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. 2000 Apr 15;524(Pt 2):317–330. doi: 10.1111/j.1469-7793.2000.t01-1-00317.x

Two mechanisms of genistein inhibition of cystic fibrosis transmembrane conductance regulator Cl− channels expressed in murine cell line

K A Lansdell 1, Z Cai 1, J F Kidd 1, D N Sheppard 1
PMCID: PMC2269882  PMID: 10766914

Abstract

  1. The isoflavone genistein may either stimulate or inhibit cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels. To investigate how genistein inhibits CFTR, we studied CFTR Cl− channels in excised inside-out membrane patches from cells expressing wild-type human CFTR.

  2. Addition of genistein (100 μM) to the intracellular solution caused a small decrease in single-channel current amplitude (i), but a large reduction in open probability (Po).

  3. Single-channel analysis of channel block suggested that genistein (100 μM) may inhibit CFTR by two mechanisms: first, it may slow the rate of channel opening and second, it may block open channels.

  4. Acidification of the intracellular solution relieved channel block, suggesting that the anionic form of genistein may inhibit CFTR.

  5. Genistein inhibition of CFTR Cl− currents was weakly voltage dependent and unaffected by changes in the extracellular Cl− concentration.

  6. Channel block was relieved by pyrophosphate (5 mM) and ATP (5 mM), two agents that interact with the nucleotide-binding domains (NBDs) of CFTR to greatly stimulate channel activity.

  7. ATP (5 mM) prevented the genistein-induced decrease in Po, but was without effect on the genistein-induced decrease in i.

  8. The genistein-induced decrease in i was voltage dependent, whereas the genistein-induced decrease in Po was voltage independent.

  9. The data suggest that genistein may inhibit CFTR by two mechanisms. First, it may interact with NBD1 to potently inhibit channel opening. Second, it may bind within the CFTR pore to weakly block Cl− permeation.

The cystic fibrosis transmembrane conductance regulator (CFTR; Riordan et al. 1989) is a unique member of the ATP-binding cassette (ABC) transporter family that forms a Cl− channel with complex regulation (for review see Hanrahan et al. 1995; Gadsby & Nairn, 1999; Sheppard & Welsh, 1999). Dysfunction of CFTR causes the genetic disease cystic fibrosis (CF; Welsh et al. 1995). CFTR is composed of five domains: two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. The MSDs contribute to the formation of the Cl−-selective pore, while the NBDs and R domain control channel activity. The activation of the cAMP-dependent protein kinase (PKA) causes the phosphorylation of multiple serine residues within the R domain. Once the R domain is phosphorylated, channel gating is controlled by a cycle of ATP hydrolysis at the NBDs. Finally, protein phosphatases dephosphorylate the R domain and return the channel to its quiescent state.

To test the hypothesis that CFTR is regulated by cAMP-independent mechanisms, Illek et al. (1995) investigated the effect on CFTR Cl− channels of the isoflavone genistein, a specific inhibitor of protein tyrosine kinases (PTKs; Akiyama et al. 1987). They demonstrated that genistein stimulated wild-type CFTR expressed in NIH 3T3 cells without altering the intracellular concentration of cAMP. Based on this and other results, Illek et al. (1995) proposed that genistein stimulates channel activity by preventing the inhibition of CFTR by PTKs. However, subsequent studies showed that genistein stimulation of channel activity requires the prior phosphorylation of CFTR by PKA (Reenstra et al. 1996; Yang et al. 1997). They also demonstrated that genistein stimulates phosphorylated CFTR Cl− channels by greatly prolonging the duration of bursts of channel activity (French et al. 1997; Hwang et al. 1997; Wang et al. 1998). Because the effect of genistein on channel gating resembled that of non-hydrolysable ATP analogues, it was proposed that genistein may directly interact with NBD2 to inhibit channel closure (French et al. 1997; Hwang et al. 1997; Wang et al. 1998). Consistent with this idea, Randak et al. (1999) demonstrated that the binding of genistein to a recombinant NBD2 protein inhibits the ATPase activity of NBD2. However, other studies showed that genistein enhances the phosphorylation of CFTR by PKA in vivo, suggesting that genistein may inhibit serine/threonine protein phosphatases that dephosphorylate CFTR and deactivate the channel (Reenstra et al. 1996; Hwang et al. 1997). A model that explains the different effects of genistein on CFTR Cl− channels has been developed by Wang et al. (1998). The authors proposed that genistein interacts with NBD2 to prolong the lifetime of the open channel conformation. Moreover, to explain the enhanced phosphorylation of CFTR by genistein, Wang et al. (1998) speculated that the open channel might have a slower rate of dephosphorylation of the R domain than the closed channel.

Elevated concentrations of genistein inhibit CFTR Cl− channels (Weinreich et al. 1997; Wang et al. 1998; Zhou et al. 1998; Obayashi et al. 1999). However, the mechanism of channel block is poorly understood. Wang et al. (1998) demonstrated that genistein inhibited CFTR Cl− channels in excised membrane patches from cells expressing recombinant CFTR both by slowing the rate of channel opening and by promoting transitions to subconductance states. In contrast, Zhou et al. (1998) and Obayashi et al. (1999) showed that genistein inhibition of CFTR Cl− currents in guinea-pig ventricular myocytes was weakly voltage dependent. To understand better how genistein inhibits CFTR, we studied CFTR Cl− channels in excised inside-out membrane patches from cells expressing wild-type human CFTR.

METHODS

Cells and cell culture

For this study, we used mouse mammary epithelial (C127) cells stably expressing wild-type human CFTR. These cells were a generous gift of Drs S. H. Cheng, C. R. O'Riordan and A. E. Smith (Genzyme, Framingham, MA, USA). C127 cells were cultured as previously described (Sheppard & Robinson, 1997). For experiments using excised inside-out membrane patches, cells were seeded onto glass coverslips and used within 48 h.

