1 Department of Physiology, McGill University, Montréal, Québec H3G 1Y6; and 2 Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 0W0
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ABSTRACT |
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Genistein and bromotetramisole (Br-t) strongly activate cystic fibrosis transmembrane conductance regulator (CFTR; ABCC7) chloride channels on Chinese hamster ovary cells and human airway epithelial cells. We have examined the possible role of phosphatases in stimulation by these drugs using patch-clamp and biochemical methods. Genistein inhibited the spontaneous rundown of channel activity that occurs after membrane patches are excised from cAMP-stimulated cells but had no effect on purified protein phosphatase type 1 (PP1), PP2A, PP2B, PP2C, or endogenous phosphatases when assayed as [32P]PO4 release from prelabeled casein, recombinant GST-R domain fusion protein, or immunoprecipitated full-length CFTR. Br-t also slowed rundown of CFTR channels, but, in marked contrast to genistein, it did inhibit all four protein phosphatases tested. Half-maximal inhibition of PP2A and PP2C was observed with 0.5 and 1.5 mM Br-t, respectively. Protein phosphatases were also sensitive to (+)-p-Br-t, a stereoisomer of Br-t that does not inhibit alkaline phosphatases. Br-t appeared to act exclusively through phosphatases since it did not affect CFTR channels in patches that had low apparent endogenous phosphatase activity (i.e., those lacking spontaneous rundown). We conclude that genistein and Br-t act through different mechanisms. Genistein stimulates CFTR without inhibiting phosphatases, whereas Br-t acts by inhibiting a membrane-associated protein phosphatase (probably PP2C) that presumably allows basal phosphorylation to accumulate.
cystic fibrosis; phosphorylation; PP2C; (iso)flavonoids; phenylimidazothiazoles; cystic fibrosis transmembrane conductance regulator
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INTRODUCTION |
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THE CYSTIC FIBROSIS transmembrane conductance regulator (CFTR; ABCC7-human gene nomenclature committee) chloride channel is activated by phosphorylation (6, 43) and is gated by hydrolyzable nucleotides (1, 2, 30). Phosphorylation by protein kinase A (PKA) provides the primary stimulus for channel activation (10, 11, 39). Protein kinase C phosphorylation modulates responsiveness of the channel to PKA (26, 31, 34, 43). Less is known regarding the dephosphorylation of CFTR, although it is equally important for controlling CFTR activity under physiological conditions. A membrane-associated phosphatase reduces the open probability in cell-attached membrane patches and causes CFTR channels to run down rapidly when excised from cAMP-stimulated Chinese hamster ovary (CHO), airway epithelial, or human colon cells (4, 16, 32, 43, 44). Protein phosphatases type 2A and especially 2C (PP2A and PP2C, respectively) have been implicated in regulating CFTR (7, 19, 32, 43, 45). CFTR and PP2C are specifically coimmunoprecipitated by antibodies against the other protein, and PP2C can be copurified by nickel chelate chromatography of histidine-tagged CFTR, suggesting that they form a stable regulatory complex (52).
The (iso)flavonoid genistein has attracted interest because of its ability to stimulate CFTR channels, including those with disease-causing mutations (14, 21, 23, 24, 41, 42, 50, 51). In most preparations, genistein stimulation requires elevated PKA activity. Genistein stimulation is associated with increased phosphorylation of tryptic peptides, which also become phosphorylated during forskolin stimulation (41). These data are consistent with phosphatase inhibition, although recent patch-clamp and biochemical studies indicate that activation of genistein is at least partially independent of phosphatase and demonstrate direct interaction of genistein with the channel (12, 14, 36, 40, 47-49).
Wild-type and mutant CFTR channels in transfected cells are also
activated by phenylimidazothiazoles such as
()-p-bromotetramisole (Br-t) and levamisole, which are
well-known inhibitors of liver, bone, and kidney (LBK) alkaline
phosphatases. Although most recent data suggest that CFTR is regulated
by protein phosphatases (notably PP2C and PP2A) rather than by an
alkaline phosphatase, the sensitivities of protein phosphatases to Br-t
have not been reported. Consequently, it remains unclear whether
phenylimidazothiazoles could act through inhibition of protein
phosphatases. Understanding how genistein and Br-t stimulate CFTR
should clarify aspects of its regulation and could be relevant to the
design of pharmacotherapies for cystic fibrosis.
