1 Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-0641; and 2 Laboratoire Jean Maetz, Commissariat a l'Energie Atomique-Centre National de la Recherche Scientifique, 06230 Villefranche Sur Mer, France
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ABSTRACT |
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Several proteins belonging to the ATP-binding cassette
superfamily can affect ion channel function. These include the cystic fibrosis transmembrane conductance regulator, the sulfonylurea receptor, and the multidrug resistance protein P-glycoprotein (MDR1).
We measured whole cell swelling-activated
Cl currents
(ICl,swell) in
parental cells and cells expressing wild-type MDR1 or a
phosphorylation-defective mutant (Ser-661, Ser-667, and Ser-671
replaced by Ala). Stimulation of protein kinase C (PKC) with a phorbol
ester reduced the rate of increase in
ICl,swell only in
cells that express MDR1. PKC stimulation had no effect on steady-state
ICl,swell.
Stimulation of protein kinase A (PKA) with 8-bromoadenosine
3',5'-cyclic monophosphate reduced steady-state ICl,swell only in
MDR1-expressing cells. PKA stimulation had no effect on the rate of
ICl,swell
activation. The effects of stimulation of PKA and PKC on
ICl,swell were
additive (i.e., decrease in the rate of activation and reduction in
steady-state
ICl,swell). The effects of PKA and PKC stimulation were absent in cells expressing the
phosphorylation-defective mutant. In summary, it is likely that
phosphorylation of MDR1 by PKA and by PKC alters swelling-activated Cl
channels by independent
mechanisms and that Ser-661, Ser-667, and Ser-671 are involved in the
responses of
ICl,swell to
stimulation of PKA and PKC. These results support the notion that MDR1
phosphorylation affects
ICl,swell.
multidrug resistance protein; adenosine 5'-triphosphate-binding cassette proteins; cystic fibrosis transmembrane conductance regulator; sulfonylurea receptor; chloride channels
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INTRODUCTION |
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THE ATP-BINDING CASSETTE (ABC) superfamily of membrane proteins includes proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR), the sulfonylurea receptor (SUR), and the multidrug resistance protein P-glycoprotein (MDR1). Most members of the ABC superfamily expressed in mammalian cells have two membrane-spanning regions, each followed by a nucleotide binding fold (27). A linker region that is a phosphorylation target in CFTR [regulatory (R) domain; see Ref. 16] and MDR1 (mini-linker domain; see Ref. 19) joins the two membrane spanning region-nucleotide binding domain tandems. The CFTR R domain is required for proper CFTR regulation by protein kinases (11, 16).
MDR1 is a plasma membrane ATPase that extrudes seemingly unrelated hydrophobic compounds from the cell interior, thus conferring resistance to a large variety of drugs (19). In addition to its pump function, MDR1 may also serve as a membrane transport regulator or modifier. A number of studies suggest that MDR1 expression alters membrane transport processes, as evidenced by changes in intracellular pH (4, 38, 43), cell membrane depolarization (25, 47), and an increase in Na+ channel activity (49).
Some members of the ABC family have been proposed to regulate the
activity of ion channels and other membrane proteins (e.g., CFTR, MDR1)
or form part of ion channels as subunits (e.g., SUR). CFTR is an ion
channel (5, 6) that has also been implicated in the regulation of both
outwardly rectifying Cl
channels and epithelial Na+
channels (39, 41). Activation of protein kinase A (PKA) has been shown
to activate outwardly rectifying
Cl
channels and inhibit
epithelial Na+ channels only in
the presence of CFTR (14, 41). SUR seems to be a subunit of
ATP-sensitive K+ channels
[KATP; formed by SUR and
inwardly rectifying K+ channels
(Kir)]. SUR confers
sulfonylurea sensitivity to Kir and is involved in the response to nucleotides (1). Several studies
have shown a relationship between swelling-activated
Cl
currents
(ICl,swell) and
MDR1 expression (2, 32, 35, 45, 46). Most importantly, protein kinase C
(PKC)-mediated phosphorylation of MDR1 was shown to either inhibit
ICl,swell (24) or
reduce its activation rate (7). Although recent studies contradict these observations (35), Hardy et al. (24) showed that mutation of all
consensus PKC phosphorylation sites in the MDR1 mini-linker domain
abolished the effect of phosphorylation. However, only Ser-661,
Ser-667, and Ser-671 of the eight Ser and Thr in the MDR1 mini-linker
domain are phosphorylated by PKC (9, 19). PKA phosphorylates two of the
PKC sites, Ser-667 and Ser-671, and also Ser-683, a site not
phosphorylated by PKC (9, 19). There is no information on a possible
role of regulation of
ICl,swell by
PKA-mediated phosphorylation of MDR1. With respect to a possible regulation of drug transport, the current evidence does not support a
major role of MDR1 phosphorylation (18, 20). However, an increase in
the apparent affinity to some drugs (i.e., verapamil, vinblastine, and
rhodamine 123) of the drug-stimulated ATPase activity by MDR1
phosphorylation has been suggested (42).
