Introduction

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Cystic fibrosis (CF) is caused by mutations in the single gene encoding the cystic fibrosis transmembrane conductance regulator protein CFTR (Riordan et al., 1989). CFTR is an epithelial chloride channel, activated by protein kinase A (PKA)-dependent phosphorylation, and gated by ATP hydrolysis at two nucleotide binding domains, NBD1 and NBD2 (Riordan et al., 1989; Anderson et al., 1991; Cheng et al., 1991; Tabcharani et al., 1991; Gadsby et al., 1995). A consequence of CFTR mutations is defective electrolyte transport in airway epithelia, resulting in chronic lung infection and premature mortality in CF patients (Welsh et al., 1995).

The most prevalent CF-associated mutation is F508 (deletion of the phenylalanine amino acid at position 508 in NBD1). This mutation, accounting for ~70% of all disease-associated mutations, causes protein trafficking defects (Welsh and Smith, 1993); mutant protein is synthesized and inserted into the endoplasmic reticulum but most protein fails to progress to the Golgi apparatus and cell membrane (Cheng et al., 1990; Welsh and Smith, 1993). F508 CFTR also has a functional defect; although a small fraction of F508 CFTR reaches the plasma membrane, its open probability (Po) is lower than that of wild-type (wt) channels, even in the presence of maximally effective concentrations of cAMP (Dalemans et al., 1991; Haws et al., 1996; Hwang et al., 1997; Al-Nakkash and Hwang, 1999). It remains unclear which defect contributes to CF pathogenesis in vivo (Kälin et al., 1999; Wang et al., 2000), since these defects may play distinct roles in different tissues. Nevertheless, research efforts have focused on pharmacological methods to rectify either the F508-CFTR defective trafficking or defective function. Restoration of F508-CFTR defective trafficking can be accomplished by either incubation of the cells at lowered temperatures (Denning et al., 1992) or through the use of chemical chaperones (Sato et al., 1996). The defective function of F508-CFTR, i.e., a reduced cAMP-dependent Po, can be restored by numerous compounds, such as genistein and 5-trifluoromethyl-1-(5-chloro-2-hydroxyphenyl)-1,3-dihydro-2H-benzimidazole-2-one (NS004) (for review, see Hwang and Sheppard, 1999).

Genistein and benzimidazolone analogs (Fig. 1) have attracted significant attention because of their high potency, i.e., both work at nanomolar to micromolar concentrations (for review, see Hwang and Sheppard, 1999; Schultz et al., 1999b). Elucidation of their mechanism of action could potentially be useful in generating pharmacological agents in CF therapeutics. Genistein, an isoflavonoid, has been shown to activate F508-CFTR chloride current at micromolar concentrations, by increasing channel Po, via an increased channel open time and a decreased channel closed time (Illek et al., 1996; Hwang et al., 1997; Yang et al., 1997). Furthermore, it has been hypothesized that genistein's stimulatory effect is mediated by direct binding of genistein to the NBD of phosphorylated CFTR (Wang et al., 1998). On the other hand, the benzimidazolone analog NS004 has also been described to activate F508-CFTR (Gribkoff et al., 1994), although its mechanism of action has yet to be elucidated. It is of note that both genistein and NS004 have been shown to increase CFTR channel activity without changing intracellular cAMP levels (Gribkoff et al., 1994; Illek et al., 1995; He et al., 1998).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Chemical structures of genistein, NS004, and NS1619.

We investigated the mechanism of action of two benzimidazolone analogs (NS004 and NS1619; Fig. 1) on F508-CFTR using both whole-cell and cell-attached patch-clamp techniques. Their effects are compared with those of genistein. We have determined that NS004 or NS1619 activates F508-CFTR at nanomolar concentrations, as previously described by Gribkoff et al. (1994). We demonstrate that saturating concentrations of genistein and benzimidazolone analogs generate similar whole-cell macroscopic F508-CFTR channel current. Furthermore, these benzimidazolone analogs act by increasing F508-CFTR chloride channel Po (via an increased open time and a decreased closed time) without affecting the number of functional channels. These effects on F508-CFTR channel kinetics are similar to those reported for genistein (Hwang et al., 1997). Neither benzimidazolone analogs nor genistein potentiate cAMP-dependent K1250A-CFTR activity, a nucleotide binding domain 2 mutant with a prolonged open time due to diminished ATP hydrolysis activity. Although the F508-CFTR channel open time is increased by introducing the K1250A mutation into the F508 background, the prolonged closed time is unaffected, suggesting that the prolonged closed time associated with the F508 mutation is independent of channel open time. Since benzimidazolone analogs and genistein enhance F508/K1250A-CFTR activity by shortening the channel closed time, we conclude that their rectification of the F508-CFTR prolonged closed time is independent of their effects on the channel open time. Future studies using structurally related compounds should shed light on the molecular nature of their binding site(s).

