- also stimulated current in a CFTR variant that lacked a large part of the R domain, suggesting that the effect was not mediated via this domain. Studies of single channels showed that F- increased the open-state probability by slowing channel closure from bursts of activity; the mean closed time between bursts and single-channel conductance was not altered. These results suggested that F- influenced regulation by the cytosolic domains, most likely the nucleotide-binding domains (NBDs). Consistent with this, we found that mutation of a conserved Walker lysine in NBD2 changed the relative stimulatory effect of F- compared with wild-type CFTR, whereas mutation of the Walker lysine in NBD1 had no effect. Based on these and previous data, we speculate that F- interacts with CFTR, possibly via NBD2, and slows the rate of channel closure.

nucleotide-binding domain; adenosine 5'-triphosphate; patch clamp; channel gating

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

THE CYSTIC FIBROSIS transmembrane conductance regulator (CFTR) is an epithelial Cl- channel with novel structure and regulation (for review, see Refs. 31, 41). CFTR is composed of two sets of membrane-spanning domains that contribute to the formation of the ion-conducting pore and three intracellular domains that regulate channel activity: two nucleotide-binding domains (NBDs) and the R domain. Loss of CFTR Cl- channel function causes cystic fibrosis, a common lethal genetic disease (42).

Regulation of CFTR Cl- channel activity is complex. Phosphorylation of the R domain by an adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PK) catalytic subunit (PKA) is necessary but not sufficient for channel activity. Once the R domain has been phosphorylated, ATP is required to open the channel (1). Functional studies of ATP regulation of variant channels and nucleotide binding to full-length CFTR and to NBD peptides suggest that ATP interacts with both NBDs (3, 9, 19, 25, 35, 38-40). The inability of nonhydrolyzable analogs to open the channel suggests that ATP hydrolysis is required for activity (1, 6, 9, 16) and that isolated NBD1 and whole CFTR hydrolyze ATP (24, 27). However, several studies indicate that the two NBDs may have different roles in controlling channel activity (3, 9, 35). Such observations have led to the development of models in which hydrolysis at NBD1 opens the channel and hydrolysis at NBD2 closes the channel (9, 22).

While studying the regulation of CFTR, we found that F- stimulated CFTR channel activity. The purpose of this study was to investigate the mechanism by which F- activates CFTR to better understand regulation of the channel. We were also interested in studying the effect of F- because in patch-clamp studies of CFTR, F- is often added to the cytosolic bathing solution to inhibit phosphatase activity (28, 29, 32, 33).

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Chemicals and solutions. PKA was obtained from Promega (Madison, WI). ATP, guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S), and all other reagents were obtained from Sigma (St. Louis, MO).

The pipette (external) solution contained (in mM) 140 N-methyl-D-glucamine, 2 MgCl2, 5 CaCl2, 100 L-aspartic acid, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.3 with HCl (Cl- concentration, 50 mM). The bath (internal) solution contained (in mM) 140 N-methyl-D-glucamine, 3 MgCl2 (except when 13 mM MgCl2 is specifically indicated), 1 cesium ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 10 HEPES, pH 7.3 with HCl (Cl- concentration, 140 mM). The estimated free Ca2+ concentration in the internal solution was <10 nM. Additions to the bath solution are indicated for individual experiments. The Al3+ concentration of the bath solution was measured by atomic-emission spectroscopy (Environmental Protection Agency 200.7 method, Univ. of Iowa Hygienic Laboratory, Des Moines, IA).

Site-directed mutagenesis. CFTR mutants were constructed in the vaccinia virus expression plasmid pTM-CFTR4 (13) by the method of Kunkel (26). Mutants were verified by restriction enzyme analysis, DNA sequencing around the sites of mutation, and in vitro transcription and translation assays (15). Mutants are named by the amino acid residue number preceded by the wild-type amino acid and followed by the amino acid to which the residue was changed using the single-letter amino acid code.

