From the * Department of Physiology, Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, Missouri
65211, Laboratory of Molecular Neuroscience, Rockefeller University, New York 10021
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ABSTRACT |
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Previous studies have shown that genistein increased cystic fibrosis transmembrane conductance regulator (CFTR) channel activity in the presence of saturating concentrations of forskolin and calyculin A in intact
cells. Possible molecular mechanisms for genistein's action include inhibition of tyrosine kinases, inhibition of
serine/threonine protein phosphatases, or direct binding of genistein to CFTR. Since genistein inhibits several
enzymes that hydrolyze ATP, and ATP hydrolysis is an intrinsic property of CFTR, we examined the effect of
genistein on CFTR gating in excised inside-out patches from Hi-5 insect cells and NIH3T3 cells expressing recombinant CFTR. Genistein (50 µM) did not open phosphorylated CFTR channels by itself, but increased the ATP-
induced CFTR channel current by approximately twofold. A similar magnitude of enhancement was observed
when genistein was applied with PKI, a specific inhibitor of protein kinase A, or vanadate, a tyrosine phosphatase
inhibitor, suggesting that inhibition of protein phosphatases or tyrosine kinases does not account for genistein's
effects. The enhancement of channel current increased with increasing concentrations of genistein and reached a
maximum at 35 µM genistein. At higher concentrations of genistein concentration, CFTR channel current decreased, resulting in a bell-shaped dose-response relationship. In the absence of genistein, both open- and closed-time histograms could be fitted with a single exponential function, yielding a mean open time (O) of 0.302 ± 0.002 s, and a mean closed time (
C) of 0.406 ± 0.003 s. In the presence of 50 µM genistein, the open time histogram could be fitted with a double exponential function with
O1 = 0.429 ± 0.003 s and
O2 = 2.033 ± 0.173 s. Thus, genistein induced a prolonged open state, an effect that mimics that of nonhydrolyzable ATP analogs.
Closed time analysis showed that 50 µM genistein caused a prolonged closed state with a time constant of 2.410 ± 0.035 s. We thus conclude that (a) the effects of genistein are likely caused by a direct binding of the drug to the
CFTR protein, and (b) at least two binding sites are required to explain the effects of genistein: a high affinity site
that decreases the closing rate and a low affinity site that reduces the opening rate.
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INTRODUCTION |
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Cystic fibrosis is caused by mutations in the single gene
encoding the cystic fibrosis transmembrane conductance regulator (CFTR).1 The predicted structure of
CFTR contains five parts: two repeats of six membrane-spanning segments, two nucleotide binding folds (NBF1
and NBF2), and a regulatory (R) domain with several
consensus serine residues for cAMP-dependent protein
kinase (PKA) phosphorylation (Riordan et al., 1989).
Its predicted topology places CFTR in the ATP binding
cassette transporter superfamily (Riordan et al., 1989
;
Hyde et al., 1990
; Ames et al., 1992). It is believed that members of this family harvest free energy from ATP
hydrolysis to pump substrates across the cell membrane, but CFTR is an ohmic chloride channel with a
single channel conductance of 8 to ~13 pS.
The major mechanism for regulation of CFTR activity involves the classical cAMP-PKA pathway. PKA phosphorylation of the R domain is essential for CFTR function (Anderson et al., 1991; Tabcharani et al., 1991
;
Berger et al., 1991
; Rich et al., 1991
; Cheng et al.,
1991
). However, phosphorylation by PKA is not adequate to open the CFTR channel. After phosphorylation, ATP binding and hydrolysis at NBF1 and NBF2
are coupled to the opening and closing of the channel.
Previous work on phosphorylation regulation and ATP
gating of CFTR chloride channels has established a
preliminary model in which ATP binding and hydrolysis at NBF1 controls channel opening while ATP binding and hydrolysis at NBF2 controls channel closing
(review by Gadsby et al., 1995
). This model proposes
that ATP hydrolysis provides the free energy for protein conformational changes in gating transitions.
Genistein, an isoflavonoid found mainly in legumes,
has been shown to inhibit tyrosine kinase (Akiyama et
al., 1987), topoisomerase (Markovits et al., 1989
), and
histidine kinase (Huang et al., 1992
) activity. The effect
of genistein on wild-type CFTR channels was first reported by Illek et al. (1995)
. In their studies, genistein was shown to activate CFTR Cl
channels in NIH3T3
cells and its effect could be abolished by vanadate
(VO4), a tyrosine phosphatase inhibitor. They therefore proposed that, in addition to the cAMP-dependent
pathway, CFTR is regulated by a cAMP-independent
mechanism that might involve tyrosine phosphorylation. However, more recent reports (Illek et al., 1996
;
Reenstra et al., 1996
; Yang et al., 1997
) suggest that
genistein may act via inhibition of protein phosphatases. Evidence supporting this latter theory includes the fact that in situ CFTR phosphorylation is increased after treatment with genistein (Reenstra et al.,
1996
). The importance of genistein or similar compounds is underscored by the demonstration that genistein potentiates cAMP-dependent channel activity from the most common disease-associated mutant
CFTR; i.e., deletion of phenylalanine 508 (
F508). The
open probability (Po) of this mutant channel in cell-attached patches is lower than that of wild-type CFTR
in the presence of cAMP stimulation (Dalemans et al.,
1991
; Haws et al., 1996
; Hwang et al., 1997
). However,
our previous work has demonstrated that the Po in the
presence of genistein is almost identical to that of wild-type CFTR (Hwang et al., 1997
). Although this latest report also provides preliminary evidence for a direct
binding of genistein to CFTR molecules (see Weinreich et al., 1997
), kinetic analysis at the single channel
level is still lacking. Since genistein has been shown to
act on ATP binding sites (Akiyama et al., 1987
) and
ATP hydrolysis is coupled to CFTR gating (Gadsby et
al., 1995
), more detailed studies could provide mechanistic information on how genistein modulates CFTR function.