In this study, we show that genistein and Br-t activate CFTR channels through distinct mechanisms. Genistein does not inhibit protein phosphatases, whereas both enantiomers of Br-t inhibit protein phosphatases at concentrations found previously to activate CFTR channels, and this probably accounts for their stimulatory effect.
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MATERIALS AND METHODS |
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Cell culture. CHO cells stably expressing wild-type CFTR were plated at low density on glass coverslips 3-5 days before patch-clamp experiments. Baby hamster kidney (BHK) cells stably expressing wild-type CFTR were plated in 10-cm-diameter plates for use in immunoprecipitations. Cells were grown at 37°C with 5% CO2 in modified minimal essential medium (Life Technologies) containing 5% dialyzed FBS.
Patch-clamp studies. CHO cells were placed in a recording chamber (200 µl) containing (in mM) 150 NaCl, 2 MgCl2, and 10 N-[Tris(hydroxymethyl)methyl]-2-aminoethane-sulfonic acid (TES), pH 7.4. In most experiments, this solution also contained 0.5 mM Mg2+-ATP. Single-channel currents were recorded from cell-attached and excised patches as described previously (32) and were analyzed using DRSCAN, a pCLAMP-compatible program for analyzing long records (15).
Phosphatases.
Recombinant PP1 was kindly provided by Dr. P. T. W. Cohen,
University of Dundee. PP2A and PP2C
were prepared from turkey gizzard smooth muscle, as described previously (37,
38). PP2B was from Calbiochem (La Jolla, CA).
GST-R domain.
cDNA corresponding to amino acids M645-M837 was amplified using
Vent polymerase and PCR primers that contained BamH I sites for subcloning into the pGEX-2T vector. Kozak consensus sequences were
also added for future studies involving expression in mammalian cells. The pGEX-2T-R domain plasmid was confirmed by DNA
sequencing. HB101 cells were transformed and grown to late log
phase at 30°C. GST-R domain fusion protein expression was induced for
2-3 h with 0.1 mM isopropyl
-D-thiogalactopyranoside. GST-R domain was
purified on glutathione-Sepharose 4B (Sigma) and eluted by addition of 20 mM glutathione at 4°C according to the manufacturer's instructions.
[32P]PO4 labeling of casein or GST-R
domain.
Casein (10 mg; Sigma) or GST-R domain fusion protein (200 µg) was
labeled by incubation with PKA (600 units; see Ref. 43 for details) and
[-32P]ATP (50 µCi; Amersham) in 1 ml reaction buffer
containing 50 mM Tris·HCl, 0.1 mM EGTA, 10 mM magnesium acetate, and
0.1%
-mecaptoethanol (pH = 7.0) for 5-12 h at 30°C. The
reaction was stopped by adding 100 µl of solution containing 100 mM
EDTA and 100 mM sodium pyrophosphate. Free [
-32P]ATP
was removed by passing the reaction mix through Sepharose G-50
(Pharmacia Biotech, Sweden) that had been preequilibrated with 50 mM
Tris·HCl, 0.1 mM EGTA, 5% glycerol, and 0.1%
-mercaptoethanol (pH = 7.0). To measure radioactivity of the purified substrates, 100 µl TCA (20% wt/vol) were added to 5- or 10-µl aliquots and centrifuged at 10,000 rpm for 1 min at 4°C, and the supernatant was
counted by liquid scintillation. The amount of free
[
-32P]ATP was always <1% of the total radioactivity.
Dephosphorylation of [32P]PO4-casein
and [32P]PO4-GST-R domain fusion protein.
Release of [32P]PO4 from prelabeled casein by
PP1 and PP2A was assayed in duplicate as follows. Purified phosphatase
(20 µl) was added to 10 µl Tris buffer containing phosphorylated
substrate (prepared as described above). The final assay solution
contained 100 mM NaCl, 1.34 mM MgCl2, 33 mM Tris-Cl, pH
7.0, and 0.5-40 nM phosphatase as indicated. The mixture was
incubated at 22°C, and the reaction was stopped at timed intervals by
the addition of 20% TCA. Samples were centrifuged for 2 min at 4,000 rpm at 4°C, and the supernatant was taken for liquid scintillation
counting to determine the amount of free
[32P]PO4 released from casein. Spontaneous
release of [32P]PO4 from phosphocasein was
low in the absence of phosphatase (typically <1%) and was subtracted
from the total quantity released with phosphatase present. The same
assay solution was supplemented with 1 mM Ca2+, 2 mM
Mn2+, and 10 µg/ml calmodulin (Boehringer Mannheim) when
measuring PP2B activity. For PP2C assays, 10 mM MgCl2 was
added to the solution described for PP1 and PP2A. Dephosphorylation of
[32P]PO4-GST-R fusion protein was carried out
under similar conditions except the reaction was terminated by adding
SDS sample buffer (3% SDS, 5% -mercaptoethanol, 10% glycerol, and
62.5 mM Tris · HCl, pH 6.8), and samples were subjected to
SDS-PAGE (12%) and autoradiography.