Cells and CFTR expression systems. We used NIH/3T3 fibroblasts that stably express CFTR after infection with a retrovirus expressing wild-type human CFTR (2). The cells were maintained as previously described (7). Functional studies performed on these cells have revealed hundreds of CFTR Cl- channels per excised, inside-out cell-free patch (1, 7). We transiently expressed wild-type or mutant CFTR in HeLa cells using the vaccinia virus-T7 hybrid expression system described previously (30).

Patch-clamp technique. The methods used for patch-clamp recording are similar to those previously described (17). The excised, inside-out configuration was used in all patch-clamp experiments. All cells and baths were maintained at 34-36°C by a temperature-controlled microscope stage (Brook Industries, Lake Villa, IL) except when ~25°C bath conditions are specifically indicated. The bath electrode consisted of an Ag-AgCl pellet connected to the bathing solution via an agar bridge filled with 1 M KCl. The junction potentials introduced after addition of 20 mM NaF were less than +1 mV. Pipette resistance was 2-6 MOmega , and seal resistance was 2-25 GOmega . An Axopatch 200-A amplifier (Axon Instruments, Foster City, CA) was used for voltage clamping and current amplification. A Gateway 2000 computer, system P5-90 (N. Sioux City, SD), and the pClamp 6.0.3 software package (Axon Instruments) were used for data acquisition and analysis. Data were recorded on digital audiotape on a DTR-1203 digital tape recorder (Biologic Science Instruments, Molecular Kinetics, Pullman, WA). Some of the data acquisition and analysis required use of the Indec Systems laboratory computer system (Sunnyvale, CA) as previously described (8).

For excised macropatch data, replayed records were filtered at 1 kHz using a variable 8-pole Bessel filter (Frequency Devices, Haverhill, MA) and digitized at 2 kHz. Voltage was referenced to the external surface of the membrane patch: a depolarizing voltage is positive. Membrane voltage was held at -40 mV unless otherwise stated. Data points during time-course experiments are mean current values during 2-s sweeps. Average currents for an intervention were determined as the average of at least the last 1 min of that intervention. To compensate for any "rundown" of current during an experiment, specific interventions were bracketed by similar conditions without the test compound; the intervention current was then compared with the average of pre- and postintervention 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 <1 ms in duration were not included in the analysis. For illustration, single-channel traces were digitally filtered at 200 Hz.

Burst analysis was performed as described previously (10, 12) using a tc (time separating interburst closures from intraburst closures) of 20 ms. Burst data were from patches containing three or fewer active channels; bursts in which there were no superimposed openings and that were separated from other bursts by >20 ms were included in the analysis. As previously described (11), we calculated the mean closed time between bursts (Tc) from the equation Po = (Tb × open-state probability within a burst)/(Tb + Tc), where Tb is mean burst duration and Po is single-channel open-state probability. Po, Tb, and open probability within a burst were determined directly from the experimental data. To calculate Po, the number of channels in a membrane patch was determined by the greatest number of simultaneous open channels observed during the entire experiment. Addition of 10 mM F- did not alter the open-state probability within a burst. Data from single- and multichannel patches gave similar results.

[alpha -32P]8-azidoadenosine 5'-triphosphate photolabeling. Photolabeling of membrane-associated CFTR was performed as described previously (40). Monolayer cultures of Spodoptera frugiperda cells were infected with a baculovirus containing the entire coding sequence for wild-type human CFTR (gift of R. J. Gregory and A. E. Smith, Genzyme, Framingham, MA). Membranes were prepared by differential centrifugation and resuspended in 20 mM HEPES, pH 7.5, 50 mM NaCl, and 3 mM MgSO4, with 2 µg/ml each of leupeptin, aprotinin, and pepstatin. 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 membrane protein/sample) on ice with [alpha -32P]8-azidoadenosine 5'-triphosphate ([alpha -32P]8-N3ATP; 80 µM, 6-12 Ci/mmol) and ATP or 10 mM NaF as indicated. After 60 s of ultraviolet irradiation, CFTR was solubilized and immunoprecipitated as described (40) using antibodies raised against the R domain (M13-1) and against the COOH terminus (M1-4). Immunocomplexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and incorporation of [alpha -32P]8-N3ATP was quantitated with an AMBIS radioanalytic imaging system (AMBIS Systems, San Diego, CA). Data are expressed as the percentage of radiolabel incorporation relative to control that had no added NaF. The specificity of the labeling has previously been shown (40).