Electrophysiology

CFTR Cl− channels were recorded in excised inside-out membrane patches using an Axopatch 200A patch-clamp amplifier (Axon Instruments Inc.) and pCLAMP data acquisition and analysis software (version 6.03, Axon Instruments Inc.) as previously described (Hamill et al. 1981; Sheppard & Robinson, 1997). The established sign convention was used throughout; currents produced by positive charge moving from intra- to extracellular solutions (anions moving in the opposite direction) are shown as positive currents.

The pipette (extracellular) solution contained (mM): 140 N-methyl-D-glucamine (NMDG), 140 aspartic acid, 5 CaCl2, 2 MgSO4 and 10 Tes; adjusted to pH 7.3 with Tris ([Cl−], 10 mM). Patch pipettes had resistances of 10–20 MΩ when filled with this solution. The bath (intracellular) solution contained (mM): 140 NMDG, 3 MgCl2, 1 CsEGTA and 10 Tes; adjusted to pH 7.3 with HCl ([Cl−], 147 mM; free [Ca2+], < 10−8 M). For pH experiments, 5 mM Bis-Tris + 5 mM Trizma base replaced 10 mM Tes in the intracellular solution. To ensure that the Cl− concentrations of the pH 7.3 and pH 6.0 solutions were identical, both solutions were first titrated to pH 7.3 with HCl before the pH 6.0 solution was further titrated to pH 6.0 with H2SO4. The intracellular solution was maintained at 37°C using a temperature-controlled microscope stage (Brook Industries, Lake Villa, IL, USA).

After excision of inside-out membrane patches, CFTR Cl− channels were activated by the addition of the catalytic subunit of PKA (75 nM) and ATP (1 mM) to the intracellular solution within 5 min of patch excision. In most experiments, the ATP concentration was subsequently reduced to 0.3 mM (∼EC50 for the activation of wild-type CFTR Cl− channels by intracellular ATP (Anderson et al. 1991)) before the addition of genistein to the intracellular solution; PKA was added to all intracellular solutions. Unless otherwise indicated, membrane patches were voltage clamped at -50 mV.

In this investigation, we used membrane patches containing large numbers of active channels for time course studies and voltage ramp protocols and membrane patches containing five or less active channels for single-channel studies. The number of channels in a membrane patch was determined from the maximum number of simultaneous channel openings observed during the course of an experiment, as previously described (Lansdell et al. 1998a). An experiment typically lasted 30–90 min and included multiple interventions of 4–5 min duration that significantly stimulated channel activity (e.g. ATP (0.3-5 mM) + PKA (75 nM)). In most experiments, to compensate for any channel run-down, specific interventions were bracketed by control periods made with the same concentrations of ATP and PKA, but without test compounds; the intervention data were then compared with the average data of pre- and postintervention control periods. To investigate whether genistein inhibition of CFTR was voltage dependent and enhanced when the external Cl− concentration was reduced, we used either voltage ramp or voltage step protocols. Macroscopic current-voltage (I–V) relationships were obtained in the absence and presence of genistein by averaging currents generated by 15–30 ramps of voltage, each of 2 s duration; holding voltage was -50 mV. Basal currents recorded in the absence of PKA and ATP were subtracted from those recorded in the absence and presence of genistein to determine the effect of genistein on CFTR Cl− currents. For single-channel studies of the voltage dependence of genistein inhibition of CFTR, voltage was stepped from -80 to +60 mV in 20 mV increments of 30 s duration in the absence and presence of the drug, and single-channel current amplitude (i) and open-probability (Po) measured at each step.

CFTR Cl− currents were initially recorded on digital audiotape using a digital tape recorder (Biologic Scientific Instruments, model DTR-1204; Intracel Ltd, Royston, UK) at a bandwidth of 10 kHz. On playback, records were filtered with an eight-pole Bessel filter (Frequency Devices, model 902LPF2; Scensys Ltd, Aylesbury, UK) at a corner frequency of 500 Hz and acquired using a Digidata 1200 interface (Axon Instruments Inc.) and pCLAMP at sampling rates of either 2.5 kHz (time course studies) or 5 kHz (single-channel studies). For the purpose of illustration, single-channel records were filtered at 500 Hz and digitized at 1 kHz.

In time course studies, each data point is the average current for a 4 s period with data points collected continuously; no data were collected while solutions were changed. Average current (I) for a specific intervention was determined as the average of all the data points collected during the intervention. To measure i, Gaussian distributions were fitted to current amplitude histograms. For Po and kinetic analyses, lists of open and closed times were created using a half-amplitude crossing criterion for event detection. Transitions < 1 ms in duration were excluded from the analyses. To analyse the voltage dependence of genistein inhibition of single channels, we filtered single-channel records at 100 Hz using a digital Gaussian filter and excluded transitions < 10 ms in duration from lists of open and closed times. Single-channel open and closed time histograms were created using logarithmic x-axes with 10 bins decade−1. Histograms were fitted with one or more component exponential functions using the maximum likelihood method. To determine which component function fitted best, the log-likelihood ratio test was used and considered statistically significant at a value of 2.0 or greater (Winter et al. 1994). Only membrane patches that contained a single active channel were used for single-channel kinetic analyses. Po was calculated using the equation:

graphic file with name tjp0524-0317-m1.jpg (1)

where N is the number of channels, Ttot is the total time analysed, and T1 is the time that one or more channels are open and T2 is the time two or more channels are open and so on.

Reagents

The catalytic subunit of PKA was purchased from Promega Ltd. ATP (disodium salt), glibenclamide, pyrophosphate (tetrasodium salt) and Tes were obtained from Sigma-Aldrich Company Ltd (Poole, UK). Genistein was purchased from either Semat Technical (UK) Ltd (St Albans, UK) or Sigma-Aldrich Company Ltd. As similar results were obtained with both sources of genistein, the data have been combined. The chemical structure of genistein is shown in Fig. 1 of Hwang & Sheppard (1999). All other chemicals were of reagent grade.

Figure 1. Effect of genistein on the activity of CFTR Cl− channels following phosphorylation by PKA.