In this study, genistein's effects on CFTR gating were
examined in Hi-5 insect cells and NIH3T3 cells expressing wild-type CFTR. In cell-attached mode, low micromolar concentrations of genistein increased CFTR channel currents induced by saturating concentrations of
forskolin and calyculin A, a membrane permeant phosphatase inhibitor (Suganuma et al., 1991). After activation of CFTR in cell-attached patches with forskolin
and calyculin A, genistein did not open CFTR channels
by itself in excised inside-out patches, but increased
ATP-elicited CFTR channel currents. Neither PKI, a specific PKA inhibitor, nor VO4, a tyrosine phosphatase inhibitor, affected the ability of genistein to enhance CFTR
channel current. Notably, the ability of genistein to enhance CFTR channel activity appears to depend on the
phosphorylation level of CFTR. Although genistein increases the activity of both partially phosphorylated and fully phosphorylated CFTR, the level of enhancement is greater on partially phosphorylated channels.
At low micromolar concentrations, genistein increased
the Po of ATP-stimulated CFTR. However, an inhibitory
effect was observed at genistein concentrations higher than 35 µM. Kinetic analysis indicated that the enhancement of CFTR activity resulted from prolongation of the open time, whereas the inhibitory effect was
through prolongation of the closed time. The two opposite effects of genistein resulted in a bell-shaped dose-response curve. These results may be explained
by a model in which there are two binding sites for
genistein: a high affinity site responsible for the enhancement effect, and a low affinity site for the inhibitory effect.
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MATERIALS AND METHODS |
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Cell Culture and Electrophysiology
Wild-type CFTR Cl channels were expressed in Hi-5 insect cells
(Invitrogen Corp., San Diego, CA) by infecting the cells with baculovirus containing recombinant CFTR cDNA (Yang et al., 1997
), while NIH3T3 cells were stably transfected with wild-type CFTR cDNA (Berger et al., 1991
). Insect cells were passaged and grown onto small cover glass chips in 35-mm tissue culture dishes with TNM-FH insect medium (Sigma Chemical Co., St. Louis, MO)
supplemented with 10% fetal bovine serum (FBS), 2 mM
glutamine, 50 mg/liter gentamicin, and 4 mM sodium bicarbonate at 27°C. 2-3 d before experiments, an aliquot of the baculovirus (~5 µl) was added to the Hi-5 cells. CFTR-containing
NIH3T3 cells were grown at 37°C in Dulbecco's modified Eagle's
medium (DMEM) supplemented with 10% FBS and 2 mM
glutamine. Immediately before recording, cover glass chips containing either cell type were transferred to a continuously perfused chamber located on the stage of an inverted microscope
(Olympus Corp., Tokyo, Japan).
For recording of CFTR channel current, glass capillary pipette
electrodes were made using a two-stage, vertical puller (Narishige, Tokyo, Japan). The pipette tips were fire-polished with a
homemade microforge to ~1 µm external diameter. Pipette resistance was 5-7 M for Hi-5 cells and 3-4 M
for NIH3T3 cells
in the bath solution. CFTR channel currents were recorded at
room temperature with an EPC-9 patch-clamp amplifier (HEKA,
Lambrecht, Germany), filtered at 100 Hz with a built-in three-pole Bessel filter (Frequency Devices Inc., Haverhill, MA), and
stored on videotapes. Data were subsequently playbacked at a refiltered frequency of 25 Hz with an eight-pole Bessel filter and
captured onto a hard disk at a sampling rate of 50 Hz. For spectral analysis, data were filtered at 50 Hz and digitized at 100 Hz.
Pipette potential was held at 50 mV in reference to the bath.
Downward deflections represent channel openings.
The pipette solution contained (mM): 140 N-methyl-D-glucamine chloride (NMDG-Cl), 2 MgCl2, 5 CaCl2, and 10 HEPES (pH 7.4 with NMDG). The superfusion solution for Hi-5 insect cells contained (mM): 150 NaCl, 2 MgCl2, 1 EGTA, 5 glucose, and 5 HEPES (pH 6.5 with 1 N NaOH). NIH3T3 cells were perfused with (mM): 150 NaCl, 2 MgCl2, 1 EGTA, 5 glucose, and 5 HEPES (pH 7.4 with 1 N NaOH). In excised inside-out patch experiments, the superfusion solution contained (mM): 150 NMDG-Cl, 10 EGTA, 10 HEPES, 8 Tris, and 2 MgCl2 (pH 7.4 with NMDG). After excision, patch pipettes were moved to a small homemade chamber where a complete solution change could be accomplished within 1 s.