Statistical analysis. Values are presented as means ± SE. The unpaired Student's t-test was used when comparing values. Values were considered statistically significant at P < 0.05.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Effect of F- on CFTR Cl- current in excised patches of membrane. While studying phosphorylation-dependent regulation of CFTR, we observed that F- stimulated CFTR Cl- current. Figure 1A shows that PKA and ATP activated CFTR Cl- channels in excised patches of membrane and that addition of F- further increased current. The increase in current was concentration dependent, as shown in Fig. 1, A and B.


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Fig. 1.   Effect of F- on cystic fibrosis transmembrane conductance regulator (CFTR) Cl- current. A: data show time course of current (I) in an excised, inside-out membrane patch from NIH/3T3 cells expressing CFTR. B: effect of increasing concentrations of F- on current. Data are means ± SE of 4-20 experiments. C: effect of F- before addition and after removal of cAMP-dependent protein kinase A (PKA) and ATP. F- (10, 20, or 100 mM), ATP (1 mM), and PKA (75 nM) were present in cytosolic solution during times indicated by bars.

When we applied 20 mM F- before addition of PKA, it had no effect, even in the presence of 1 mM ATP (Fig. 1C) (n = 6). After addition of PKA, CFTR channels activated. When PKA and ATP were removed, the current returned to baseline even in the continued presence of F-. These experiments indicate that F- alone did not stimulate Cl- current and that stimulation by F- required the presence of ATP and phosphorylation of the channel.

Many studies of CFTR activity, including previous studies of F- (6), are performed at room temperature. Figure 2, A and B, shows that stimulation by 10 mM F- at room temperature (~25°C) was not significantly different from stimulation at 37°C. We considered the possibility that added F- might reduce the free Mg2+ concentration. However, increasing cytosolic Mg2+ (13 mM) had no significant effect on F- (10 mM) stimulation of CFTR current (Fig. 2, A and B). Stimulation by F- was not cell type specific, since it occurred in two different cell types: in HeLa cells, using a transient expression system, and in stably transfected NIH/3T3 cells. As a control, baseline current did not significantly change with addition of 10 mM NaCl (Fig. 2B).


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Fig. 2.   Effect of F- on CFTR Cl- current at different temperatures and Mg2+ concentrations. A: data show time course of current at 25°C in an excised, inside-out membrane patch from NIH/3T3 cells expressing CFTR. F- (10 mM), Mg2+ (3 and 13 mM), ATP (1 mM), and PKA (75 nM) were present in cytosolic solution during times indicated by bars. B: effect of NaCl (10 mM), NaF (10 mM), MgCl2 (3 and 13 mM), or temperature (25 and 37°C) on current. All values were obtained in presence of 1 mM ATP and are expressed as percentage of current supported by 1 mM ATP. Data are means ± SE of 3-11 experiments.

Effect of deferoxamine on stimulation by F-. Previous work by Baukrowitz et al. (6) has shown that BeF3 and orthovanadate (VO4) can stimulate CFTR channel activity by slowing the rate of closure from bursts of activity, i.e., by "locking" CFTR open. They proposed that these agents adopt a conformation similar to the gamma -phosphate of ATP associated with an active CFTR channel. Therefore, we considered the possibility that the effects of F- may have been due to AlF3 because solutions are frequently contaminated by Al3+. We found that our bath solution contained up to 10 µM Al3+. To chelate the Al3+, we added 1 mM deferoxamine (44) to the bathing solution. Figure 3 shows that deferoxamine had no effect on the current stimulated by F- (n = 4). Moreover, when deferoxamine was added first, F- still stimulated current (n = 3, data not shown). In the solutions we used, it can be calculated that with 10 µM AlCl3 and 20 mM NaF, the free Al3+ concentration is 3.2 × 10-27 M and the AlF<SUP>−</SUP><SUB>4</SUB> concentration is 5.1 × 10-10 M (44). For comparison, Sternweis and Gilman (36) reported that in the absence of deferoxamine, 1 µM Al3+ with 5 mM F- was required to begin to observe G protein stimulation of adenylate cyclase. In CFTR, Baukrowitz et al. (6) found that 5 mM CsF added with 0.5 mM BeSO4 stimulated CFTR. These considerations and our results suggest that F- stimulated CFTR independently of Al3+.


