We have investigated the mechanism of action of two benzimidazolone analogs (NS004 and NS1619) on ΔF508-CFTR using both whole-cell and cell-attached patch-clamp techniques and compared their effects with those of genistein. We conclude that benzimidazolone analogs and genistein act through a common mechanism, based on the following evidence: 1) both act only on phosphorylated CFTR, 2) the maximal ΔF508-CFTR current activated by benzimidazolone analogs is identical to that induced by genistein, 3) benzimidazolone analogs increase the open probability of the forskolin-dependent ΔF508-CFTR channel activity through an increase of the channel open time and a decrease of the channel closed time (effects indistinct from those reported for genistein), and 4) the prolonged K1250A-CFTR channel open time (in the presence of 10 μM forskolin) is unaffected by benzimidazolone analogs or genistein, supporting the hypothesis that these compounds stabilize the open state by inhibiting ATP hydrolysis at nucleotide binding domain 2 (NBD2). In addition, we demonstrate that NS004 and NS1619 are more potent CFTR activators than genistein (EC50 values are 87 ± 14 nM, 472 ± 88 nM, and 4.4 ± 0.5 μM, respectively). From our studies with the double mutant ΔF508/K1250A-CFTR, we conclude that benzimidazolone analogs and genistein rectify the ΔF508-CFTR prolonged closed time independent of their effects on channel open time, since these agonists enhance ΔF508/K1250A-CFTR activity by shortening the channel closed time. These studies should pave the way toward understanding the agonist binding sites at a molecular level.
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 ΔF508 (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). ΔF508 CFTR also has a functional defect; although a small fraction of ΔF508 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 ΔF508-CFTR defective trafficking or defective function. Restoration of ΔF508-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 ΔF508-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 ΔF508-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 ΔF508-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).
We investigated the mechanism of action of two benzimidazolone analogs (NS004 and NS1619; Fig. 1) on ΔF508-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 ΔF508-CFTR at nanomolar concentrations, as previously described byGribkoff et al. (1994). We demonstrate that saturating concentrations of genistein and benzimidazolone analogs generate similar whole-cell macroscopic ΔF508-CFTR channel current. Furthermore, these benzimidazolone analogs act by increasing ΔF508-CFTR chloride channel Po (via an increased open time and a decreased closed time) without affecting the number of functional channels. These effects on ΔF508-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 ΔF508-CFTR channel open time is increased by introducing the K1250A mutation into the ΔF508 background, the prolonged closed time is unaffected, suggesting that the prolonged closed time associated with the ΔF508 mutation is independent of channel open time. Since benzimidazolone analogs and genistein enhance ΔF508/K1250A-CFTR activity by shortening the channel closed time, we conclude that their rectification of the ΔF508-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
NIH3T3 mouse fibroblast cells stably expressing either ΔF508-CFTR or K1250A-CFTR were prepared as described previously (Berger et al., 1991; Zeltwanger et al., 1999). The ΔF508/K1250A-CFTR double mutation was generated as follows. Plasmids containing ΔF508-CFTR (ΔF508pRBG4) or K1250A-CFTR (K1250ApRBG4) were generously provided by Dr. R. R. Kopito (Stanford University, Stanford, CA). The PstI-SphI fragment containing the ΔF508 mutation was swapped with the same fragment in the K1250ApRBG4, thus generating the ΔF508/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 ΔF508/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-ΔF508 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 ΔF508-CFTR channel density in the cell membrane, does not influence channel function (Hwang et al., 1997).
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 MΩ (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 withN-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 MΩ 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 = Σj ·t j/n, wheret j is the time that jchannels 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 (ttests) were performed using Sigmaplot software with significance given at P < 0.05.
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.
Enhancement of Macroscopic ΔF508-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 ΔF508-CFTR current from NIH3T3-ΔF508 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 ΔF508-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 ECl (−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.