Figure 1

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A, representative single-channel recordings are from an excised inside-out membrane patch from a C127 cell stably expressing wild-type human CFTR. ATP (0.3 mM) and the catalytic subunit of PKA (75 nM) were continuously present in the intracellular solution to which the indicated concentrations of genistein were added. Voltage was -50 mV, and there was a Cl− concentration gradient across the membrane patch (internal [Cl−]= 147 mM; external [Cl−]= 10 mM). Dashed lines indicate the closed state and downward deflections correspond to channel openings. B and C, effect of genistein concentration on i and Po, respectively. Columns and error bars indicate means +s.e.m. of n = 5–6 observations at each concentration. The asterisks indicate values that are significantly different from the control value (P < 0.01). Genistein (100 μM) decreased i to 82 ± 3 % of the control value (n = 5; P < 0.01) and Po to 38 ± 5 % of the control value (n = 6; P < 0.001). Other details as in A.

Stock solutions of genistein (100 mM) and glibenclamide (25 mM) were prepared in DMSO and stored at -20°C. Immediately before use, stock solutions were diluted in intracellular solution to achieve final concentrations. The vehicle did not affect the activity of CFTR Cl− channels (Lansdell et al. 1998b).

Statistics

Results are expressed as means ± s.e.m. of n observations. To compare sets of data, we used either an analysis of variance (ANOVA) or Student's t test. Differences were considered statistically significant when P < 0.05. All tests were performed using SigmaStat (version 1.03; Jandel Scientific GmbH, Erkrath, Germany).

RESULTS

Effect of genistein on the activity of CFTR Cl− channels phosphorylated by PKA

To investigate how genistein inhibits CFTR, we studied CFTR Cl− channels in excised inside-out membrane patches from C127 cells expressing wild-type human CFTR. Figure 1A shows the effect of genistein on the activity of a single CFTR Cl− channel following phosphorylation by PKA. In the continued presence of PKA and ATP, genistein was added to the intracellular solution. As previously described (Winter et al. 1994), under control conditions the gating behaviour of CFTR was characterised by bursts of channel activity, interrupted by brief closures, separated by longer closures between bursts (Fig. 1A, top trace). Visual inspection of single-channel records suggested that as the concentration of genistein increased, i decreased and the gating behaviour of CFTR Cl− channels changed in two ways. First, there was a large increase in flickering closures interrupting bursts of channel activity (Fig. 1A). Second, the duration of long closures separating bursts of channel activity increased dramatically (Fig. 1A). To quantify these effects of genistein, we measured i and Po. Figure 1B and C shows that genistein (100 μM) caused a small reduction in i (P < 0.01), but a large decrease in Po (P < 0.001). Genistein inhibition of CFTR was readily reversible (Fig. 2).

Figure 2. Open (A) and closed (B) time histograms for a single CFTR Cl− channel inhibited by genistein.

Figure 2

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Representative histograms for the indicated interventions from a single experiment are shown. ATP (0.3 mM) and the catalytic subunit of PKA (75 nM) were continuously present in the intracellular solution to which the indicated concentrations of genistein (gen) were added. Other details as in Fig. 1. For open time histograms, the continuous line is the fit of a one-component exponential function. For closed time histograms, the continuous line is the fit of a two (Control, Wash)-, three (30 μM genistein)- or four (100 μM genistein)-component exponential function. The dashed lines show the individual components of the exponential functions. Logarithmic x-axes with 10 bins decade−1 were used for both open and closed time histograms.

To determine how genistein decreased Po, we investigated the effect of genistein on the gating kinetics of CFTR Cl− channels following phosphorylation by PKA using membrane patches that contained only a single active channel. We analysed histograms of open and closed times in the absence and presence of genistein to determine whether we could detect new populations of channel closures that represented channels blocked by genistein. Consistent with previous results, the open and closed time histograms of CFTR were best fitted with one- and two-component functions, respectively (Fig. 2 and Table 1; Winter et al. 1994).

Table 1. Effect of genistein on the open and closed time constants of wild-type CFTR Cl−channels.

[Genistein] (μm) 0 30 100
τO (ms) 54.5 ± 4.7 44.8 ± 2.2 16.1 ± 3.6
τC1 (ms) — 0.85 ± 0.10 1.66 ± 0.12
τC2 (ms) 2.68 ± 0.27 2.88 ± 0.29 7.40 ± 0.83
τC3 (ms) 172.2 ± 15.3 159.4 ± 15.6 164.7 ± 27.1
τC4 (ms) — — 1704 ± 255
Total time (s) 967 965 835
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Open and closed time constants were measured at the indicated genistein concentrations by fitting one or more component exponential functions to open and closed time histograms as described in the Methods. The total time analysed for each concentration of genistein is shown, and in each patch approximately 5000 events were analysed per intervention. Values are means ± s.e.m. of n = 4 observations. Measurements were made in the presence of the catalytic subunit of PKA (75 nm) and ATP (0.3 mm) in the intracellular solution; voltage was —50 mV.

In the presence of genistein open time histograms were best fitted with one-component functions (Fig. 2 and Table 1). However, closed time histograms were best fitted with three- and four-component functions in the presence of genistein (30 μM) and genistein (100 μM), respectively (Fig. 2 and Table 1). The new populations of channel closures were described by very fast (τC1; 30 and 100 μM) and very slow (τC4; 100 μM) closed time constants. To determine whether open and closed time constants changed with genistein concentration, we performed an analysis of variance. At genistein (100 μM), τO decreased significantly (P < 0.05), whereas τC1 and τC2 increased significantly (P < 0.05). However, τC3 did not change significantly with genistein concentration (P > 0.05). These data indicate that genistein has complex effects on channel gating. They suggest that genistein (100 μM) decreased the Po of CFTR by first, reducing channel residence in the open state, second, increasing the fast closed time constant (τC2) by 3-fold, and third, promoting transitions to two new populations of channel closures, described by very fast (τC1) and very slow (τC4) closed time constants. Thus, genistein may inhibit CFTR by two mechanisms: first, it may slow the rate of channel opening and second, it may block open channels.