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Fig. 3.   Effect of deferoxamine on current stimulated by F-. Data show time course of current in an excised, inside-out membrane patch from NIH/3T3 cells expressing CFTR. F- (20 mM), ATP (1 mM), PKA (75 nM), and deferoxamine (1 mM) were present in cytosolic solution during times indicated by bars.

Effect of GTPgamma S on CFTR Cl- current. Because F- can have some stimulatory effects on G proteins that do not require Al3+ (4, 5, 20), we tested the possibility that the stimulatory effects might have resulted from G protein activation. It was possible that F- may have activated a G protein that stimulated CFTR. Therefore, we asked whether GTPgamma S would stimulate CFTR. Figure 4 shows that addition of up to 250 µM GTPgamma S did not stimulate CFTR current (1.5 ± 4.5% increase, n = 5, P = 0.77). When 20 mM F- was added to the same membrane patches, CFTR current increased. These data suggesting that the effect of F- was not via a G protein are consistent with the report of Ismailov et al. (23), who found that 100 µM GTPgamma S had no effect on CFTR channels incorporated into planar lipid bilayers.


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Fig. 4.   CFTR Cl- current in presence of guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) in an excised, inside-out membrane patch from NIH/3T3 cells expressing CFTR. GTPgamma S (250 µM), F- (20 mM), ATP (1 mM), and PKA (75 nM) were present in cytosolic solution during times indicated by bars.

Effect of F- on CFTRDelta R-S660A current. Because CFTR requires phosphorylation for activation and because F- is known to inhibit several protein phosphatases (34), we asked whether F- might stimulate CFTR activity by inhibiting a phosphatase. To test this possibility, we studied a CFTR variant (CFTRDelta R-S660A) in which part of the R domain (amino acids 708-835) is deleted and a remaining phosphorylation site (serine 660) is mutated to alanine (30). This channel shows constitutive activity in the presence of ATP and is not further activated by PKA. Figure 5 shows that addition of F- increased the current in CFTRDelta R-S660A (n = 3), suggesting that F- did not stimulate the channel by inhibiting a phosphatase.


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Fig. 5.   Effect of F- on Cl- current in CFTRDelta R-S660A. Data show time course of current in an excised, inside-out membrane patch from NIH/3T3 cells expressing CFTRDelta R-S660A. Cl- channels were constitutively active in presence of ATP before addition of F-. No PKA was added to this membrane patch. F- (20 mM) and ATP (1 mM) were present in cytosolic solution during times indicated by bars.

Effect of F- on 8-N3 ATP photolabeling. The stimulation by F- is similar to the effects of some other agents that stimulate CFTR. For example, both inorganic pyrophosphate (PPi) and F- stimulate CFTR only after phosphorylation and in the presence of ATP (12, 16). PPi increased photolabeling of CFTR by 8-N3ATP, suggesting that PPi modulates channel gating by altering the interaction with nucleotides (12). However, in contrast to the effect of PPi, 10 mM F- had no measurable effect on 8-N3ATP photolabeling (Fig. 6). This result suggests that F- modulates channel gating by a mechanism distinct from that of PPi.