Dephosphorylation of immunoprecipitated (full-length) CFTR.
After cells were lysed with 1% Triton X-100, 1% deoxycholic acid,
0.1% SDS, 150 mM NaCl, 20 mM Tris · HCl (pH = 8), 0.25 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and 2 µg/ml aprotinin (RIPA buffer), CFTR was immunoprecipitated on protein G-Sepharose 4B beads using the monoclonal antibody M3A7
(27). CFTR was phosphorylated while still on the beads by
incubation with 180 nM PKA catalytic subunit, 20 µM ATP, 10 µCi
[-32P]ATP, 10 µg BSA, 140 mM NaCl, 4 mM KCl, 2 mM
MgCl2, 0.5 mM CaCl2, and 10 mM Tris, pH 7.4, for 1 h at 22°C. The reaction was stopped as before, and the
beads were carefully washed with RIPA buffer to remove PKA and then
incubated in buffer (140 mM NaCl and 10 mM TES) containing 2 nM PP2A
and 2 mM MgCl2 or, alternatively, 10 nM PP2C and 10 mM
MgCl2. Immunoprecipitates from ~1.5 plates (10 cm2) of confluent BHK cells overexpressing wild-type CFTR
were used in each experiment. The reaction was stopped by adding sample loading buffer. Samples were subjected to 6% SDS-PAGE and
autoradiography to assess the amount of
[32P]PO4 remaining on CFTR. Aliquots were
taken from the same immunoprecipitated sample when examining
dephosphorylation under various test conditions, and identical volumes
were used in each lane during SDS-PAGE. This procedure provided
consistent loading, as indicated by several experiments in which
Western blots were carried out on the same nitrocellulose membranes
used for autoradiography (e.g., see Fig. 4B).
Preparation of CHO cell fractions for phosphatase assays. Cells were lysed by sonication in 2× lysis buffer containing 100 mM Tris · HCl, pH 7.4, 4 mM EDTA, 4 mM EGTA, 2% wt/vol Nonidet P-40, and protease inhibitors. The insoluble fraction was removed by centrifugation at 3,000 g for 15 min to remove unbroken cells and nuclei. The supernatant was centrifuged at 7,800 g for 10 min to pellet the mitochondrial fraction, which was washed with lysis buffer, resuspended in 2× buffer, and mixed with an equal volume of 100% glycerol. The supernatant was centrifuged again at 100,000 g for 60 min. The pellet from this spin (crude membrane fraction) was treated as described for the mitochondrial fraction. The supernatant was taken as the cytosolic fraction and was mixed with an equal volume of 100% glycerol.
Statistics. Values are presented as means ± SE. Significance was assessed at the 95% confidence level using a Student's t-test.
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RESULTS |
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Effect of genistein on CFTR channel rundown.
Rapid deactivation of CFTR channels in excised patches can be reversed
by exposure to PKA and therefore reflects dephosphorylation of PKA
sites by a robust, membrane-associated phosphatase activity (43). If genistein stimulates CFTR at the level of
phosphatases, it would be expected to slow the rundown of channel
activity in excised patches (e.g., see Ref. 4). To test this
prediction, patches were excised from CHO cells that had been
prestimulated using 10 µM forskolin. Under control conditions,
channel activity declined by >90% within 100 s after excision in
bath solution containing 1 mM Mg2+-ATP (Fig.
1A), as described previously
(4, 43). However, if genistein (50 µM) was
added to the bath once forskolin-stimulated channel activity had
increased to a stable level, then CFTR channels remained active for >5
min after excision (Fig. 1B). Genistein is a well-known
tyrosine kinase inhibitor; however, tyrosine kinases were probably not
involved in prolonging channel activity in isolated patches. Another
broad-range tyrosine kinase antagonist, erbstatin (IC50 = 780 nM), had no effect on rundown when used at
the same concentration (Fig. 1C), nor did two other
inhibitors (tyrphostin A47 and lavendustin A; data not shown).