	Materials and Methods

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Cell Culture

NIH3T3 mouse fibroblast cells stably expressing either F508-CFTR or K1250A-CFTR were prepared as described previously (Berger et al., 1991; Zeltwanger et al., 1999). The F508/K1250A-CFTR double mutation was generated as follows. Plasmids containing F508-CFTR (F508pRBG4) or K1250A-CFTR (K1250ApRBG4) were generously provided by Dr. R. R. Kopito (Stanford University, Stanford, CA). The PstI-SphI fragment containing the F508 mutation was swapped with the same fragment in the K1250ApRBG4, thus generating the F508/K1250ApRBG4 construct. The double mutant CFTR cDNA was then further subcloned into a mammalian expression vector, pcDNA3.1 Zeo (+) (Invitrogen, Carlsbad, CA). Both mutations were confirmed by DNA sequencing (DNA Core; University of Missouri, Columbia, MO). Using Superfect reagent (Qiagen, Valencia, CA), the F508/K1250A-CFTR double mutant was transiently transfected into NIH3T3 mouse fibroblast cells, according to the manufacturer's protocol. Briefly, NIH3T3 cells were seeded at ~2 × 10⁵ cells/60-mm dish. The cells were transfected 18 h later, during a 3-h period, with 5.5 µg of total DNA (5 µg of double mutant DNA and 0.5 µg of pEGF-C3 DNA encoding green fluorescent protein). The cells were passaged into dishes containing glass coverslips, for use with the patch-clamp studies after a further 16-h incubation. Cells expressing CFTR were identified under a fluorescent microscope. Because of the 1:10 ratio of plasmids used for cotransfection, almost all visible "green" cells express CFTR.

All cell lines were grown in Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum and maintained under standard tissue culture conditions. NIH3T3-F508 cells were placed at 27°C for 2 to 3 days before use. Previous work from our laboratory has demonstrated that lowering the culturing temperature, while increasing F508-CFTR channel density in the cell membrane, does not influence channel function (Hwang et al., 1997).

Electrophysiology

Cell-Attached Patch-Clamp Experiments. Cells were grown on small glass chips, placed in tissue culture dishes. The glass chips were transferred to a continuously perfused chamber located on the stage of an inverted microscope (Olympus, Tokyo, Japan). Pipette electrodes were made from Corning 7056 borosilicate glass capillaries (Warner Instrument Corp., Hammed, CT) with a two-stage vertical puller (Narishige, Tokyo, Japan). Pipette tips were fire polished with a homemade microforge and had resistances of 4.46 ± 0.08 M (n = 100) in the bath solution. CFTR channel currents were recorded at room temperature (~22°C) with an EPC-9 patch-clamp amplifier (HEKA, Lambrecht/Pfalz, Germany), filtered at 100 Hz with a built-in four-pole Bessel filter, and stored on videotapes. Data were refiltered at 50 Hz with an eight-pole Bessel filter (Frequency Device, Haverill, MA) and captured onto a hard disk (Quadra 650, Macintosh computer) at a sampling rate of 100 Hz. Solution changes were effected via parallel silastic tubings descending from separate gravity-feed reservoirs into a common perfusion manifold. The pipette potential was held at +50 mV with reference to the bath. Downward deflections in the recordings represent channel openings. The pipette solution contained 140 mM N-methyl-D-glucamine chloride, 2 mM MgCl₂, 5 mM CaCl₂, and 10 mM HEPES (pH 7.4 with N-methyl-D-glucamine). All cell-attached experiments were performed in a perfusion solution containing 145 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 1 mM CaCl₂, 5 mM glucose, 5 mM HEPES, and 20% sucrose (pH 7.4 with 1 N NaOH). Addition of sucrose to the perfusion solution circumvented activation of swelling-induced chloride currents (Worrell et al., 1989).