Forskolin was purchased from Calbiochem Corp. (San Diego, CA). Calyculin A and genistein were purchased from LC Laboratories (Woburn, MA). Sodium vanadate was purchased from Aldrich Chemical Co. (Milwaukee, WI). MgATP, all salts and buffers were purchased from Sigma Chemical Co. Genistein, forskolin, and calyculin A were dissolved in dimethylsulfoxide at a stock concentration of 100 mM, 20 mM, and 100 µM, respectively. PKI, MgATP, and orthovanadate were dissolved in distilled water as stock solutions. Orthovanadate (VO4) was boiled for 15-20 min before dilution into superfusion solution immediately before use.
Kinetic Analysis
In most of our experiments in the inside-out configuration, channels were first activated with forskolin plus calyculin A in the cell-attached mode. After excision, variable rundown, presumably due to dephosphorylation by membrane-associated protein phosphatases, was observed. For kinetic analysis, recordings with minimal rundown were selected. In experiments for estimation of genistein's effects, 0.5 mM ATP was applied before and after addition of genistein. This bracketing helps in detection of any time-dependent changes in CFTR channel current. Potential effects of rundown were then corrected accordingly. In most cases, the period over which the measurement was made was short; therefore, rundown was negligible.
Macroscopic kinetic analysis.
The mean current amplitude was
calculated with the Igor software (Wavemetrics, Lake Oswego,
OR). Curve fitting of the dwell time histogram was performed
with the Igor software. The power spectrum density was constructed and analyzed to compare the current fluctuations of
CFTR Cl channels activated by ATP and ATP plus genistein.
The data were fast Fourier transformed to generate noise spectrum, and then further analysis was performed by the curve fitting using the Lorentzian function: S = S0/[1 + (f/fc)2], where fc
and S0 are the corner frequency and the zero frequency asymptotes, respectively.
Single-channel kinetic analysis.
Dwell time analysis was performed
using Igor software. After measuring the open time for each single channel open event, the data were sorted on a basis of duration from shortest to longest and plotted against the upper cumulative probability that a particular opening event will last at
least time (t) (see Figs. 5, 8, and 11). Therefore, the probability
of an event lasting for at least 60 ms, the shortest defined t for an
open state, is 1. This method is similar to that used by Baukrowitz
et al. (1994). All open duration data could be fitted with either a
single exponential or double exponential function, yielding either one or two time constants, respectively. The same method
was applied to analyze the single channel closed events for the
closed time constant(s).
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RESULTS |
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Enhancement of CFTR Activity by Genistein
Our previous studies (Yang et al., 1997) have shown
that, in Hi-5 insect cells transiently expressing wild-type
CFTR, genistein further increased CFTR channel currents activated with forskolin and calyculin A, a membrane-permeant inhibitor of phosphatases 1 and 2A.
Fig. 1 A shows a representative experiment from a cell-attached patch in Hi-5 cells. Macroscopic CFTR currents were elicited by bath application of saturating
concentrations of forskolin (10 µM) and calyculin A
(25 nM). Application of 30 µM genistein in the presence of forskolin and calyculin A significantly increased CFTR channel activity by 3.20 ± 1.14-fold (n = 7). A
similar magnitude (3.16 ± 0.57-fold, n = 6) of enhancement of forskolin- and calyculin A-induced CFTR
currents by genistein was observed in NIH3T3 cells (Fig. 1 B). Upon removal of genistein, this enhancement of cAMP-dependent CFTR channel activity was
readily reversible. Thus, genistein increases CFTR activity even with maximal cAMP stimulation and inhibition
of phosphatase 1 and 2A. These results and the fact that genistein does not activate CFTR in the absence of
cAMP stimulation (Yang et al., 1997
) suggest that
genistein only potentiates the activity of CFTR channels
that have been phosphorylated by PKA.
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Since it has been demonstrated that genistein can bind to the ATP binding site in tyrosine kinases, we first tested whether genistein can mimic ATP to open phosphorylated CFTR channels. Channels were first activated with 10 µM forskolin plus 25 nM calyculin A in cell-attached patches from Hi-5 insect cells. Gating of CFTR by ATP was then examined in cell-free, excised inside-out patches. Fig. 2 A shows that opening of phosphorylated CFTR depends on the presence of ATP and that 50 µM genistein alone, in the absence of ATP, did not open CFTR channels. However, when 50 µM genistein was applied together with 0.5 mM ATP, the elicited CFTR current was higher than that in the presence of ATP alone (Fig. 2 B).