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Effect of genistein on activities of purified PP2A and PP2C.
To determine if genistein directly inhibits protein phosphatases
implicated in CFTR regulation, we assayed the effect of genistein on
PP2A and PP2C. The amount of each phosphatase used in this and
subsequent experiments was chosen based on their activities in
preliminary experiments. Under control conditions, purified PP2A (0.5 nM) and PP2C (2 nM) both caused significant
[32P]PO4 release from radiolabeled casein
within 10 min (7%-17%, Fig. 2,
A and B). Genistein (50 µM) had no effect on
PP2A-catalyzed release, which was inhibited ~90% by calyculin A (10 nM) as expected, nor on the residual 10% phosphatase activity
remaining in the presence of calyculin A. PP2C-catalyzed
[32P]PO4 release from phosphocasein was also
genistein insensitive (Fig. 2B), although it was inhibited
by 90% when free Mg2+ concentration ([Mg2+])
was reduced (i.e., in nominally Mg2+-free solution
containing 10 mM EGTA). Finally, to determine if genistein alters the
time course of dephosphorylation, we compared [32P]PO4 release by PP2C at eight different
time points. Genistein (50 µM) did not affect PP2C-catalyzed
dephosphorylation at any time during the 30-min period (Fig.
2C).
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Effect of genistein on protein phosphatase activity in cell
fractions.
Although most studies suggest that CFTR is regulated by PP2C- and
PP2A-like phosphatases, particular isoforms of these enzymes or
unidentified endogenous phosphatases could potentially be sensitive to
genistein and could mediate its stimulatory action in intact cells. We
therefore examined the effect of genistein on phosphatase activities in
different CHO cell fractions (crude membrane extract, cytosol, and
mitochondria). Under control conditions, all three cell fractions
caused significant [32P]PO4 release from
phosphocasein after 10 min of exposure at 22°C, but none was affected
by genistein (50 µM; Fig. 3). Thus we
could find no evidence for genistein-sensitive protein phosphatases in
CHO cells.
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Effect of genistein on dephosphorylation of GST-R domain fusion protein and immunoprecipitated CFTR. The experiments described above indicate that genistein slows channel rundown in excised patches without directly inhibiting protein phosphatases. We considered another possibility; i.e., that genistein inhibits dephosphorylation at the level of CFTR by making it a less effective substrate for phosphatases. Because most phosphorylation of CFTR is on the R domain, we first examined dephosphorylation of the GST-R domain fusion protein.
GST-R was radiolabeled, and its dephosphorylation was assessed by autoradiography as described in MATERIALS AND METHODS. The entire fusion protein was used since the R domain portion contains >97% of the radioactive phosphate after incubation with PKA and [
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Effect of Br-t on CFTR channel rundown.
The alkaline phosphatase inhibitor ()-p-Br-t (2 mM) also
slowed deactivation of CFTR channels in excised patches (Fig.
5). Channel activity was sustained for
>5 min in seven out of seven patches, although it eventually declined
to zero, consistent with previous results (4). However,
the stereoisomer (+)-p-Br-t, which is often used as a
negative control when assaying alkaline phosphatases, also slowed
rundown. These data are consistent with recent patch-clamp studies
(28) and provide further evidence that channel
deactivation is not mediated by one of the "LBK" alkaline
phosphatases, which are specifically inhibited by (
)-Br-t.
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Effect of Br-t on purified PP2A and PP2C.
Because PP2C and PP2A are the most likely candidates to be CFTR
phosphatases, we investigated their sensitivities to Br-t using the
protocol described above for genistein. As shown in Fig.
6A, both enantiomers reduced
PP2A (0.5 nM) activity by >90%. Similar results were obtained in PP2C
(2 nM) assays, although inhibition by (+)-Br-t was somewhat stronger.
Some dephosphorylation occurred even at the highest Br-t concentrations
used, which probably explains why Br-t slows deactivation approximately
fourfold in patch-clamp experiments but does not completely abolish
rundown (4).
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Effect of Br-t on dephosphorylation of CFTR and GST-R domain fusion
protein.
The ability of Br-t to inhibit dephosphorylation could depend on the
particular substrate used in the assay; therefore, we examined its
effect on dephosphorylation of both GST-R domain fusion protein and
full-length CFTR. GST-R and immunoprecipitated CFTR were
incubated with PKA and [-32P]ATP and then were exposed
to purified PP2A (2 nM) or PP2C (10 nM) in the absence or presence of
Br-t, as described in MATERIALS AND METHODS. Br-t strongly
inhibited dephosphorylation of GST-R by all four types of protein
phosphatase under these conditions (Fig.