The dose-response relationships of genistein, NS004, and NS1619, summarized from multiple whole-cell patches are shown in Fig.3. The ΔF508-CFTR current generated at each concentration of agonist tested is normalized to that obtained with 20 μM genistein in the same cell, and EC50values for genistein, NS004, and NS1619 were found to be 4.4 ± 0.5 μM, 87 ± 14 nM, and 472 ± 88 nM, respectively. It is worth noting from the whole-cell dose-response relationship that the maximal whole-cell ΔF508-CFTR current enhanced by genistein, NS004, or NS1619 is very similar. These data suggest that the efficacy of these benzimidazolone analogs is the same as that of genistein, whereas the potency of these benzimidazolone analogs is greater than that of genistein.
These effects of benzimidazolone analogs on the forskolin-induced ΔF508-CFTR chloride current were confirmed in cell-attached patches. In the continued presence of 10 μM forskolin, 20 μM NS004 potentiated the macroscopic ΔF508-CFTR channel current by 11.09 ± 1.76-fold (n = 9). Of note, 20 μM genistein stimulated the ΔF508-CFTR current by a similar magnitude to that observed with 20 μM NS004 (fold increase was 11.95 ± 2.34,n = 8). Similar results were observed with 20 μM NS1619 (15.74 ± 3.37 fold, n = 4). Thus, artificial dialysis of the cell with the whole-cell configuration does not modify the effects of genistein and benzimidazolone analogs. Since the tight seal between the glass pipette and the cell membrane in the cell-attached configuration effectively prevents diffusion of the drug onto the external surface of the channel, the fact that similar magnitudes of enhancement were obtained for whole-cell and cell-attached configurations suggests that these chemicals can diffuse through the lipid bilayer into the cell and act on the cytoplasmic side of the membrane.
NS004 and NS1619 Increase ΔF508-CFTR Channel Open Probability.
To further dissect the mechanism by which benzimidazolone analogs increase mean ΔF508-CFTR current, we analyzed the effects of these compounds on ΔF508-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 ΔF508-CFTR current must be attributed to an increase in Po.
We then quantified the Po of ΔF508-CFTR in the presence of various agonists in those patches that show less than four channel opening steps. In the presence of forskolin alone (10 μM), generally there is little channel activity and the Po is therefore low (0.11 ± 0.05,n = 3). Under such conditions the channels mostly reside in the closed state (Fig. 4). Addition of 50 nM and 20 μM NS004 to the bath solution increases the ΔF508-CFTR channel Po to 0.45 ± 0.12 (n = 3) and 0.77 ± 0.04 (n = 3), respectively (Fig. 4). From binomial analysis of all-point histograms (Fig. 4, legend), our results suggest that ΔF508-CFTR channels in the cell-attached patches behave independently of each other. In the presence of 50 nM and 20 μM NS1619, the ΔF508-CFTR channel Po increased to 0.26 ± 0.09 and 0.73 ± 0.06 (n = 4), respectively. The maximal Po values obtained for benzimidazolone analogs are similar to those described for genistein by Hwang et al. (1997). Furthermore, following the application of a maximally effective concentration of NS004, the inclusion of 20 μM genistein in the bath solution exerts no additional change in the ΔF508-CFTR channel current. Figure5 shows a cell-attached recording, demonstrating this lack of effect of genistein (20 μM) upon ΔF508-CFTR current activated by 20 μM NS004 (in the continued presence of 10 μM forskolin). In five experiments, the current in the presence of forskolin plus NS004 is not significantly different from that obtained with forskolin plus NS004 and genistein (p = 0.11, paired t test). Comparable results were observed with NS1619 (p = 0.13, pairedt test). Thus, maximal enhancement of ΔF508-CFTR by the benzimidazolone analogs NS004 and NS1619 occludes further enhancement by genistein.
NS004 and NS1619 Prolong the ΔF508-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 ΔF508-CFTR. Figure 6A shows a cell-attached recording from a patch containing one single channel. As reported previously (Haws et al., 1996), ΔF508-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 ΔF508-CFTR is reduced in the presence of benzimidazolone analogs or genistein.