Whole-Cell Patch-Clamp Experiments. Cell suspensions were prepared by brief trypsinization (0.25% trypsin in phosphate-buffered saline). Pipette electrodes were made from Kimax 51 brand thin-walled capillaries (Fischer Scientific, Pittsburgh, PA), with a two-stage vertical puller (Narishige). Pipette tips were not fire polished and had resistances of ~3 M in the bath solution. The membrane potential was held with an Axopatch 1D amplifier (Axon Instrument, Foster City, CA) at 0 mV (12 mV after correction of the junction potential), following break-in with suction. I-V relationships were generated using Igor software (Wavemetrics, Lake Oswego, OR) and XOP (developed by Dr. R. Bookman, University of Miami, Miami, FL). Current traces in response to voltage pulses (±100 mV in 12-mV increments and 100-ms duration) were filtered at 1 kHz with a built-in four-pole Bessel filter and then digitized (at 2 kHz) directly into the computer hard drive (7100/80, Macintosh computer) through an ITC-16 interface (Instrutech Corp., Greatneck, NY). CFTR channel currents were recorded at room temperature (~22°C). The pipette solution contained 85 mM aspartic acid, 5 mM pyruvic acid, 10 mM EGTA, 20 mM tetraethylammonium-chloride, 5 mM triscreatine phosphate, 10 mM MgATP, 2 mM MgCl₂, 5.5 mM glucose, and 10 mM HEPES (pH 7.4 with 8 N CsOH). The bath solution contained 150 mM NaCl, 2 mM MgCl₂, 1 mM CaCl₂, 5 mM glucose, 5 mM HEPES, and 20% sucrose (pH 7.4 with 1 N NaOH).

Data Analysis and Statistics

Dose-response relationships between NS004, NS1619, and genistein concentrations versus CFTR channel activity were fitted with the Hill equation using Sigmaplot software (Jandel Scientific, San Rafael, CA). Mean steady-state current amplitudes were calculated with Igor software (Wavemetrics Inc.), from a 1- to 2-min segment of the steady-state CFTR current. All-point histograms were generated with Igor software to determine single-channel amplitudes. Gaussian functions were used to fit the histogram data, and single-channel amplitudes were obtained by measuring the difference between two adjacent peaks (representing the channel opening and closing). Dwell time analysis (with exponential fits of the distributions) was performed using Igor software. After measuring the open time for each single-channel open event, the data were sorted in terms of duration, from shortest to longest, and data plotted as number of events versus the duration of opening. Open duration data could be fit with either a single or double exponential function, yielding either one or two time constants, respectively. For multiple-channel recordings, openings and closings of a single channel cannot be paired, and therefore estimations of mean open times are more complicated. Estimations of the mean channel open time were obtained by examining the average gating behavior of the channels. The mean open time for individual channels in multiple-channel patches can be calculated using the following formula: t_n = j · t_j/n, where t_j is the time that j channels open at the same time and n is the total number of transitions from open to close. Therefore, the multiple-channel open event is transferred to n single-channel open events with open time t_n for each event. The open time constant can thus be determined using the same method as described for the single-channel recordings. This method was originally described by Fenwick et al. (1982) and has been previously used to characterize CFTR open events (Wang et al., 1998; Zeltwanger et al., 1999). Data are presented as mean ± S.E.M. Statistical analyses (t tests) were performed using Sigmaplot software with significance given at P < 0.05.

Reagents

Forskolin was purchased from Calbiochem (La Jolla, CA) and stored as 20 mM stock in dimethyl sulfoxide (DMSO) at 20°C. Genistein was purchased from Alexis Corp. (San Diego, CA) and stored as 100 mM stock in DMSO at 20°C. All other chemicals were purchased from Sigma. The benzimidazolone analogs (NS004 and NS1619) were a generous gift from Dr. S.-P. Olesen (Neurosearch, Glostrup, Denmark), and were stored as 50 mM stock in DMSO at 20°C. Nanomolar concentrations of NS004 and NS1619 were made by serial dilution of the 50 mM stock.