8). Dephosphorylation of full-length CFTR
by PP2A (2 nM) and PP2C (10 nM) was also inhibited by both enantiomers
(Fig. 9). To compare the concentration
dependence of Br-t inhibition more quantitatively, the IC50
for inhibition of PP2C by each enantiomer was assessed using
radiolabeled GST-R as the substrate. As shown in Fig.
10A, elevating (
)-Br-t
concentration caused progressively stronger inhibition of PP2C,
yielding an IC50 ~0.5 mM and maximal inhibition at 2 mM.
Similar results were obtained with (+)-p-Br-t, although the
IC50 was somewhat lower (0.2 mM; Fig. 10B).
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DISCUSSION |
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Only a few potent activators of the CFTR channel have been described to date, and their mechanisms of action remain obscure. In this paper, we have examined the role of protein phosphatases in stimulation by two of these, genistein and Br-t. In addition to activating wild-type CFTR in epithelial cells, both drugs have been shown to stimulate channels with disease-associated mutations. Genistein potentiates forskolin activation of CFTR that have the most common CF mutation, a deletion of phenylalanine 508 (21), whereas Br-t activates several mutants, including G551D, when cells have only basal PKA activity (4).
Mechanisms of CFTR activation by genistein. Early evidence for genistein stimulation of CFTR came from studies of NIH/3T3 cells transfected with CFTR (24). Addition of genistein alone stimulated 125I efflux and CFTR channel activity in cell-attached patches, and these responses were blocked by the tyrosine phosphatase inhibitor orthovanadate. Genistein did not elevate cAMP but rather decreased the concentration of forskolin required for half-maximal stimulation. Similar results were obtained with shark rectal glands (SRG) and primary cell cultures derived from SRG (29). The T84 cell line also responded to tyrphostin-47, another tyrosine kinase inhibitor, strengthening the idea that CFTR channels are tonically inhibited by tyrosine kinases (42); however, subsequent studies indicated that genistein has some other mode of action. The tyrosine kinase inhibitors erbastatin, herbimycin A, tyrphostin-51, and tyrphostin-47 did not mimick genistein stimulation of CFTR channels in other cell types (12, 23, 49). Moreover, subsequent studies with the tyrosine kinase src revealed that tyrosine phosphorylation stimulates rather than inhibits channel activity. Exposing CFTR to the tyrosine kinase p60c-src potentiated stimulation by PKA (13) or strongly activated CFTR channels when added alone (25). The latter stimulation was associated with phosphorylation of tyrosines on CFTR.
Genistein has been proposed to stimulate CFTR by inhibiting protein phosphatases that normally counteract its activation by PKA. This hypothesis was supported by the finding that genistein's effect on CFTR channels in NIH/3T3 and Hi-5 insect cells was greatly enhanced by cAMP stimulation (41, 50). Genistein exposure increased CFTR phosphorylation in vivo, as assessed by metabolic labeling of cells with [32P]PO4. Subsequent digestion by trypsin, SDS-PAGE, and TLC yielded phosphopeptide maps during genistein treatment that resembled those during forskolin stimulation (41). Because genistein does not stimulate PKA, these results were most easily explained by inhibition of a CFTR phosphatase. Sustained CFTR-mediated currents across permeabilized T84 and HT-29/B6 cell monolayers after cAMP washout provided further support for the phosphatase hypothesis (23). Most recently, genistein has been shown to increase the mean open time of single CFTR channels in patches with little phosphatase activity (22). Moreover, repeated genistein stimulations were obtained in the absence of PKA, which argues against a mechanism that requires phosphorylation/dephosphorylation (49). Genistein further increased the activity of channels that had been stimulated by PKA and Mg2+-adenosine 5'-O-(3-thiotriphosphate), although the thiophosphoryl moiety would be expected to resist phosphatases (14). On the basis of these findings, it was proposed that genistein alters CFTR gating by directly interacting with the channel, perhaps at a nucleotide binding fold (48). Indeed, direct interaction of genistein with a fusion protein composed of the second nucleotide binding fold (NBF2) and maltose binding protein, and ATPase inhibition, have recently been demonstrated (40). This is compatible with long open times, since inhibitors of hydrolysis at NBF2 are thought to stabilize the open state of the channel (20). Genistein stimulation of CFTR by such a mechanism contrasts with that proposed for multidrug resistance-associated