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The simplest explanation for these results is that
genistein affects CFTR gating through direct binding
to the CFTR protein. However, two other possibilities
may be considered. First, it is likely that a local network
of PKA and phosphatases exists in excised membrane
patches. Based on this scenario, besides opening CFTR
channels, ATP would also be used by PKA to phosphorylate CFTR. Membrane-bound phosphatases acting in
concert with PKA would establish an equilibrium between phosphorylation and dephosphorylation. Thus,
when ATP was applied alone, the fraction of phosphorylated channels in the patch would be less than unity. If
genistein acts via inhibition of phosphatases, then its
application with ATP will shift the phosphorylation/dephosphorylation equilibrium in the direction of a higher
phosphorylation, thereby increasing the current. This
model predicts that application of the PKA inhibitor, PKI, should decrease ATP-induced CFTR channel current in excised patches and that PKI should abolish
genistein's effects. However, 3 µM PKI (a saturating
concentration; Cheng et al., 1986) did not decrease channel activity induced by 0.5 mM ATP, and CFTR channel
activity was still increased after application of genistein in
the presence of ATP and PKI (Fig. 3 A).
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We next consider the possibility that both tyrosine kinases and phosphatases are present in the membrane
patches since tyrosine kinases and tyrosine phosphatases
both exist as intrinsic membrane proteins and as membrane-associated proteins (Lee and Pilch, 1994; Tonks
and Neel, 1996
). Based on this scenario, tyrosine phosphorylation of CFTR or a regulatory substrate would inhibit CFTR activity, and application of genistein would
increase CFTR current via inhibition of tyrosine kinases.
Application of genistein with ATP should shift the equilibrium of tyrosine phosphorylation/dephosphorylation such that the steady state level of tyrosine-phosphorylated substrate would be decreased by the actions
of endogenous tyrosine phosphatases. This model predicts that application of orthovanadate (VO4), a nonspecific tyrosine phosphatase inhibitor, should decrease
ATP-induced CFTR channel current in excised patches
and that VO4 should abolish genistein's effects. However, addition of 50 µM VO4 did not decrease the ATP-dependent CFTR channel activity. Moreover, 50 µM
genistein still increased the ATP-induced CFTR channel current by 2.2 ± 0.3-fold (n = 5) in the presence of
50 µM VO4. Thus, though other, more complicated,
mechanisms might explain our results, the simplest interpretation is that genistein binds to CFTR and
thereby affects CFTR gating.
Genistein Prolongs the Channel Open Time
To further understand how genistein affects CFTR gating, the following experiments and analysis were performed. In excised patches containing multiple CFTR channels, the macroscopic current amplitude is determined by three factors: the total number of phosphorylated channels, n, the single channel amplitude, i, and the single channel open probability, Po. Since CFTR channels had been activated (and therefore phosphorylated) in cell-attached patches, and PKI had no effect on ATP-dependent CFTR currents, n is relatively constant during the short period the experiments were performed. Only an increase in i or Po can account for the increase of the macroscopic current by genistein.
Fig. 4 A shows a current trace in an excised inside-out patch containing a single channel from a Hi-5 insect cell. In the presence of ATP alone (Fig. 4 A, top), both openings and closings are in the range from several hundred milliseconds to seconds. In the presence of genistein, prolonged openings are clearly discernible (Fig. 4 A, bottom). Genistein also changed the single-channel current amplitude in such a way that there were more subconductance states with variable single-channel amplitudes compared with that in the presence of ATP alone. The corresponding amplitude histograms for ATP alone and ATP plus genistein are shown in Fig. 4 B. The single channel amplitude (the distance between the two peaks of the histogram) is smaller in the presence of genistein. The peak representing the open state in the presence of genistein is also wider than the peak representing the open state with ATP alone. This is due to the multiple subconductance states of the channel in the presence of genistein because full conductance openings can still be seen. The single channel amplitude, calculated from amplitude histograms, is 13 ± 3% smaller (n = 4) in the presence of genistein plus ATP than in the presence of ATP alone. Therefore, enhancement of ATP-induced macroscopic CFTR channel current by genistein is due to an increase in Po.
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To collect a large enough number of events for dwell-time analysis, we pooled all the open and closed times
from multiple single channel recordings. Fig. 5 shows
the resulting open-time histogram (see Fig. 8 A for the
closed-time histogram). In the presence of ATP alone,
the open-time histogram was fitted with a single exponential function with the open time constant O = 0.302 ± 0.002 s. In the presence of ATP and genistein,
the open-time histogram was fitted with a double exponential function with two open time constants:
O1 = 0.429 ± 0.003 s and
O2 = 2.033 ± 0.173 s. These results suggest that genistein induces a prolonged open state, an effect that mimics that of nonhydrolyzable
ATP analogues (Gunderson and Kopito, 1994
; Hwang
et al., 1994
). A similar prolongation of the open time
by genistein was observed by French et al. (1997)
. Support for the conclusion made from the single-channel
kinetic analysis was obtained from spectral analysis of
macroscopic noise. Time-domain macroscopic currents
were transformed to power-frequency spectra using fast
Fourier transformation. The resulting spectra were fitted with a Lorentzian function. The average corner frequency obtained for CFTR activity activated by ATP
alone (fc = 1.135 ± 0.164 Hz, n = 10) was significantly
higher than that by ATP plus genistein (fc = 0.640 ± 0.047 Hz, n = 10).