The aims of this study were to determine whether 1) activation of PKC alters ICl,swell in MDR1-expressing cells, 2) stimulation of PKA affects ICl,swell in MDR1-expressing cells, and 3) Ser-661, Ser-667, and/or Ser-671 of MDR1 are required for the effects of PKA and PKC stimulation on ICl,swell.
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MATERIALS AND METHODS |
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General procedures. Mouse fibroblast cells (BALB/c-3T3; a gift from Dr. E. B. Mechetner) were grown in DMEM supplemented with 10% fetal bovine serum (GIBCO, Rockville, MD) and 1% (vol/vol) streptomycin-penicillin (1 unit penicillin-1 µg streptomycin; GIBCO) at 37°C in 5% CO2. The cells transfected with MDR1 cDNA (BALB-MDR1) and MDR1-3SA cDNA (BALB-MDR1-3SA) were grown in the continuous presence of the antibiotic geneticin (G418; GIBCO) at a concentration of 600 µg/ml.
Generation of MDR1- and MDR1-3SA-expressing clones.
BALB/c-3T3 cells were transfected with either wild-type MDR1 or mutant
MDR1 (MDR1-3SA) cDNA in the vector pLK444M. This vector contains a
-actin promoter and was derived from pLK444 (a gift of Dr. P. Melera; see Ref. 12). Site-directed mutagenesis was used to substitute
Ser-661, Ser-667, and Ser-671 with Ala, and both MDR1 and MDR1-3SA
contain six histidine residues at the COOH-terminal end. This results
in a modified MDR1 that has drug-stimulated ATPase activity and
transports drugs (Ref. 31; see Fig. 1). Detailed
information on the cDNA engineering and development of the cell lines
is described elsewhere (A. Castro, J. Horton, and G. Altenberg,
unpublished observations). Functional expression of MDR1 in the plasma
membrane was determined by measuring the efflux of the MDR1 substrate
rhodamine 123, as previously described (3, 46).
Electrophysiology. BALB/c-3T3, BALB-MDR1, and BALB-MDR1-3SA were detached the day of the experiment by exposure to a Ca2+-free PBS (in mM: 137 NaCl, 8 sodium phosphate, 1.5 potassium phosphate, and 2.7 KCl, pH ~7.4) containing 0.5 mM EDTA. The cells were allowed to recover for ~30 min at room temperature (22-23°C) in HEPES-buffered solution (HBS; in mM: 135 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 7.8 glucose, and 5 HEPES-NaOH, pH 7.4, osmolality ~280 mosmol/kg). The cells were detached because when grown at low confluence they spread out and their thinness makes them difficult to patch. Detachment of MDR1-expressing BALB/c-3T3 cells did not affect MDR1 functional expression in the plasma membrane, i.e., rhodamine 123 efflux was similar in attached and detached cells (data not shown). The isolated cells were placed in a plastic chamber (volume ~700 µl) and allowed to settle to the bottom and attach (~20 min). The chamber was mounted on the stage of an inverted microscope (Nikon Diaphot, Nikon, Tokyo, Japan).