Acidifying the intracellular solution relieves genistein inhibition of CFTR

Previous studies have demonstrated that CFTR is inhibited by large anions that occlude the intracellular end of the CFTR pore (Linsdell & Hanrahan, 1996a, b; Ishihara & Welsh, 1997; Sheppard & Robinson, 1997). To investigate whether the anionic form of genistein inhibits CFTR, we compared the effect of genistein (100 μM) on CFTR Cl− currents at pH 7.3 and 6.0. Acidifying the intracellular solution from pH 7.3 to 6.0 decreases the concentration of genistein present in the anionic form. We previously showed that acidifying the intracellular solution to pH 6.3 was without effect on the activity of wild-type CFTR (Sheppard & Robinson, 1997). Although the data presented in Fig. 3A and C suggest that acidifying the intracellular solution to pH 6.0 stimulates CFTR Cl− channels, when all the data were analysed the magnitude of CFTR Cl− currents did not change significantly at pH 6.0 (see Fig. 3 legend).

Figure 3. Acidification of the intracellular solution relieves genistein inhibition of CFTR and facilitates genistein stimulation of CFTR.

Figure 3

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A, time course of current in an excised inside-out membrane patch. ATP (0.3 mM), PKA (75 nM) and genistein (100 μM) were present in the intracellular solution during the times indicated by the filled bars. During the period indicated by the hatched bar, the pH of the intracellular solution was pH 6.0. Each point is the average current for a 4 s period and no data were collected while solutions were changed. For the purpose of illustration, the time course has been inverted so that an upward deflection represents an inward current. B, effect of intracellular pH on genistein (100 μM) inhibition of CFTR Cl− currents. Columns and error bars indicate means +s.e.m. (n = 6) for each condition. Values represent the average current recorded during the indicated interventions normalized to that measured under control conditions at pH 7.3 at the start of the experiment. Other details as in A. The asterisk indicates a value that is significantly different from the control value (P < 0.05). At pH 7.3, genistein (100 μM) decreased CFTR Cl− current to 38 ± 6 % of the control value (n = 6; P < 0.05). In contrast, at pH 6.0, genistein (100 μM) increased CFTR Cl− current to 111 ± 6 % of the control value (n = 6; P > 0.05). C, time course of current in an excised inside-out membrane patch. Details as in A, except that genistein (30 μM) was used. D, effect of intracellular pH on genistein (30 μM) stimulation of CFTR Cl− currents. Columns and error bars indicate means +s.e.m. (n = 6) for each condition. Other details as in B. At pH 7.3, genistein (30 μM) was without effect on CFTR Cl− current (n = 6; P > 0.05). In contrast, at pH 6.0, genistein (30 μM) increased CFTR Cl− current to 145 ± 8 % of the control value (n = 6; P < 0.01). Acidifying the intracellular solution to pH 6.0 was without effect on the magnitude of CFTR Cl− current (pH 7.3, I = 100 ± 0 %; pH 6.0, I = 106 ± 8 %; n = 12; P > 0.5).

Figure 3A and B shows that at pH 7.3, genistein (100 μM) significantly inhibited CFTR Cl− currents (P < 0.001). In contrast, at pH 6.0, genistein (100 μM) was without effect on CFTR Cl− currents (P > 0.05; Fig. 3A and B). Genistein (100 μM) inhibition of CFTR was completely relieved. Based on this result, we were interested to learn whether the undissociated form of genistein stimulates CFTR. To test this idea, we compared the effect of genistein (30 μM) on CFTR Cl− currents at pH 7.3 and pH 6.0. Figure 3C and D shows that at pH 7.3, genistein (30 μM) was without effect on CFTR Cl− currents (P > 0.5). In contrast, at pH 6.0, genistein (30 μM) significantly stimulated CFTR Cl− currents (P < 0.01; Fig. 3C and D). Thus, these results suggest that the anionic form of genistein may inhibit CFTR, while the undissociated form of genistein may stimulate CFTR.

Genistein inhibition of CFTR is weakly voltage dependent and unaffected by changing the external Cl− concentration

The observation that the anionic form of genistein may inhibit CFTR suggests that genistein may bind within the CFTR pore, where it may occlude the pore and prevent Cl− permeation. To test this hypothesis, we performed two types of experiments. First, we examined whether genistein inhibition of CFTR is voltage dependent and second, we investigated whether genistein and Cl− ions compete for a common binding site. To examine the voltage dependence of genistein inhibition of CFTR, membrane patches were bathed in symmetrical 147 mM Cl− solutions and CFTR Cl− currents recorded in the absence and presence of genistein (100 μM) over the voltage range ±100 mV using a voltage ramp protocol. Figure 4A demonstrates that genistein (100 μM) decreased CFTR Cl− currents at both negative and positive voltages. In contrast to CFTR inhibition by arylaminobenzoates, disulphonic stilbenes and sulphonylureas (McCarty et al. 1993; Linsdell & Hanrahan, 1996b; Sheppard & Robinson, 1997), channel block by genistein was not relieved at positive voltages (Fig. 4A).

Figure 4. Effect of changing the external Cl− concentration on the voltage dependence of genistein inhibition of CFTR Cl− currents.

Figure 4

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A, I-V relationships of CFTR Cl− currents recorded in the absence and presence of genistein (100 μM) when the membrane patch was bathed in symmetrical 147 mM Cl− solutions. ATP (1 mM) and the catalytic subunit of PKA (75 nM) were continuously present in the intracellular solution. I–V relationships are the average of 15 voltage ramps each of 2 s duration; holding voltage was -50 mV. Basal currents recorded in the absence of PKA and ATP have been subtracted. At -75 mV, genistein (100 μM) decreased I to 42 ± 4 % of the control value (n = 5) and at +75 mV, genistein (100 μM) decreased I to 48 ± 4 % of the control value (n = 5; P > 0.05). B, relationship between Kd and voltage when the external Cl− concentration was either 147 mM (^) or 10 mM (•). Data points are means ± s.e.m. (n = 4-5) at each voltage. Kd was calculated as described in the Results. The continuous lines are the fits of first-order regressions to the data.