	Results

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Enhancement of Macroscopic F508-CFTR Channel Current by NS004 and NS1619. We first quantified the potency and efficacy of benzimidazolone analogs by using the whole-cell patch-clamp technique to measure F508-CFTR current from NIH3T3-F508 cells. Since the mechanism of action of genistein on CFTR is well understood (Illek et al., 1995, 1996; Hwang et al., 1997; Wang et al., 1998), we used the effect of genistein as a gauge to estimate the effects of benzimidazolone analogs. Figure 2A shows that genistein potentiates the forskolin-activated F508-CFTR whole-cell chloride current in a dose-dependent manner. In the absence of forskolin, basal conductance is minimal (Wang et al., 2000). The application of forskolin (10 µM) alone induced slight increase in membrane conductance with a reversal potential of 33.9 ± 2.8 (n = 4), which is close to the equilibrium potential for chloride E_Cl (47 mV). The addition of various concentrations of genistein in the presence of 10 µM forskolin produces incremental increases in the holding current, with parallel increases in the chloride conductance (the adjacent I-V relationship shows an example of the increase in conductance, compared with forskolin alone, at one given concentration of agonist). From these experiments we establish that 20 µM is a maximally effective concentration of genistein. Figure 2B shows a typical whole-cell trace recording upon application of various concentrations of NS004 in the continued presence of 10 µM forskolin. For comparison, 20 µM genistein was used as the final manipulation. Addition of various concentrations of NS004 (Fig. 2B), elicit incremental increases in the holding current, with concomitant increases of the chloride conductance. Similar results were observed for NS1619 (data not shown). Reversal potentials for NS004 and NS1619 are 35.7 ± 4.2 (n = 5) and 34.7 ± 7.2 (n = 4), respectively.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Dose-response relationships of genistein and NS004 in NIH3T3-F508 cells. A, concentration-dependent effects of genistein. Numbers 1 to 6 represent step increases of genistein concentration (2.5, 5, 10, 20, 30, and 50 µM, respectively). The I-V relationship to the right shows the data at points b and c as marked (basal current is subtracted); forskolin activated current () and forskolin plus 10 µM genistein (). B, concentration-dependent effects of NS004. Numbers 1 to 5 represent step increases of the NS004 concentration (100 fM, 63 pM, 50 nM, 1 µM, and 20 µM, respectively). Number 6 is 20 µM genistein. The I-V relationship to the right shows the data at points b and c as marked (basal current is subtracted); forskolin-activated current () and forskolin plus 50 nM NS004 ().

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Dose-response relationship between NS004, NS1619, and genistein concentration. Whole-cell F508-CFTR currents at each concentration were normalized to that obtained with 20 µM genistein (n = 5-6 for each data point). The current density for genistein, NS004, and NS1619 (each at 20 µM) was found to be 8.19 ± 5.3, 7.46 ± 4.26, and 7.16 ± 4.44 pA/pF, respectively. Smooth curves represent nonlinear least-squares fits of the data with the Hill equation.

NS004 and NS1619 Increase F508-CFTR Channel Open Probability. To further dissect the mechanism by which benzimidazolone analogs increase mean F508-CFTR current, we analyzed the effects of these compounds on F508-CFTR channel activity in cell-attached patches, where microscopic current is obtained. In patches containing few channels (less than four channels open at any given time throughout the recording), the number of channel open steps can be readily discerned, and thus the number of functional channels can be estimated. Figure 4 shows a representative example of such a patch containing only two channel open steps throughout a 37-min recording period. Using Gaussian fits of all-points amplitude histograms, we obtained the single-channel amplitude, i, under different experimental conditions. The single-channel amplitude in the presence of 10 µM forskolin alone (0.28 ± 0.01 pA, n = 3) is not significantly different from those measured in the presence of forskolin plus 50 nM NS004 (0.27 ± 0.02 pA, n = 3, p = 0.58, paired t test) or forskolin plus 20 µM NS004 (0.27 ± 0.02 pA, n = 3, p = 0.50, paired t test). Analogous to the NS004 data, there was no effect of either 50 nM or 20 µM NS1619 upon the single-channel current amplitude (data not shown). Since there is little effect of benzimidazolone analogs upon the single-channel amplitude or the number of functional channels we predicted that the potentiative effects of NS004 and NS1619 upon the F508-CFTR current must be attributed to an increase in Po.

View larger version (40K): [in this window] [in a new window]   Fig. 4.   Potentiation of forskolin-dependent F508-CFTR chloride current by NS004. A representative cell-attached recording (lasting 37 min) from an NIH3T3-F508 cell. In the presence of forskolin alone (10 µM) the channels are mostly closed, as indicated by the arrow. In this condition calculated Po is 0.08 and the relative areas in the amplitude histograms at closed level (A0), one channel open level (A1) and two channel open level (A2) are 0.86, 0.13, and 0.005, respectively (theoretical areas from binomial analysis: 0.85, 0.15, and 0.006). In the presence of 50 nM NS004, the calculated Po = 0.56 and A0:A1:A2 = 0.16:0.48:0.36 (theoretical ratio = 0.14:0.51:0.34). In the presence of 20 µM NS004, the calculated Po = 0.81 and A0:A1:A2 = 0.012:0.19:0.79 (theoretical ratio = 0:0.23:0.78).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Genistein has little effect on the mean F508-CFTR current amplitude in the presence of benzimidazolone analogs. A 30-min continuous recording of F508-CFTR in the cell-attached mode. Closed state is indicated by the arrow. Downward deflections are channel openings.