High Concentrations of Genistein Inhibit ATP-dependent CFTR Gating
The enhancement of macroscopic CFTR channel current by genistein increased with increasing concentrations of the drug, reaching a maximum of 3.31 ± 0.62-fold (n = 7) at 35 µM genistein. Surprisingly, at concentrations of genistein higher than 35 µM, the enhancement of macroscopic CFTR current was decreased (Fig. 6 A). Notably, at concentrations of genistein higher than 75 µM, channel current was lower than that obtained with ATP alone. The bell-shaped dose-response relationship suggests two opposite effects of genistein in excised patches, enhancement at lower concentrations and inhibitory at higher concentrations.
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Since genistein decreased single channel amplitude (Fig. 4 B), this dose-response relationship obtained from analysis of macroscopic CFTR channel current does not reliably reflect the effects of genistein on CFTR gating. We thus analyze the effects of genistein on single channel Po using recordings from patches that contained less than four channel steps. In those patches where individual steps of channel opening and closing can be discerned clearly, we were able to estimate Po accurately without any interference from genistein effects on the single channel amplitude. The Po of CFTR exhibited a bell-shaped dose- response relationship to genistein that was very similar to that obtained from measurement of macroscopic channel current (Fig. 6 B).
Fig. 7 A shows a representative recording in an excised patch from Hi-5 insect cells. The mean current amplitude in the presence of 20 µM genistein plus 0.5 mM ATP is approximately twofold higher than that with ATP alone. In the same patch, application of 100 µM genistein generated a current that is only ~93% of the current with ATP. Both enhancement and inhibitory effects were readily reversible upon removal of genistein. In patches containing multiple channels, the net current generated by ATP plus 100 µM genistein resulted from a combination of potentiation and inhibition effects. If genistein binds to two different sites to produce these effects, one might expect that the enhancement effect coexists with the inhibitory effect even at 100 µM genistein. In other words, with multi-channel patches, some channels are potentiated and some are inhibited at any instant of the recording. Therefore, the inhibitory effect is likely underestimated if one simply calculates the percent decrease of the macroscopic current by genistein.
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At the single molecule level, the channel may be either potentiated or inhibited. This was substantiated
when observing effects of genistein in patches containing a single channel (Fig. 7 B). Prolonged closed events
were rarely observed in the presence of 10 µM genistein,
but at 60 µM genistein only a few bursts of openings interrupted by long closed events were seen. This result
also suggests that the inhibitory effect of genistein was
due to a prolonged closed state. Dwell-time analysis of
the closed-time distribution revealed a closed time constant C = 0.406 ± 0.003 s in the presence of ATP alone
(Fig. 8). In the presence of genistein, the closed time
histogram can be fitted with a double exponential function with
C1 = 0.449 ± 0.003 s and
C2 = 2.410 ± 0.035 s. Thus, genistein can either increase CFTR channel activity through prolongation of the open time (i.e., a decrease of the closing rate) or decrease CFTR channel
activity through prolongation of the closed time (i.e., a
decrease of the opening rate).
The inhibitory effect of genistein was also observed
in cell-attached patches. Fig. 9 A shows a biphasic response to 100 µM genistein in a cell-attached patch
from Hi-5 cells. In the presence of saturating concentrations of forskolin (10 µM) and calyculin A (20 nM),
application of 100 µM genistein first increased CFTR channel activity. After 3-4 min, the channel activity
reached a maximum level, and then declined. The
channel activity was transiently recovered upon removal of 100 µM genistein. Fig. 9 B shows both the potentiation and the inhibition of a single channel by 100 µM genistein in a cell-attached patch from a Hi-5 insect
cell in the presence of forskolin (10 µM) and calyculin
A (20 nM). Note the appearance of relatively long
openings (enhancement effect) soon after application
of 100 µM genistein. After several minutes of genistein
application, prolonged closings (inhibition effect) appear, although the longer openings still remain. The
slow time course for inhibition by higher concentrations of genistein could be due to a rate-limiting diffusion of the compound to its site(s) of action. This slow
time course may explain why we previously reported
only enhancement of CFTR channel activity using 50 µM genistein (Yang et al., 1997).
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Enhancement by Genistein Depends on the Phosphorylation Level of CFTR
In excised inside-out patches, an average increase of
the ATP-induced CFTR channel activity of two- to
threefold was observed with 20-50 µM genistein (Fig.
6). However, we noted a relatively large variation in the
fold increase. For example, in experiments where the
ATP-dependent CFTR activity was relatively low (see MATERIALS AND METHODS), genistein produced an apparently greater enhancement (e.g., Fig. 3 A). Interestingly, in these patches the closed time is much longer
than 400 ms, a value obtained from patches with apparently higher Po. We hypothesize that the variable CFTR
activity in excised patches was due to variable phosphatase activity associated with the membrane patch,
resulting in channels from different patches having different levels of phosphorylation and that genistein's effects might depend on the degree of CFTR phosphorylation. In cell-attached patches, different levels of CFTR
phosphorylation may be obtained by using different
concentrations of forskolin. In excised patches, partial
or more complete phosphorylation of CFTR may be obtained by modulating the amount or duration of exogenous PKA application. Since the relationship between
forskolin and cAMP concentrations in NIH3T3 cells
has been well characterized (Illek et al., 1995), and similar effects of genistein (both qualitatively and quantitatively) were observed in the NIH3T3 expression system (Hwang et al., 1997
), experiments were designed using
NIH3T3 cells to test our hypothesis.