To estimate channel open times we collected and combined events obtained from several recordings with few channels (i.e., less than four channel open steps at any given time throughout the recording). Open time histograms were constructed and analyzed as described underMaterials and Methods. Figure 6B shows the resulting open time histograms from pooled data obtained with patches containing few channels. Fitting the data with double exponential functions yielded the following time constants; τo1 = 0.35 s and τo2 = 2.13 s in the presence of 10 μM forskolin, τo1 = 0.46 s and τo2 = 13.89 s in the presence of forskolin plus 50 nM NS004. Similar time constants were observed with forskolin plus 50 nM NS1619 (data not shown). These results are consistent with the idea that benzimidazolone analogs, like genistein, stabilize an open state of CFTR.
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). Figure7 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.
We further examined the effect of benzimidazolone analogs and genistein upon the open time of K1250A-CFTR. In cell-attached patches, steady-state macroscopic K1250A-CFTR current was generated by forskolin alone or forskolin plus either genistein or NS004 (each 20 μM); the patch was then excised into an ATP-free bath. The time constant of the current decay, reflecting the channel open time, was obtained by fitting the time course with a simple exponential function. The time constant in the presence of forskolin (10 μM) alone was found to be 81.8 ± 13.7 s (n = 5). There was no significant difference in the time constants obtained in the presence of forskolin plus either 20 μM genistein (72.8 ± 11.2 s,n = 5) or 20 μM NS004 (52.3 ± 7.06 s,n = 4). These data demonstrate that the open time of the K1250A-CFTR channels activated with a maximally effective concentration of forskolin (10 μM) is not affected by either genistein or benzimidazolone analogs. Since neither the Po nor the open time of K1250A-CFTR is affected by genistein or benzimidazolone analogs, we conclude that the closed time of K1250A-CFTR is not affected when the cAMP pathway is maximally activated. The fact that these drugs do not change the open time of K1250A-CFTR is consistent with the idea that genistein acts by inhibiting ATP hydrolysis at NBD2 (Wang et al., 1998; Randak et al., 1999).
However, a lack of effect on the K1250A-CFTR mutant could be caused by an obliteration of the binding site for these compounds by the mutation. This is perhaps not the case, since these reagents increase K1250A-CFTR channel activity when the cAMP stimulation is submaximal. Like wt-CFTR, the activity of K1250A-CFTR can be manipulated using different concentrations of forskolin (Al-Nakkash and Hwang, 1999). For example, sequential addition of 100 nM and then 10 μM forskolin to the bath solution generates an incremental increase in the steady-state macroscopic K1250A-CFTR current (data not shown). Under conditions that produce submaximal stimulation of the cAMP-dependent K1250A-CFTR channel activation (i.e., 100 nM forskolin), benzimidazolone analogs enhanced mean K1250A-CFTR current. In the presence of 100 nM forskolin, 50 nM and 20 μM NS004 increased the K1250A-CFTR current by 6.22 ± 2.55-fold (n = 6) and 19.09 ± 9.71-fold (n = 6), respectively. Similar results were produced with 50 nM and 20 μM NS1619. These data suggest, for those K1250A-CFTR channels that are not maximally stimulated (i.e., have not attained a maximum Po), that benzimidazolone analogs can potentiate the cAMP-dependent channel current.
Effect of NS004 and NS1619 upon the ΔF508/K1250A Double Mutation.
It is well established that the major functional defect associated with ΔF508-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 ΔF508/K1250A-CFTR.