NS004 and NS1619 Prolong the F508-CFTR Channel Open Time and Shorten Channel Closed Time. Similar to genistein, benzimidazolone analogs also increase the open time and decrease the closed time of F508-CFTR. Figure 6A shows a cell-attached recording from a patch containing one single channel. As reported previously (Haws et al., 1996), F508-CFTR opens in bursts with long closed events separating individual burst openings. Those flickery closings within a burst are voltage-dependent and likely due to flickery blockade (Z. Zhou, S. Hu, and T.-C. Hwang, submitted). Upon inspection, NS1619 and genistein apparently increase the Po by affecting both the bursting open time and closed time. In the presence of forskolin alone (10 µM) the open time is short and the time the channel spends closed is long (when flickery closings are ignored). Upon application of either NS1619 (20 µM) or genistein (20 µM) the open time becomes longer and the closed time shorter. Although closed times do affect Po, they are more difficult to determine because of the rarity of single-channel patches and of the slow gating of the channel (i.e., to obtain enough closed events to perform dwell time analysis one would have to record for many hours in a given patch). However, for the one long-lasting single-channel recording obtained, the mean closed time in the presence of forskolin (10 µM), forskolin plus NS1619 (20 µM), or forskolin plus genistein (20 µM) was found to be 87.16 ± 21.63 (16 events), 13.85 ± 1.89 (51 events), and 17.22 ± 3.96 (28 events), respectively. These data demonstrate that the prolonged closed time of F508-CFTR is reduced in the presence of benzimidazolone analogs or genistein.

View larger version (37K): [in this window] [in a new window]   Fig. 6.   Effect of benzimidazolone analogs and genistein upon F508-CFTR channel kinetics. A, recordings of one single F508-CFTR channel in response to different agonists as marked. Each trace lasts for 150 s. Closed state is denoted by the arrow. Downward deflections are channel openings. B, dwell time analysis of pooled open events from patches containing few channels. The data were fit with double exponential functions to yield the open time constants.

Effects of NS004 and NS1619 on K1250A-CFTR. To further corroborate our evidence that NS004 and NS1619 act to stabilize the channel open state, we examined the effect of these drugs upon the K1250A-CFTR mutant channel. This mutant channel, once opened, can stay open for minutes (Zeltwanger et al., 1999). Figure 7 shows a representative recording of K1250A-CFTR in a cell-attached patch. A macroscopic current was elicited by a maximal concentration of forskolin (10 µM), once a maximal level of current was achieved, subsequent addition of 50 nM or 20 µM NS1619 failed to increase the current. Fold increases in mean K1250A-CFTR current amplitude were 1.09 ± 0.11 (n = 3) and 1.15 ± 0.18 (n = 3), respectively. Removal of forskolin and NS1619 resulted in a slow, stepwise deactivation of all channels. In the same patch forskolin alone can reactivate the K1250A-CFTR current, and subsequent addition of 20 µM genistein had minimal effect on the current (fold increase = 1.03 ± 0.02, n = 5). Similar results were obtained with NS004 (1.15 ± 0.18-fold, n = 3). Thus, neither benzimidazolone analogs nor genistein altered the Po of K1250A-CFTR in the presence of a maximal concentration of forskolin.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Effect of NS1619 and genistein upon forskolin-dependent K1250A-CFTR channel current. In this continuous cell-attached recording (lasting 50 min), the application of 10 µM forskolin elicits a macroscopic K1250A-CFTR current. There is no effect on the mean current amplitude by the addition of either 50 nM or 20 µM NS1619. Similarly, there is no effect of 20 µM genistein upon the steady-state macroscopic K1250A-CFTR current generated by 10 µM forskolin. The expanded section shows the closure of all K1250A channels upon removal of agonists and a return to basal activity (expanded trace lasts for 200 s). Closed state is denoted by the arrow. Downward deflections are channel openings.