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Fig. 6.   Effect of 10 mM F- on 8-azidoadenosine 5'-triphosphate (8-N3ATP) photolabeling of membrane-associated CFTR. Membranes of Spodoptera frugiperda cells infected with CFTR baculovirus were photolabeled with [alpha -32P]8-N3ATP under standard conditions in presence of 10 mM F- and in presence of 100 mM ATP [which has been previously shown to inhibit photolabeling (40)]. Incorporation is expressed as percentage of photolabeling observed without added ATP or F-. Values are means ± SE; n = 6-9.

Kinetic effects of F- on CFTR single-channel activity. These data suggest that F- might interact directly with CFTR. F- could stimulate current by increasing single-channel conductance, Po, or the number of channels. To evaluate these possibilities, we examined the effect of F- on single-channel currents; an example is shown in Fig. 7A. The single-channel current-voltage relationships are shown in Fig. 7B. The channels were Cl- selective as indicated by the reversal potentials (reversal potential = 24 mV, expected Cl- reversal potential = 27 mV). Single-channel conductance in the absence of F- was 9.87 ± 0.39 pS and in the presence of 20 mM NaF was 8.45 ± 0.44 pS (P = 0.055). This small decrease in single-channel conductance is not unexpected, since F- is less conductive than Cl- (2, 37). F- stimulation did not change the linear current-voltage relationship of single CFTR channels or macroscopic CFTR current (Fig. 7B) and showed no voltage dependence (n = 8, data not shown). These data indicate that the increase in current produced by F- could not be explained by an increase in conductance.


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Fig. 7.   Gating behavior of CFTR Cl- channels in presence of F-. A: effect of 10 mM F- on a single channel in an excised, inside-out patch from NIH/3T3 cells expressing CFTR. Broken line represents closed state of channel, and downward deflections correspond to openings. Membrane voltage was clamped at -100 mV. Scale bar, 1 s and 100 ms for prolonged and expanded traces, respectively. B: single-channel current-voltage relationship for CFTR in absence and presence of 20 mM F-. Each data point represents the mean ± SE of 2-39 experiments; in many cases, error bars are hidden by symbol. All data were obtained in presence of ATP (1 mM) and PKA (75 nM). C: effect of 10 mM F- on single-channel open-state probability (Po), burst duration, and closed time between bursts. Measurements were obtained in presence of ATP (1 mM) and PKA (75 nM) with (n = 6) or without (n = 14) 10 mM F-. All data points are means ± SE. * P < 0.005.

We found that 10 mM F- increased the Po of activated CFTR channels (Fig. 7C). Twenty millimolar F- also increased Po from 0.29 ± 0.07 to 0.65 ± 0.04 (n = 3, P = 0.01). The increase in Po was due to an increase in burst duration, with no change in the Tc (Fig. 7C). Moreover, F- produced no change in gating within a burst. Stimulation by F- was not due to an increase in the number of activated channels. Figure 7A shows one channel activated after addition of PKA and ATP. The number of channels did not increase further with F- present for up to 5 min. Similar results were obtained in nine other excised membrane patches.

Effect of F- on CFTR with Walker lysine mutations. Previous work had suggested that NBD2 is a primary determinant of burst duration (9, 11, 22). This suggested that F- might interact with CFTR at NBD2. To investigate this possibility further, we studied channels in which the conserved Walker lysine in NBD2 (lysine 1250) was mutated. We found that F- stimulates CFTR-K1250M channels to a greater extent than wild-type CFTR (Fig. 8). In contrast, channels containing a mutation of the conserved Walker lysine of NBD1 (lysine 464) had a response similar to that of wild-type CFTR. These data suggest that F- may have altered gating by NBD2.


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Fig. 8.   Effect of increasing F- concentration on current in wild-type CFTR, CFTR-K464A, and CFTR-K1250M. All values were obtained in presence of 1 mM ATP and are expressed as percentage of current supported by 1 mM ATP. Data are means ± SE of 3-20 experiments. * P < 0.005 compared with wild-type CFTR and * P < 0.026 compared with CFTR-K464A.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Our results show that F- stimulated CFTR Cl- channels but only after they had been phosphorylated and only in the presence of ATP. The effect of F- was likely independent of phosphatase inhibition, since F- increased current in CFTRDelta R-S660A. Moreover, stimulation by F- was probably not via G protein activation, since GTPgamma S had no effect on CFTR. The evidence suggests that F- was interacting with CFTR.