The voltage-dependent dissociation constant (Kd) for genistein inhibition of CFTR can be calculated using the relationship:

graphic file with name tjp0524-0317-m2.jpg (2)

where Kd(V) is the voltage-dependent dissociation constant at voltage V, and I and Io are the current values in the presence and absence of drug, respectively. Figure 4B shows that Kd was weakly voltage dependent. The data also suggest that genistein (Kd(0 mV) (the voltage-dependent dissociation constant at 0 mV) = 87 ± 11 μM; n = 5) is a less potent inhibitor of CFTR than the sulphonylurea glibenclamide (Kd(0 mV) = 37 μM; Sheppard & Robinson, 1997), of similar potency to the disulphonic stilbene 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS; Kd(0 mV) = 77 μM; Linsdell & Hanrahan, 1996b), but a more potent inhibitor than the arylaminobenzoate diphenylamine-2-carboxylate (DPC; Kd(0 mV) = 1116 μM; McCarty et al. 1993).

The electrical distance across the membrane sensed by blocking ions can be calculated using the relationship (Woodhull, 1973):

graphic file with name tjp0524-0317-m3.jpg (3)

where z’ is the apparent valency of the blocking ion (defined as the actual valency of the blocking ion (z) multiplied by the electrical distance across the membrane experienced by the blocking ion (δ)), and F, R and T are the Faraday constant, gas constant and absolute temperature, respectively. Although genistein has a net negative charge of -1, our data and those of Wang et al. (1998), Zhou et al. (1998) and Obayashi et al. (1999) suggest that genistein may interact with CFTR at more than one site. This complicates calculation of the electrical distance across the membrane sensed by genistein. Using the data in Fig. 4B, z′ = 0.10 ± 0.01 (n = 5) measured from the inside of the membrane over the voltage range -100 to -40 mV.

To test further the hypothesis that genistein inhibits CFTR by blocking the pathway for Cl− permeation, we investigated whether the passage of Cl− ions through the CFTR pore interferes with genistein inhibition. We reduced the external Cl− concentration to 10 mM and recorded CFTR Cl− currents in the absence and presence of genistein (100 μM) over the voltage range -100 to +25 mV using a voltage ramp protocol. Figure 4B shows that when the external Cl− concentration was reduced to 10 mM, the potency of genistein inhibition of CFTR did not change (external [Cl−] = 147 mM, Kd(0 mV) = 87 ± 11 μM (n = 5); external [Cl−] = 10 mM, Kd(0 mV) = 81 ± 9 μM (n = 5); P > 0.05). Reducing the external Cl− concentration also did not alter the electrical distance sensed by genistein (z′ = 0.12 ± 0.01 (n = 5) measured from the inside of the membrane over the voltage range -100 to -40 mV; P > 0.05). Thus, these data indicate that genistein inhibition of CFTR was weakly voltage dependent and unaffected by reducing the external Cl− concentration. This suggests that genistein may interact with a site located outside the CFTR pore to block the channel.

Genistein inhibition of CFTR is relieved by pyrophosphate and elevated concentrations of intracellular ATP

The observation that high concentrations of genistein dramatically increase the duration of long closures separating bursts of channel activity (Wang et al. 1998; present study), suggests that genistein may interact with the closed state to inhibit channel opening. To test this hypothesis, we investigated whether agents that potently stimulate channel activity relieve genistein inhibition of CFTR. One such agent is the inorganic phosphate analogue pyrophosphate (PPi). PPi binds with high affinity to NBD2 to stimulate channel activity in two ways. First, it increases the rate of channel opening and second, it greatly slows the rate of channel closure (Carson et al. 1995b; Gunderson & Kopito, 1995; Cotten et al. 1996).

Figure 5A and B shows that addition of genistein (100 μM) to the intracellular solution in the presence of PKA (75 nM) and ATP (0.3 mM) significantly inhibited CFTR Cl− currents (P < 0.001). Subsequent addition of PPi (5 mM) to the intracellular solution dramatically stimulated CFTR Cl− currents. Not only was channel block by genistein completely relieved, but the magnitude of CFTR Cl− current observed in the presence of genistein and PPi was significantly greater than that observed under control conditions (P < 0.05; Fig. 5A and B). After washout of PPi, genistein failed to inhibit CFTR. Instead, the magnitude of CFTR Cl− current remained elevated until PKA and ATP were removed from the intracellular solution (Fig. 5A).

Figure 5. Pyrophosphate relieves genistein, but not glibenclamide, inhibition of CFTR Cl− currents.

Figure 5

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A, time course of current in an excised inside-out membrane patch. ATP (0.3 mM), the catalytic subunit of PKA (75 nM), genistein (100 μM) and pyrophosphate (PPi; 5 mM) were present in the intracellular solution during the times indicated by the filled bars. Other details as in Fig. 3A. B, effect of PPi (5 mM) on genistein (100 μM) inhibition of CFTR Cl− currents. Columns and error bars indicate means +s.e.m. (n = 6). Values represent the average current recorded during the indicated interventions normalized to that measured under control (C) conditions at the start of the experiment. Other details as in A. C, time course of current in an excised inside-out membrane patch. ATP (0.3 mM), PKA (75 nM), glibenclamide (Glib; 50 μM) and PPi (5 mM) were present in the intracellular solution during the times indicated by the filled bars. D, effect of PPi (5 mM) on glibenclamide (50 μM) inhibition of CFTR Cl− currents. Columns and error bars indicate means +s.e.m. (n = 6). Other details as in C.

As a control, we tested whether PPi relieves glibenclamide inhibition of CFTR; glibenclamide is an open-channel blocker of CFTR (Schultz et al. 1996; Sheppard & Robinson, 1997). As previously observed, addition of glibenclamide (50 μM) to the intracellular solution significantly decreased CFTR Cl− currents (P < 0.001; Fig. 5C and D). When PPi (5 mM) was subsequently added to the intracellular solution, the magnitude of CFTR Cl− current was increased. However, glibenclamide inhibition of CFTR was not relieved (Fig. 5C and D). Moreover, the effect of PPi was reversible and only after glibenclamide was removed from the intracellular solution did PPi (5 mM) stimulate CFTR Cl− currents (n = 3; Fig. 5C). Thus, these results suggest that PPi may relieve CFTR inhibition by genistein, but not by glibenclamide.