As a control, Fig. 10 A shows a typical gating behavior
in an excised patch from NIH3T3 cells where a single
CFTR channel was strongly activated by saturating concentrations of forskolin (10 µM) and calyculin A (50 nM) in the cell-attached configuration. The open and
closed time constants (data not shown) from this single-channel recording are consistent with those derived
from multiple experiments (Figs. 5 A and 8). These results also suggest that the gating parameters of CFTR in
excised inside-out patches from NIH3T3 cells are similar
to those from Hi-5 insect cells, although CFTR expressed
in these two systems assumes a somewhat different gating pattern in cell-attached patches (Yang et al., 1997).
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Fig. 10 B shows an experiment where CFTR channels
were first activated with 50 nM forskolin (a submaximal
concentration; Illek et al., 1995) in a cell-attached configuration. The patch was then excised into an inside-out mode. The Po was ~0.03 in the presence of 0.5 mM
ATP with long closed events. Neither the Po nor the kinetic parameters were changed when [ATP] was increased from 0.5 to 2 mM. However, addition of 10 µM
genistein caused an ~10-fold (9.90 ± 1.65, n = 6) increase of Po. Not only was the mean open time increased with genistein (3.56 ± 0.65 fold, n = 6), but
also the mean closed time was only ~25% (24.5 ± 5.8%, n = 6) of that in the presence of 0.5 mM ATP
alone. Similar results were obtained in excised patches
where CFTR channels were activated with brief pulses
of PKA and ATP (data not shown). Assuming most channels in the patch preactivated with submaximal
concentrations of forskolin were phosphorylated incompletely, this result suggests that partially phosphorylated CFTR has a lower opening rate and that
genistein increases the opening rate and decreases the
closing rate of the partially phosphorylated CFTR. Fig.
10 C summarizes the effects of genistein on presumably
partially phosphorylated CFTR in six patches prestimulated with submaximal concentrations of forskolin (50 nM). In contrast to partially phosphorylated channels,
when CFTR in excised patches were activated with exogenous PKA (to enhance phosphorylation) plus ATP,
only an approximately twofold (2.19 ± 0.33, n = 7) increase of the ATP-dependent CFTR activity by 10 µM
genistein was observed.
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DISCUSSION |
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Direct Effects of Genistein on CFTR Gating
Coupling of ATP hydrolysis to gating transitions is a
hallmark feature of CFTR Cl channels (Anderson et
al., 1991
). Several models of CFTR gating have since
been proposed (Hwang et al., 1994
; Gunderson and
Kopito, 1994
; Carson et al., 1995
). It is generally agreed
that hydrolysis of ATP at NBF1 is coupled to (Hwang et
al., 1994
; Carson et al., 1995
) or is required for (Gunderson and Kopito, 1994
) the opening of PKA-phosphorylated CFTR, whereas binding and hydrolysis of ATP at NBF2 control the closing. Thus, the rate of ATP
hydrolysis at NBF1 modulates how long channels stay
in the closed state and the rate of ATP hydrolysis at
NBF2 determines how long channels remain in the
open state. Our single-channel kinetic analysis shows
that for strongly phosphorylated CFTR both the mean
open and the mean closed times are in the range of several hundred milliseconds in the presence of 0.5 mM
ATP. These values are very close to those reported by Li et
al. (1993)
for purified CFTR incorporated in black lipid
membranes. Measuring ATP hydrolysis from purified
CFTR protein, Li et al. (1996)
recently suggest that one
ATP molecule is hydrolyzed by CFTR per second. Their data are consistent quantitatively with the idea that ATP
hydrolysis at NBFs is coupled to the gating transitions that
yield time constants of several hundred milliseconds.
Short-lived closings within individual openings, observed
in several reports (Winter et al., 1994
; Gunderson and Kopito, 1994
), could represent open channel blockade (e.g., compare Fischer and Machen, 1994
, 1996
).
Our results show that effects of genistein on highly
phosphorylated CFTR are through prolongation of
channel open or closed time, depending on the concentration of genistein. Since genistein is a known inhibitor of several enzymes that hydrolyze ATP, the simplest explanation for our observations is that, at low
concentrations, genistein decreases the ATP hydrolysis
rate at NBF2, whereas, at high concentrations, it reduces the ATP hydrolysis rate at NBF1. The bell-shaped
dose-response curve is a result of genistein binding to
two sites distinguished by their different affinities and
effects on CFTR gating. But where does genistein bind?