protein (MRP1), another ATP-binding cassette transport protein (18). Genistein is thought to stimulate ATPase activity of MRP1 by interacting at the substrate binding site for drug transport rather than a NBF. There is also evidence that genistein acts on CFTR preferentially from the extracellular side (51). Regardless of the site of interaction, direct binding of genistein to CFTR would not preclude other modes of action such as phosphatase inhibition (17, 48).Genistein does not inhibit protein phosphatases or dephosphorylation of immunoprecipitated CFTR. The present results argue strongly against a role of phosphatases in the activation of CFTR by genistein. Genistein slowed channel rundown in excised patches but did not inhibit any of the purified protein phosphatases tested (PP1, PP2A, PP2B, and PP2C), nor did it affect the endogenous phosphatase activities of cell fractions. We could find no evidence that the ability of CFTR to serve as a phosphatase substrate was affected by genistein; however, extrapolating from immunoprecipitated CFTR protein to native membrane is problematic because genistein could inhibit dephosphorylation through some affect on channel gating that is disrupted by solubilization. In preliminary experiments, we found that genistein alters the digestion pattern of CFTR produced by limited proteolysis with chymotrypsin (data not shown), further evidence for genistein inducing a conformational change (40). Although a secondary (gating-dependent) effect on phosphatase sensitivity cannot be excluded, the present data are most consistent with stimulation via direct binding of genistein to CFTR and stabilization of the open state.
Mechanism of CFTR activation by Br-t.
Br-t has long been known as a potent and specific inhibitor of
particular types of alkaline phosphatases (8). LBK
alkaline phosphatase activities are abolished by 0.1 mM
()-p-Br-t, whereas intestinal and placental alkaline
phosphatases are not inhibited significantly (46). The
stereoisomer (+)-p-Br-t does not inhibit alkaline
phosphatases and is widely used as an inactive analog. In previous
work, we found that (
)-Br-t inhibits CFTR rundown in patches from CHO
and human airway epithelial cells [NP34 (4, 5)] and also activates quiescent G551D mutant channels
that are normally unresponsive to forskolin. Dephosphorylation of CFTR in crude CHO cell membrane preparations was also sensitive to millimolar concentrations of (
)-Br-t. Inhibition of rundown by 1 mM
(+)-Br-t was not detected; however, the effects of this enantiomer on
dephosphorylation of CFTR protein were not assessed.
Direct evidence that Br-t stimulates CFTR by inhibiting
phosphatases.
Phenylimidazothiazoles (0.1-2 mM) stimulate iodide efflux and
activate single CFTR channels in CFTR-expressing CHO and airway cells
without elevating cAMP (4, 5). ()-Br-t also
stimulates CFTR conductance in other preparations (28,
35), although the response seems somewhat weaker [e.g.,
15% of the forskolin response in permeabilized T84 cell monolayers
(35)]. Such variations would be expected if Br-t
stimulation is indirect and mediated by inhibition of phosphatase(s) as
originally proposed (4), since the responsiveness of
different preparations would depend on their basal PKA activity, which
in turn would reflect adenylyl cyclase and phosphodiesterase activities
and perhaps other factors. The present results support the proposal
that Br-t stimulates channel activity by inhibiting CFTR
dephosphorylation, since it inhibited all four major types of protein
phosphatases examined.
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ACKNOWLEDGEMENTS |
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We thank X.-B. Chang and J. Riordan, Mayo Clinic Scottsdale, for providing BHK cell lines and T. Jensen, N. Kartner, and J. Riordan for providing M3A7 anti-CFTR antibody. We also thank J. Liao for maintaining the cultures.
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FOOTNOTES |
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This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (to J. W. Hanrahan) and the Medical Research Council of Canada (MRC; to M. D. Pato). J. Luo and T. Zhu received a studentship and postdoctoral fellowship from the Canadian Cystic Fibrosis Foundation, respectively. J. W. Hanrahan is a senior scientist of the MRC.
Address for reprint requests and other correspondence: J. W. Hanrahan, Dept. of Physiology, McGill Univ., 3655 Drummond St., Montréal, Québec, Canada H3G 1Y6 (E-mail: hanrahan{at}med.mcgill.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 5 October 1999; accepted in final form 20 January 2000.
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