To test further whether genistein interacts with the closed state to inhibit channel opening, we investigated whether elevated concentrations of intracellular ATP relieve genistein inhibition of CFTR. The rate of transition from the closed state to a burst of channel activity is regulated by ATP (Winter et al. 1994). As the ATP concentration is increased, the rate of channel opening is enhanced. This suggests that increasing the intracellular ATP concentration may alleviate genistein inhibition of CFTR. Consistent with this idea, Fig. 6 demonstrates that when the intracellular ATP concentration was increased from 0.3 to 5 mM genistein (100 μM) inhibition of CFTR Cl− currents was relieved.

Figure 6. Elevating the intracellular ATP concentration relieves genistein inhibition of CFTR Cl− currents.

Figure 6

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A, time course of current in an excised inside-out membrane patch. ATP (0.3 or 5 mM), the catalytic subunit of PKA (75 nM) and genistein (100 μM) were present in the intracellular solution during the times indicated by the filled bars. Other details as in Fig. 3A. B, effect of ATP concentration on genistein (100 μM) inhibition of CFTR Cl− currents. Column and error bars indicate means +s.e.m. (n = 5). Values represent the average current recorded during the indicated interventions normalized to that measured in the presence of ATP (0.3 mM) at the start of the experiment. Other details as in A. When [ATP]= 0.3 mM, genistein (100 μM) decreased CFTR Cl− current to 32 ± 3 % of the control value (n = 5). However, when [ATP]= 5 mM, genistein (100 μM) only decreased CFTR Cl− current to 76 ± 4 % of the control value (n = 5; P < 0.01).

To investigate how elevated concentrations of ATP alleviate genistein inhibition of CFTR, we studied single channels. Figure 7A and B demonstrates that in the presence of ATP (0.3 or 5 mM), genistein (100 μM) induced a large increase in flickering closures and decreased i. However, ATP (5 mM) prevented both the increase in the duration of long closures separating bursts of channel activity and the decrease in Po induced by genistein (100 μM; Fig. 7A and C).

Figure 7. Effect of ATP (5 mM) on genistein inhibition of CFTR Cl− channels.

Figure 7

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A, representative recordings show the effect of genistein (100 μM) on the activity of two CFTR Cl− channels when the intracellular ATP concentration was either 0.3 mM (top) or 5 mM (bottom). Other details as in Fig. 1A. B and C effect of genistein (100 μM) on i and Po, respectively, when the intracellular ATP concentration was either 0.3 or 5 mM. Column and error bars indicate means +s.e.m. of n = 5 observations at each ATP concentration. The asterisks indicate values that are significantly different from the control value (P < 0.01). Other details as in A. When [ATP]= 0.3 mM, genistein (100 μM) decreased i to 82 ± 2 % of the control value (n = 5; P < 0.01) and Po to 36 ± 9 % of the control value (n = 5; P < 0.01). In contrast when [ATP]= 5 mM, genistein (100 μM) decreased i to 78 ± 3 % of the control value (n = 5; P < 0.01), but was without effect on Po (n = 5; P > 0.05).

Two mechanisms of genistein inhibition of CFTR

Based on our observation that ATP (5 mM) relieved the genistein-induced decrease in Po, but not i, we reinvestigated the voltage dependence of genistein inhibition of CFTR Cl− channels. We bathed membrane patches in symmetrical 147 mM Cl− solutions and measured i and Po over the voltage range -80 to +60 mV. Figure 8A and B demonstrates that genistein (100 μM) decreased i at negative voltages, but at positive voltages inhibition was relieved. In contrast, both the increase in the duration of long closures separating bursts of channel activity and the decrease in Po induced by genistein (100 μM) were voltage independent (Fig. 8A and C).

Figure 8. Effect of voltage on genistein inhibition of CFTR Cl− channels.

Figure 8

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A, representative recordings show the effect of genistein (100 μM) on the activity of two CFTR Cl− channels at -60 mV (top) and +60 mV (bottom). The membrane patch was bathed in symmetrical 147 mM Cl− solutions. ATP (0.3 mM) and the catalytic subunit of PKA (75 nM) were continuously present in the intracellular solution. B, single-channel I–V relationships in the absence (•) and presence (^) of genistein (100 μM). Data points are means ± s.e.m. (n = 4-5) at each voltage. The continuous lines are the fits of first- and second-order regressions to the control and genistein data, respectively. Other details as in A. C, relationship between Po and voltage in the absence (•) and presence (^) of genistein (100 μM). Data points are means ± s.e.m. (n = 3-4) at each voltage. Other details as in A. D, relationship between Kd and voltage for the data shown in B (i;^) and C (Po; •). Data points are means ± s.e.m. (i: n = 4-5; Po: n = 3-4). The continuous lines are the fits of first-order regressions to the data. For comparison, the dotted line shows the line fitted to the Kd-voltage data for genistein inhibition of CFTR Cl− currents when the external [Cl−] was 147 mM and the intracellular [ATP] was 1 mM. Kd values for genistein inhibition of i were calculated using eqn (2), and those for genistein inhibition of Po were calculated as described in Sheppard & Robinson (1997). In B-D, we combined data from 3 experiments where [ATP]= 0.3 mM and 2 experiments where [ATP]= 1 mM. We pooled data at different [ATP] values because changing the [ATP] between 0.3 and 1 mM was without effect on i and only had a small effect on Po (Z. Cai, K. A. Lansdell & D. N. Sheppard, unpublished observations).

Using the data in Fig. 8B and C, we calculated Kd values for genistein inhibition of i and Po. Figure 8D demonstrates that Kd values for genistein inhibition of i were strongly voltage dependent. In contrast, Kd values for genistein inhibition of Po were voltage independent (Fig. 8D). The data also suggest that genistein more potently inhibited Po (Kd(0 mV) = 64 ± 13 μM; n = 3) than i (Kd(0 mV) = 1820 ± 176 μM; n = 5). Consistent with these data, Kd values for genistein inhibition of CFTR Cl− currents were very similar to those of genistein inhibition of Po, but very different from those of genistein inhibition of i (Fig. 8D). Thus, these data explain why genistein inhibition of CFTR Cl− currents was only weakly voltage dependent.