Considering the fact that the ATP binding motif is the
common feature among those enzymes that are inhibited by genistein, we argue that genistein interacts directly with NBFs. However, genistein does not compete with ATP since a similar magnitude of potentiation was
observed when [ATP] was increased fourfold (Wang,
F., and T.-C. Hwang, unpublished observations). Moreover, genistein had no effect on the ATP dose-response
relationship (Weinreich et al., 1997), suggesting genistein-
and ATP-binding sites are probably different. Pharmacological results suggest that genistein binds to the
ATP-binding site of tyrosine kinases (Akiyama et al.,
1987
), but genistein and ATP are not likely to share the
same binding site in topoisomerases (Markovits et al.,
1989
). Lack of competition between ATP and genistein
in our experiments suggests that genistein binding to CFTR allosterically inhibits ATP hydrolysis. Although we
favor a direct action of genistein on CFTR molecules, it
is entirely possible that genistein may not bind directly
to the NBFs. Alternatively, the molecular targets for
genistein could be a protein that is associated with CFTR.
Testing genistein on purified CFTR in bilayers could provide direct evidence that genistein interacts with CFTR. Development of high-affinity genistein analogs will hopefully shed light on where genistein binds. Thus, genistein
and its analogs could provide a useful chemical probe to
study the structure and function of NBFs.
Effects of Genistein on Single Channel Conductance
At least three discrete subconductance states have been
shown for the CFTR Cl channel incorporated in bilayers (Tao et al., 1996
). Although the mechanism for
these subconductance states is unknown, it has been
suggested that they might be caused by different subunit aggregation (Tao et al., 1996
), or reflect different
protein conformations in the ATP hydrolysis cycle
(Gunderson and Kopito, 1995
). We also have observed
discrete subconductance states of CFTR in cell-attached or excised patches from Hi-5 insect cells transiently expressing the wild-type CFTR (Wang et al., 1996
). However, the effect of genistein on the single channel current amplitude is unusual. First, genistein seems to promote the appearance of subconductance states. Even in
the presence of a high concentration of genistein, full
conductance states can be clearly seen (Fig. 4 A, bottom). This observation is inconsistent with the concept
of fast blockers, which uniformly decrease single channel amplitude (Hille, 1992
). Multiple subconductance
states with variable current amplitudes are observed in
the presence of genistein (Fig. 4 A, bottom). Second, single channel amplitudes sometimes show a gradual
change instead of a discrete transition from one state to
the other (Fig. 4 A, bottom).
Although the molecular mechanism for subconductance states of ion channels remains unknown, the
structure of the permeation pore is likely altered in different subconductance states. Effects of genistein on
the single channel amplitude and perhaps on the phosphorylation status of CFTR (Illek et al., 1996; Reenstra
et al., 1996
; Yang et al., 1997
) may suggest more than
two binding sites for genistein. However, if our hypothesis is correct that NBFs are the sole molecular targets
for genistein, our results suggest that a conformational
change after genistein binding may alter the permeation pore. This raises the interesting possibility that
part of NBFs might contribute to the formation of the
channel pore. Recent demonstrations that NBF1 has
extracellular accessibility as determined by chemical
modification with the biotinylation reagent, NHS-biotin
(Gruis and Price, 1997
), supports this possibility. Considering the close proximity of the channel gate to the
channel pore in Shaker K+ channels (Liu et al., 1997
)
and nicotinic receptor channels (Labarca et al., 1995
),
one should not be surprised that NBFs might be physically close to the CFTR pore.
Effects of Genistein on Partially Phosphorylated CFTR
The magnitude of genistein's enhancement depends
on the phosphorylation status of CFTR. When genistein
was tested on partially phosphorylated CFTR, a stronger potentiation effect was observed. Genistein not
only prolongs the open time, but also shortens the closed time of partially phosphorylated CFTR, resulting
in an ~10-fold increase of the ATP-dependent CFTR
channel activity. A similar inverse relationship between
the phosphorylation status and the degree of potentiation by genistein was recently reported (French et al.,
1997). These dramatic potentiation effects are observed even at 10 µM genistein. If the binding site for
these potentiation effects is also in NBF2, an apparent
conclusion from our data is that binding of genistein at
NBF2 can affect the function of NBF1 when CFTR is
partially phosphorylated. Interactions between different cytoplasmic domains have been implicated in several studies (e.g., Kiser et al., 1996
) and preliminary
biochemical evidence has been reported recently (Gruis,
D.B., and E.M. Price, manuscript submitted for publication). Thus, our results, together with others, strongly
suggest that NBF1, NBF2, and the R domain should be
considered as a single functional unit.