Figure 8A shows a 30-min cell-attached recording from an NIH3T3 cell expressing ΔF508/K1250A-CFTR. In the absence of agonists there is no channel activity. In the presence of 10 μM forskolin alone, only two to three channel open steps could be observed. However, addition of 20 μM NS004 elicited a large increase in the macroscopic current. This effect of NS004 is readily reversible since removal of NS004 results in the return of current levels to those observed in the presence of forskolin alone. These data suggest that introducing the K1250A mutation into the ΔF508-CFTR background does not rectify the functional defect associated with ΔF508-CFTR and that NS004 greatly potentiates the Po of ΔF508/K1250A-CFTR. This effect of NS004 is through a decrease of the closed time as demonstrated in Fig.8B. In this cell-attached patch recording of ΔF508/K1250A-CFTR, one single channel opens for 20 s in the presence of forskolin alone (a phenotype for the K1250A-CFTR mutation), but the channel is predominantly closed (a phenotype for the ΔF508-CFTR mutation). However, when we applied NS1619 (20 μM) the channel opens more frequently, and the time the channel stays closed is greatly reduced. This example clearly demonstrates that benzimidazolone analogs act by decreasing the closed time of this double mutant-CFTR.
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.
Lately, a cAMP-independent mechanism of CFTR activation has evolved (for review, see Hwang and Sheppard, 1999). Several classes of compounds, including isoflavones, benzimidazolones, and xanthines, can activate CFTR by mechanisms that may not activate any kinases regulating CFTR (Drumm et al., 1991; Gribkoff et al., 1994; Illek et al., 1995, 1999; Reenstra et al., 1996; Hwang et al., 1997; He et al., 1998; Al-Nakkash and Hwang, 1999). Of these CFTR activators, the one that has been most extensively studied is the isoflavone genistein (Fig. 1). Illek et al. (1995) first demonstrated that genistein activated wt-CFTR chloride channels in NIH3T3 fibroblast cells. It was also demonstrated that genistein does not affect intracellular cAMP concentration. Kinetically, it has previously been shown that genistein increases the Po of ΔF508-CFTR by prolonging the open time and shortening the closed time (Hwang et al., 1997). Since genistein can potentiate CFTR channel activity even in cell-free, excised inside-out membrane patches, it is postulated that genistein acts by direct binding to the NBD of phosphorylated CFTR (Wang et al., 1998). This idea is perhaps not surprising since genistein is known to inhibit tyrosine kinases through a competitive binding to the nucleotide binding sites (Akiyama and Ogawara, 1991). Furthermore, Randak et al., (1999) described the first biochemical evidence for a direct interaction between CFTR and genistein, using a fusion protein, comprised of both a maltose binding protein and NBD2. Despite the fact that genistein has been well studied and its mechanism of action better understood, a major limitation in potential application in therapeutics is its micromolar EC50 (Illek et al., 1995). However, benzimidazolone analogs, with a nanomolar EC50 (Gribkoff et al., 1994; this study), should provide a better parent compound for future drug development.
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).
Second, our whole-cell experiments demonstrate that, in the same cell, the maximal ΔF508-CFTR current activated by benzimidazolone analogs is not different from that induced by genistein (Fig. 3). In cell-attached patches, the enhancement effect of genistein on ΔF508-CFTR channel current is occluded by the presence of forskolin and a maximally effective concentration of NS004 or NS1619 (Fig. 5).
Third, like effects of genistein on ΔF508-CFTR channel kinetics (Hwang et al., 1997), benzimidazolone analogs increase the Po of forskolin-dependent ΔF508-CFTR channel activity through an increase of the open time and a decrease of the closed time. Furthermore, the single-channel Po value for ΔF508-CFTR in the presence of forskolin and benzimidazolone analogs (0.77 ± 0.04 for NS004, 0.73 ± 0.06 for NS1619) is very similar to that with forskolin and genistein (∼0.7 in Hwang et al., 1997). It should be noted that two different open states, distinguished by their differences in dwell times, can be seen in the presence of forskolin as reported previously (Hwang et al., 1997). It was suggested that these two open states reflect channels with one or both of the NBDs being occupied by nucleotides. Based on this biochemical interpretation of kinetic data, we hypothesize that benzimidazolone analogs as well as genistein stabilize the second open state by inhibiting ATP hydrolysis at NBD2.