Effect of NS004 and NS1619 upon the F508/K1250A Double Mutation. It is well established that the major functional defect associated with F508-CFTR is a prolonged closed time when measured in cell-attached patches (Haws et al., 1996; Hwang et al., 1997). It is clear that genistein and benzimidazolone analogs increase the channel open time and decrease channel closed time. However, it is not clear whether these two effects are causal as suggested previously (Schultz et al., 1999a). To address this, we examined the effects of benzimidazolone analogs and genistein on the double mutant F508/K1250A-CFTR.

View larger version (31K): [in this window] [in a new window]   Fig. 8.   Effect of NS004 and NS1619 on F508/K1250A current. A, 30-min continuous recording showing the effect of NS004 on forskolin-dependent macroscopic F508/K1250A-CFTR current. The middle part of the recording is expanded to demonstrate steady-state current in the presence of forskolin alone, where 1 to 3 channel open steps can be discerned (trace lasts for 230 s). The expansion of the latter part of the recording demonstrates the eventual closure of all channels upon washout of all agonists (trace lasts for 450 s). B, effect of NS1619 on forskolin-dependent F508/K1250A-CFTR channel current in a patch containing one single channel. Each trace lasts for 120 s. Downward deflections are channel openings. Closed state is indicated by the arrow.

	Discussion

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Cyclic AMP-Dependent and -Independent Regulation of CFTR. The cAMP-PKA-signaling pathway is well established as the major mechanism for regulation of CFTR chloride channel activity. PKA-dependent activation of CFTR likely includes phosphorylation of the regulatory domain at multiple sites (Riordan et al., 1989; Anderson et al., 1991; Cheng et al., 1991; Tabcharani et al., 1991; Gadsby et al., 1995). Direct evidence for this came from the demonstration that purified CFTR, when incorporated into bilayers, produces small conductance chloride channels activated by PKA plus ATP (Bear et al., 1992). Biochemical studies also showed that PKA can phosphorylate CFTR at multiple serine residues both in vitro and in vivo (Picciotto et al., 1992). Although phosphorylation regulation of CFTR by kinases other than PKA has been reported (Tabcharani et al., 1991; Hwang and Sheppard, 1999), their physiological role remains unclear.

A Common Mechanism for CFTR Activation. We believe that benzimidazolone analogs and genistein act through a common mechanism for the following reasons. First, both benzimidazolone analogs and genistein act only on phosphorylated CFTR. Yang et al. (1997) found that genistein potentiates forskolin-dependent CFTR activity, but does not activate CFTR by itself. NS004 (20 µM), when applied alone for 4 to 6 min, does not activate CFTR in cell-attached patches that lack basal activity, but subsequent addition of forskolin (10 µM) with NS004 (20 µM) in the same patch induced macroscopic CFTR current (L. Al-Nakkash and T. C. Hwang, unpublished observation).

Biophysical Basis for the Functional Defect of F508-CFTR. Our previous studies on CFTR regulation and gating (Hwang et al., 1997; Zeltwanger et al., 1999; Wang et al., 2000) suggest that in an intact cell, the channel open time is mostly determined by the rate of ATP hydrolysis at NBD2, whereas the closed time is determined by the level of cAMP stimulation (thus, the rate of PKA-dependent phosphorylation of CFTR). We also reported that the abnormally prolonged closed time associated with the F508 mutation is caused by a slower rate of PKA-dependent phosphorylation of F508-CFTR channels (cf. Drumm et al., 1991; Wang et al., 2000). Our biochemical interpretation of CFTR kinetics implies that the open time and closed time are somewhat independent of each other mechanistically. That is, maneuvers that change the open time do not necessarily alter the closed time. However, the fact that genistein or benzimidazolone analogs affect both the open time and the closed time of F508-CFTR raises the possibility whether these two effects are causal. Schultz et al. (1999a) explain the effects of 3-isobutyl-1-methylxanthine on F508-CFTR with the proposal that a shortening of the closed time by 3-isobutyl-1-methylxanthine is secondary to a prolongation of the open time. This apparently contradicts our findings. First, when K1250A-CFTR is submaximally stimulated with nanomolar forskolin, the closed time can still be decreased by benzimidazolone analogs. Second, when the double mutant F508/K1250A-CFTR is stimulated with a maximally effective concentration of forskolin, the prolonged open time caused by the K1250A mutation does not automatically correct the abnormally long closed time associated with the F508 mutation. Thus, these two kinetic effects (i.e., increased open time and decreased closed time) of genistein or benzimidazolone analogs are likely independent of each other. Although we cannot rule out the possibility that one single binding site can cause both effects, it is possible that two binding sites may exist to account for the effects of these compounds.