While studying effects of genistein on partially phosphorylated CFTR, we also uncovered a gating mechanism for partially phosphorylated CFTR. Our previous
studies have provided experimental evidence for regulation of CFTR gating by multiple PKA phosphorylation sites (Hwang et al., 1993). The data suggest that
when CFTR channels are partially phosphorylated, the
channel open time is shortened. Thus, it is proposed
that stronger phosphorylation promotes longer channel opening through modulating the function of NBF2
(Hwang et al., 1994
). However, those studies do not
rule out other mechanisms of CFTR modulation through
differential phosphorylation. By using different experimental strategies, we demonstrate that when CFTR channels are partially phosphorylated, the closed time
is prolonged. Since the opening rate is determined
mainly by ATP hydrolysis at NBF1, this result suggests
the presence of phosphorylation site(s) that control
the Po through modulating the function of NBF1. Furthermore, this prolonged closed time of partially phosphorylated CFTR is not affected by increasing [ATP]
from 0.5 to 2.0 mM. Since [ATP] <2 mM is already a
saturating concentration for CFTR gating (Weinreich
et al., 1997
), the step(s) that are effected by phosphorylation of those sites is likely beyond the initial binding of
ATP to the ATP-binding pocket. Our results on partially
phosphorylated CFTR also agree with the kinetic studies
on mutant CFTR where some of the PKA consensus
serines are converted to alanines (Hanrahan, J.W., personal communication). Although regulation of CFTR activity via multiple phosphorylation sites is very complicated, both electrophysiological and biochemical results
suggest that phosphorylation in the R domain likely affects the biochemical reactions at both NBFs.
Effects of Genistein on CFTR Phosphorylation/Dephosphorylation
Our results as well as others (Weinreich et al., 1997;
French et al., 1997
) demonstrate a direct interaction
between genistein and CFTR in excised inside-out
membrane patches. But can these effects of genistein
on CFTR gating quantitatively account for the potentiation action of genistein on cAMP-dependent CFTR
channel activity in cell-attached patches? When the
wild-type CFTR is strongly phosphorylated with saturating concentrations of forskolin (e.g., 10 µM) in NIH3T3
cells or Calu-3 cells, genistein increases forskolin-
dependent CFTR channel currents by approximately
threefold (Hwang et al., 1997
). Genistein causes an
~40-fold increase in the forskolin-dependent CFTR
channel activity in Hi-5 insect cells transiently expressing wild-type CFTR (Yang et al., 1997
). In Hi-5 insect
cells, when phosphatases 1 and 2A are inhibited (Fig. 1
A), an approximately threefold enhancement is seen
with genistein. In excised patches, however, genistein
increases ATP-dependent CFTR activity by 2-10-fold,
depending on the phosphorylation status of the channel. In addition to these quantitative discrepancies in results obtained from electrophysiological experiments, it is also notable that genistein increases the
steady state phosphorylation level of CFTR (Reenstra et
al., 1996
). Thus it is likely that genistein also somehow
affects the equilibrium of phosphorylation/dephosphorylation of CFTR. Since genistein does not increase
cellular [cAMP] (nor does it stimulate PKA), it is not
likely that genistein can promote CFTR phosphorylation through PKA. One possibility, first raised in our
previous report (Hwang et al., 1997
), is that genistein binding to CFTR may inhibit the dephosphorylation reactions in the R domain. It is possible that genistein affects dephosphorylation of CFTR via binding to site(s)
other than NBFs. However, an alternative, perhaps
more interesting, possibility is that the effects of genistein on CFTR dephosphorylation are secondary to a conformational change caused by binding of genistein at
NBFs. For example, promoting the open state through
genistein's effects on NBFs may increase the steady
state phosphorylation level of CFTR if the open channel conformation has a slower dephosphorylation rate
than that of the closed state.
Physiological Significance
Our results provide evidence for a direct action of
genistein on CFTR. One would predict that genistein
should enhance cAMP-dependent CFTR activity in all
systems expressing CFTR if the CFTR protein is the direct target for genistein. Indeed, genistein has been shown to potentiate cAMP-dependent CFTR channel
activity in several heterologous expression systems. These
include Xenopus oocytes (Weinreich et al., 1997), Hi-5
insect cells (Yang et al., 1997
), HEK293 cells (Hwang et
al., 1997
), and NIH3T3 cells (Illek et al., 1995
; Reenstra
et al., 1996
; Yang et al., 1997
). Genistein also enhances
cAMP-dependent CFTR channel activity in cells that express endogenous wild-type CFTR, such as Calu-3 cells
(Hwang et al., 1997
), T-84 cells (Sears et al., 1995
), and
shark rectal glands (Lehrich and Forrest, 1995
). It is
therefore likely that genistein would also be effective in
epithelial cells in vivo. Furthermore, since genistein, at
low concentrations, prolongs channel open time, we
predict that genistein may have therapeutic value in
that it may enhance channel activity of disease-associated CFTR mutants that exhibit partial function.
![]() |
FOOTNOTES |
---|
Address correspondence to Dr. Tzyh-Chang Hwang, Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO 65211. Fax: 573-884-4232; E-mail: dcrctch{at}showme.missouri.edu
Received for publication 29 July 1997 and accepted in revised form 16 January 1998.
Luis Reuss served as guest editor on this article.We thank Dr. Elmer M. Price for providing baculovirus containing CFTR cDNA.
This work was supported by the National Institutes of Health (HL-53445 and DK-51767), the Cystic Fibrosis Foundation, and the American Heart Association, Missouri Affiliate. F. Wang is a recipient of the Molecular Biology Postdoctoral Fellowship at the University of Missouri-Columbia.
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