Last, neither benzimidazolone analogs nor genistein can potentiate K1250A-CFTR channel current activated by a maximally effective concentration of forskolin. Previous reports have demonstrated that mutating this Walker A lysine residue at NBD2 greatly prolongs the channel open time (Gunderson and Kopito, 1995; Zeltwanger et al., 1999), presumably due to an abolition of the ATP hydrolysis reaction at NBD2 (Ramjeesingh et al., 1999). We show, from macroscopic relaxation analysis, that the open time for K1250A-CFTR is not changed by benzimidazolone analogs or genistein, supporting the hypothesis that these compounds stabilize the open state by inhibiting ATP hydrolysis at NBD2.
Biophysical Basis for the Functional Defect of ΔF508-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 ΔF508 mutation is caused by a slower rate of PKA-dependent phosphorylation of ΔF508-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 ΔF508-CFTR raises the possibility whether these two effects are causal. Schultz et al. (1999a) explain the effects of 3-isobutyl-1-methylxanthine on ΔF508-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 ΔF508/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 ΔF508 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.
Our conclusion for a common mechanism for effects of benzimidazolone analogs and genistein could potentially be of benefit to the formulation of pharmacotherapies for CF. If these two different groups of compounds act in such a similar manner and likely have the same site of action, then a closer look at their structures should reveal key moieties on these compounds (or structural similarities), essential for CFTR activation. Certain hydroxyl groups on the chroman ring of genistein appear to be important for CFTR activation (Fig. 1). For instance daidzein, the inactive analog of genistein (Yang et al., 1997), does not have the hydroxyl group at the C7 position (Schultz et al., 1999b). We have shown that NS004 is more potent than NS1619 (Fig.3). Subtle structural differences within the benzimidazolone analogs likely make one a more potent CFTR activator than another. For example, a halide group (Cl) is present in NS004, whereas a CF3 group is present at the equivalent position in NS1619 (Fig. 1). Comparing the structures of genistein and benzimidazolone analogs also reveals similarities and differences. For example, both groups of compounds contain substituted benzene rings. However, the spatial orientation of these rings and the specific structural features between these rings may have impact on their binding affinities. This knowledge of key moieties essential for activation of CFTR located on either genistein or benzimidazolone analogs might provide guidance for the future design of novel, even more potent compounds. Future studies using structurally related compounds should shed light on the molecular nature of their binding site(s) on CFTR.
We thank Dr. Soren Peter Olesen (Neurosearch, Glostrup, Denmark) for the generous gifts of NS004 and NS1619. We thank Dr. A. Powe for helpful discussions.
- Received September 7, 2000.
- Accepted October 20, 2000.
Send reprint requests to: Layla Al-Nakkash, Ph.D., Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO 65211. E-mail:
This work is supported by the Cystic Fibrosis Foundation (to L.A.) and the National Institutes of Health (to T.-C.H.).
This work was previously presented: Al-Nakkash L, Hu S and Hwang TC (1998) The substituted benzimidazolone, NS004 and genistein activate wt- and ΔF508-CFTR through a common mechanism. North American Cystic Fibrosis Meeting (poster presentation), 12th Annual North American Cystic Fibrosis Conference, Montreal, Quebec, Canada, October 15–18, 1998; Al-Nakkash L and Hwang TC (1999) NS004 activates ΔF508-CFTR at nanomolar concentrations. FASEB (poster presentation), Experimental Biology 99, Washington, DC, April 17–21, 1999; and Al-Nakkash L and Hwang TC (1999) Potentiation of CFTR by benzimidazolone analogs. North American Cystic Fibrosis Meeting (poster and oral presentation), 13th Annual North American Cystic Fibrosis Conference, Seattle, WA, October 7–10, 1999.
- cystic fibrosis
- cystic fibrosis transmembrane conductance regulator protein
- protein kinase A
- nucleotide binding domain
- open probability
- dimethyl sulfoxide
- The American Society for Pharmacology and Experimental Therapeutics