Some clues about how F- stimulated CFTR came from studies of single-channel currents and mutant CFTR. We cannot exclude the possibility that F- stimulated CFTR through an interaction with the membrane-spanning domains; however, the minimal effects on single-channel conductance and the lack of voltage dependence suggest that this was unlikely. The findings that F- stimulated activity when added to the cytoplasmic surface of the patch and that F- affected the gating of CFTR suggested an interaction with the cytoplasmic domains. It seemed unlikely that F- stimulated through an interaction with the R domain because F- stimulated CFTRDelta R-S660A. Instead, the data suggest that F- interacts with the NBDs. Models of CFTR gating suggest distinct functions for each NBD, with burst duration influenced primarily by NBD2 (3, 6, 9, 22, 35). The effect of F- on burst duration suggested the possibility of an interaction with NBD2. This suggestion was supported by the finding that mutation of the conserved Walker lysine of NBD2 but not NBD1 enhanced the effect of F-.

Baukrowitz et al. (6) found that BeF3 stimulated CFTR and suggested that BeF3 stabilized the open-channel state. They found that BeF3 prolonged bursts of activity by locking the channel in the open state. However, they found that F- alone was ineffective. Those studies led us to test the possibility that the effect of F- was, in fact, due to AlF3. However, our studies suggest that even in the absence of Al3+ or beryllium, F- stimulated activity. There are several possible explanations for differences between the two studies. Although most of our studies were performed at 37°C, a difference in temperature is not likely to explain the difference because we found that F- stimulated current at 25°C, the temperature used by Baukrowitz et al. (6). Another possible explanation is that different measurements were made in the two studies. Baukrowitz et al. (6) measured closing rate after removal of the stimulating agent, whereas we measured current in the presence of F- under equilibrium conditions. Another, but less likely, possibility is that Baukrowitz et al. (6) studied CFTR from guinea pig cardiac myocytes. This cardiac CFTR is alternatively spliced, deleting 30 amino acids from exon 5 (18, 21). It is possible that this structural difference may have accounted for the different effects of F-.

The suggestion that F- might stimulate CFTR by interacting with NBD2 prompts a comparison with the effects of other agents predicted to have an effect on the NBDs (Table 1). Previous studies showed that PPi increased burst duration, increased the rate of channel opening (i.e., decreased the long closed time), and increased 8-N3ATP photolabeling (12, 16). Like PPi, F- increased burst duration but did not increase channel-opening rate or 8-N3ATP photolabeling. More strikingly, PPi stimulated less current in K1250M-CFTR than in wild-type CFTR (14), whereas F- stimulated more current in K1250M-CFTR than in wild-type CFTR. Therefore, there are distinct differences between the effects of F- and PPi.

                              
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Table 1.   Comparison of the effect of F-, other nucleotides, and inorganic phosphate analogs

Like F-, 5'-adenylylimidodiphosphate (AMP-PNP) prolonged the burst duration, had no effect on the long closed time, and has been proposed to bind to NBD2 (9, 16, 22). But unlike F-, AMP-PNP decreased 8-N3ATP photolabeling and required the continued presence of PKA to have an effect (9, 22, 40). Therefore, although AMP-PNP and F- may both affect NBD2 to prolong the burst duration, there are differences between the two that suggest they interact differently. The effect of F- on CFTR was very different from the effect of inorganic phosphate (Pi). Pi decreased the long closed time, decreased 8-N3ATP photolabeling, and had no effect on burst duration (10).