Our data indicate that genistein inhibition of Po was voltage independent, whereas genistein inhibition of i was voltage dependent. This suggests that genistein may interact with CFTR at two sites to inhibit channel activity. The voltage independence of genistein inhibition of Po suggests that genistein may bind to a site located outside the electric field of the membrane. In contrast, the voltage dependence of genistein inhibition of i suggests that genistein may bind to a second site located within the electric field of the membrane. The electrical distance sensed by genistein can be calculated using eqn (3). Using the data in Fig. 8D, z′ = 0.54 ± 0.08 (n = 5) measured from the inside of the membrane over the voltage range -80 to -40 mV. This suggests that genistein traverses about half the electrical distance across the membrane to reach its binding site within the membrane electric field.

DISCUSSION

The goal of this study was to investigate how genistein inhibits CFTR. Our data suggest that genistein may inhibit CFTR Cl− channels in two ways. First, it may interact with NBD1 to strongly inhibit channel opening. Second, it may bind within the CFTR pore to weakly block Cl− permeation.

A number of agents that stimulate CFTR Cl− channels have been demonstrated to inhibit channel activity at high concentrations. These include the non-hydrolysable ATP analogue 5′-adenylylimidodiphosphate (AMP-PNP; Mathews et al. 1998), PPi (Ma et al. 1997) and genistein (Weinreich et al. 1997; Wang et al. 1998; Zhou et al. 1998; Obayashi et al. 1999; present study). Each of these agents stimulates CFTR Cl− channels by binding tightly to NBD2, greatly slowing the rate of NBD2-mediated channel closure, and hence, dramatically prolonging the duration of bursts of channel activity (Hwang et al. 1994; Carson et al. 1995a, b; Gunderson & Kopito, 1995; French et al. 1997; Mathews et al. 1998; Wang et al. 1998). In addition, both PPi and genistein decrease the closed time interval between bursts of channel activity, suggesting that they may also increase the rate of NBD1-mediated channel opening (Carson et al. 1995b; Wang et al. 1998). Ma et al. (1997) reported that high concentrations of PPi caused CFTR to undergo irreversible transitions to a 3 pS subconductance state. However, the effect of high concentrations of PPi on channel gating was not described. When Mathews et al. (1998) elevated the AMP-PNP concentration above that of ATP, AMP-PNP inhibited channel opening, decreased Po, and reduced the rate at which channels became locked in the open configuration. Based on these results, Mathews et al. (1998) proposed that non-hydrolysable ATP analogues may bind to NBD1 and inhibit the opening of CFTR Cl− channels. Consistent with this idea, Weinreich et al. (1999) recently demonstrated that AMP-PNP slows ATP-dependent activation of CFTR Cl− currents in excised membrane patches evoked by rapid changes in the concentration of nucleotides in the intracellular solution.

Several lines of evidence suggest that high concentrations of genistein inhibit CFTR by prolonging the lifetime of the closed channel conformation. First, high concentrations of genistein promoted transitions to a new closed state described by a very slow closed time constant (Wang et al. 1998; present study). Occupancy of this state greatly prolonged the closed time interval between bursts of channel activity and hence decreased the rate of channel opening. Second, channel block was relieved by PPi, an agent that locks channels open to greatly prolong the gating cycle of CFTR (Carson et al. 1995b; Gunderson & Kopito, 1995). To explain this result, we suggest that genistein interacts with the closed state to inhibit channel opening. In the absence of PPi, the gating cycle of CFTR is short and CFTR makes frequent transitions to the closed state where it is available to be blocked by genistein. In contrast, in the presence of PPi, the gating cycle is prolonged with the result that CFTR makes fewer sojourns to the closed state where it may interact with genistein. This explanation also accounts for the different effects of PPi on genistein and glibenclamide inhibition of CFTR. Since glibenclamide is an open-channel blocker of CFTR (Schultz et al. 1996; Sheppard & Robinson, 1997), it inhibits CFTR even when channels are locked open. Third, channel block was relieved when the intracellular concentration of ATP was increased. This result suggests that genistein interacts with the closed state to inhibit CFTR because the rate of transition from the closed state to a burst of channel activity is regulated by ATP (Winter et al. 1994). Thus, as first suggested by Wang et al. (1998), our data suggest that the principal mechanism by which genistein inhibits CFTR is by interacting with the closed state to prevent channel opening.

Previous studies suggest that the stimulatory genistein binding site is located within NBD2 (French et al. 1997; Hwang et al. 1997; Wang et al. 1998; Randak et al. 1999). They also suggest that the genistein binding site is close to, but distinct from, the ATP-binding site of NBD2 (Weinreich et al. 1997; Wang et al. 1998; Randak et al. 1999). Our finding that ATP (5 mM) relieved channel block suggests that genistein may inhibit CFTR by competing with ATP for a common binding site or interacting with a site close to an ATP binding site. Because ATP hydrolysis at NBD1 opens CFTR (for review see Gadsby & Nairn, 1999; Sheppard & Welsh, 1999), this result suggests that genistein may interact with either the ATP binding site of NBD1 or a closely related site to inhibit channel opening. However, in the absence of studies using site-directed mutations, it remains possible that genistein may interact with a site outside NBD1 to inhibit channel opening.

Genistein inhibits a variety of different ion channels. Block of some ion channels may involve either tyrosine phosphorylation (Voets et al. 1998) or ATP binding sites (Wang et al. 1998; present study). However, block of other ion channels may occur by different mechanisms which involve a direct interaction between genistein and the ion channel. For example, in rat brain neurons, genistein and daidzein, an inactive analogue of genistein, rapidly inhibited voltage-gated Na+ channels, whereas other tyrosine kinase inhibitors were without effect (Paillart et al. 1997). Because genistein inhibited batrachotoxin binding, Paillart et al. (1997) suggested that genistein may interact with the neurotoxin receptor site 2 of voltage-gated Na+ channels. Moreover, in cardiac myocytes, genistein and diadzein rapidly and reversibly inhibited both cAMP-regulated delayed rectifier K+ currents and L-type Ca2+ currents (Chiang et al. 1996; Yokoshiki et al. 1996; Hool et al. 1998). Based on these results, Hool et al. (1998) speculated that genistein blocks the pores of cation channels by a non-specific mechanism.