The effect of F- was qualitatively most similar to that reported for VO4 and BeF3 (6, 16). These agents prolong the burst duration; 8-N3ATP photolabeling was not evaluated. VO4 has been proposed to be a phosphate analog and to bind tightly at a site where the gamma -phosphate is released after ATP hydrolysis, resulting in a stable conformation and prolonging CFTR burst duration (6). BeF3 is thought to have a similar effect. Similarity between the effects of VO4 and BeF3 and those which we observed with F- raises the question of whether they might share a similar mechanism. There are several reports demonstrating an Al3+-independent activation of heterotrimeric G proteins by F- (4, 5, 20). Higashijima et al. (20) found that millimolar concentrations of F- plus Mg2+ were able to increase G protein fluorescence, suggesting that MgF2 could stimulate G proteins. In addition, Antonny et al. (4) found that in the presence of Mg2+, F- concentrations >3 mM could activate a G protein (transducin-guanosine diphosphate) in the absence of Al3+ or beryllium. However, in contrast to BeF3, the inactivation rate was 100-1,000 times faster for F- alone than for AlF3 or BeF3, suggesting that F- (MgF2) was less stable or that it bound less tightly to the protein than did AlF3 or BeF3. Nuclear magnetic resonance spectroscopy of G proteins revealed that activation by MgF2 was chemically distinct from that obtained with AlF3 (5), although both MgF2 and AlF3 were thought to mimic the gamma -phosphate of a GTP. Because our solutions contained Mg2+ out of necessity (MgATP is required for channel activity), we speculate that F-, perhaps MgF2, is interacting with CFTR in a manner analogous to that observed in G proteins. This scenario might also explain, in part, the difference in burst duration in the presence of F- (~450 ms) compared with that in the presence of VO4 [bursts lasted for minutes (6, 16)]. Perhaps, as proposed for VO4, F- or MgF2 functions as a phosphate analog, binding at a site where the gamma -phosphate is released after ATP hydrolysis, resulting in a stable conformation and prolonging CFTR burst duration (6). However, burst duration may be shorter with F- than with BeF- or VO4 because those agents bind more tightly.

There is also the interesting possibility that F- may interact with a site in CFTR that is different from that to which VO4 and BeF3 bind. Such a mechanism could be analogous to the inhibitory effect of F- on guanosine 5'-triphosphatase (GTPase)-activating proteins (GAP) (43). F--dependent inhibition of GTPase activity leads to prolonged GTP binding and activation of GTP binding proteins such as Rac (43). F- inhibition of GAP activity was independent of Al3+, and F- did not affect guanosine diphosphate release from Rac. Perhaps F- modification of an allosteric site on CFTR might regulate its activity in an analogous manner. Future studies with site-directed mutations will be needed to determine the residues that are important for F- interaction and stimulation of CFTR.

Finally, our results have practical implications for studies of CFTR function. Because F- is a nonspecific phosphatase inhibitor, it is sometimes added to solutions bathing CFTR in an attempt to prevent dephosphorylation. Our data suggest that F- should be added to the solutions with caution because it can affect channel gating.

    ACKNOWLEDGEMENTS

We thank Pary Weber, Phil Karp, Theresa Mayhew, and Deb Gilmere for excellent technical assistance and our laboratory colleagues for critical comments and discussions. We thank Dr. Mark Arnold, Dr. Johna Leddy, and Carolyn Green (Dept. of Chemistry, Univ. of Iowa) for calculations of Al3+ and AlF<SUP>−</SUP><SUB>4</SUB> in our solutions. We thank John Marshall and Seng Cheng (Genzyme) for the M13-1 antibody and the baculovirus encoding CFTR.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-42385.

H. A. Berger was a Parker B. Francis Fellow, and M. J. Welsh is an Investigator of the Howard Hughes Medical Institute.

Address for reprint requests: M. J. Welsh, Howard Hughes Medical Institute, Univ. of Iowa College of Medicine, 500 EMRB, Iowa City, IA 52242.

Received 13 May 1997; accepted in final form 21 November 1997.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

1.   Anderson, M. P., H. A. Berger, D. P. Rich, R. J. Gregory, A. E. Smith, and M. J. Welsh. Nucleoside triphosphates are required to open the CFTR chloride channel. Cell 67: 775-784, 1991[Medline].

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