Large anions inhibit CFTR Cl− channels by binding within a wide vestibule at the intracellular end of the CFTR pore, where they occlude the pore and prevent Cl− permeation (for review see Hwang & Sheppard, 1999). Our data suggest that genistein may weakly inhibit CFTR by a similar mechanism. Block of CFTR by genistein was due to the anionic form of the drug rather than the undissociated form (see below). Single-channel analysis of channel block indicated that genistein decreased open time and dramatically increased both the frequency and duration of flickery closures. This flickering block was voltage dependent. At negative voltages, it decreased i providing an explanation for the voltage-dependent block of CFTR Cl− currents in guinea-pig ventricular myocytes by genistein (Zhou et al. 1998; Obayashi et al. 1999). These features of genistein inhibition of CFTR are characteristic of open-channel block. Moreover, the location of the genistein binding site within the membrane electric field is similar to those of a number of large anions that occlude the CFTR pore (Hwang & Sheppard, 1999). Thus, our data suggest that genistein may enter the CFTR pore from the intracellular side of the membrane and bind to a site within the pathway for Cl− permeation. While genistein binds, its bulky size (predicted dimensions 1.26 nm × 0.58 nm; P. J. McLaughlin, personal communication) occludes the pore, preventing Cl− permeation (Cheung & Akabas, 1996; Linsdell et al. 1997). However, because genistein inhibition of CFTR Cl− currents was only weakly voltage dependent and unaffected by altering the external Cl− concentration, we propose that this site is not the principal site at which genistein binds to CFTR to inhibit channel activity.

Acidification of the intracellular solution relieved genistein inhibition of CFTR. The simplest interpretation of this result is that the anionic form of genistein may inhibit CFTR. However, we recognise that other explanations are possible. First, acid pH may cause a change in the solubility of genistein. Second, if there are residues with protonatable side-chains in the genistein binding sites, then acid pH may alter the structure of the binding sites and hence the interaction of genistein with CFTR. We consider the first explanation unlikely, because we observed no pH-dependent changes in the solubility of genistein. However, the second explanation remains an interesting possibility.

We found that low micromolar concentrations of genistein failed to stimulate the activity of phosphorylated CFTR Cl− channels in excised membrane patches, while genistein (100 μM) inhibited channel activity (control: Po = 0.32; genistein: Po = 0.12). In contrast, using similar conditions, French et al. (1997) demonstrated that genistein (100 μM) stimulated CFTR Cl− channels in excised inside-out membrane patches from NIH 3T3 cells expressing wild-type human CFTR (control: Po = 0.13; genistein: Po = 0.38). One possible explanation for these conflicting results is the different cell types used to express wild-type human CFTR. However, a more likely explanation is the phosphorylation status of CFTR and hence the level of channel activity (for discussion see Hwang & Sheppard, 1999). Wang et al. (1998) demonstrated that genistein strongly enhanced the activity of weakly phosphorylated CFTR Cl− channels, but had little or no effect on the activity of strongly phosphorylated CFTR Cl− channels. Similarly, we found that low micromolar concentrations of genistein greatly augmented cAMP-stimulated iodide efflux from cells expressing recombinant CFTR, but failed to enhance the activity of CFTR Cl− channels in excised membrane patches following phosphorylation by PKA (J. F. Kidd, K. A. Lansdell & D. N. Sheppard, unpublished observations). Thus, the effect of genistein on channel activity depends on the phosphorylation status of CFTR.

Wang et al. (1998) demonstrated that genistein caused transitions to subconductance states. In contrast, we found that genistein caused a voltage-dependent flickering block of CFTR. This difference between the data of Wang et al. (1998) and our own probably reflects the different conditions used to analyse gating kinetics. Wang et al. (1998) studied CFTR at room temperature and heavily filtered their data. In contrast, we studied CFTR at 37°C and lightly filtered our data. Similarly, the different patterns of channel gating observed in the presence of the biological buffer 3-(N-morpholino)propanesulphonic acid (Mops) were explained by the different conditions used to study CFTR Cl− channels (Gunderson & Kopito, 1995; Ishihara & Welsh, 1997).

The observation that genistein may either stimulate or inhibit CFTR Cl− channels has implications for the treatment of diseases associated with the dysfunction of CFTR. Genistein or related flavonoids that potently stimulate CFTR Cl− channels may provide a new therapeutic strategy for treating CF patients (Illek et al. 1999). In contrast, flavonoids that strongly inhibit CFTR Cl− channels may prove to be of value in the treatment of diseases such as secretory diarrhoea and polycystic kidney disease, which may involve increased activity of the CFTR Cl− channel (Gabriel et al. 1994; Sullivan et al. 1998). If flavonoids that inhibit channel opening have greater specificity than agents that block the CFTR pore, they may be used to develop therapeutically active inhibitors of CFTR with minimal adverse effects. In search of more effective activators of CFTR, some investigators have begun to examine the relationship between the chemical structure of flavonoids and their effects on CFTR Cl− channels (Illek & Fischer, 1998; Singh et al. 1998). We anticipate that these and other studies will identify potent, specific activators and inhibitors of the CFTR Cl− channel.

Acknowledgments

We thank Professors S. L. Flitsch and M. J. Welsh and Drs P. J. McLaughlin and B. D. Schultz for valuable discussions and Professor D. C. Gadsby and Drs H. Fischer and C. Randak for pre-prints of manuscripts. We thank Drs S. H. Cheng, C. R. O'Riordan and A. E. Smith (Genzyme, Framingham, MA, USA) for the generous gift of C127 cells expressing wild-type human CFTR. This work was supported by the Biotechnology and Biological Sciences Research Council, the Cystic Fibrosis Trust and the National Kidney Research Fund.

K. A. Lansdell and Z. Cai contributed equally to this work.

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