Whole cell recordings were performed at room temperature while the cells were superfused at 2-3 ml/min with the experimental solutions in an ~500-µl-volume chamber. The pipette and bath solutions [isosmotic (ISO) and 22% hyposmotic (HYPO)] were designed to have ClCell volume measurements. Detached BALB-MDR1 cells (see above) were placed on glass coverslips mounted in a Leiden microincubator (Medical Systems, Greenvale, NY). The cells were loaded for ~1 h with 15 µM 5-chloromethylfluorescein diacetate (CMFDA; Molecular Probes, Eugene, OR; a fluorescent dye not transported by MDR1; see RESULTS) at room temperature in HBS. After CMFDA loading, the cells were placed on the stage of an inverted microscope (Nikon Diaphot) coupled to a confocal laser scanning video system (Odyssey, Noran Instruments, Middleton, WI). Superfusion with CMFDA-free ISO solution (see above for composition) was initiated, and cell fluorescence was measured (excitation at 495 nm, emission at >535 nm). After recording of cell fluorescence for 6-8 min under basal conditions, the superfusate was changed to the experimental solution, i.e., ISO plus 200 nM phorbol 12-myristate 13-acetate (PMA). Changes in cell fluorescence due to changes in cell volume were calibrated by exposing control cells to either 11% NMDG-Cl HYPO solution (NMDG-Cl was reduced by 17.5 mM; ~245 mosmol/kg) or 11% NMDG-Cl HYPER (sucrose was added to NMDG-Cl ISO to obtain ~305 mosmol/kg). An increase in cell fluorescence denotes an increase in CMFDA concentration due to cell shrinkage, and a fall in cell fluorescence denotes cell swelling.
Data are presented as means ± SE. Statistical differences were calculated using Student's t-test for paired or unpaired data, as appropriate, and were considered significant at P < 0.05 (two-tailed analysis). ![]() |
RESULTS |
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BALB/c-3T3 mouse cells transfected with wild-type or mutant MDR1
cDNA express functional MDR1 in the plasma membrane.
We chose to carry out the experiments on MDR1-expressing cell lines
that had never been exposed to chemotherapeutic agents to exclude the
possibility that exposure to chemotherapeutic agents, and not MDR1
expression itself, could account for any of the unique properties of
these cells (32, 33). Cell lines expressing either wild-type MDR1 or
mutant MDR1, without selection with chemotherapeutic agents, were
generated by transfecting BALB/c-3T3 cells with MDR1 cDNA (BALB-MDR1
cells) or MDR1-3SA cDNA (BALB-MDR1-3SA). Both BALB-MDR1 and
BALB-MDR1-3SA cells displayed significant rhodamine 123 unidirectional
efflux, whereas the rhodamine 123 efflux from the parental cells was
~10-fold slower than that of the MDR1-expressing BALB/c-3T3 cells
(Fig. 1). The rhodamine 123 efflux denotes MDR1-mediated transport, as previously shown (3). Both
wild-type and mutant MDR1-expressing cell lines have equivalent levels
of expression of MDR1 and MDR1-3SA in the plasma membrane, and MDR1,
but not MDR1-3SA, is phosphorylated in vitro by PKC (A.F. Castro, J.K. Horton, C.G. Vanoye, and G.A. Altenberg, unpublished observations). In
addition, we previously showed that MDR1 expression is undetectable in
the parental cells (8, 23). Figure 1 shows that BALB-MDR1 and
BALB-MDR1-3SA cells express functional MDR1 at the plasma membrane, whereas BALB/c-3T3 does not.
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Expression of MDR1 or MDR1-3SA does not alter the magnitude,
activation rate, or current-voltage
(I-V)
relationship of ICl,swell
in BALB/c-3T3 cells.
Representative records of
Cl currents under basal
conditions (ISO) and after activation by cell swelling (HYPO) are shown in Fig. 2. Whole cell
Cl
currents were measured 2 min after breaking of the seal in ISO solution and then 6 min after
exposure to HYPO solution. Figures 2 and 3
show that the three cell lines exhibited sizable swelling-activated Cl
currents with similar
outward rectification. The ratio of the absolute currents measured at
40 ms, at +80 and
80 mV, used to assess the degree of
rectification, was not significantly different among all cell lines and
was on average 1.25 ± 0.02 (n = 35). The three cell
lines showed reversibility of
ICl,swell when
the bath solution was changed back to ISO (data not shown). Figure 4 shows the swelling-activated currents
measured, as a function of time, in parental and in MDR1- and
MDR1-3SA-expressing cells. As seen in Fig. 4, expression of either
wild-type or mutant MDR1 did not alter the activation rate of
ICl,swell in
BALB/c-3T3 cells. Figures 2-4 show that expression of MDR1 or
MDR1-3SA does not alter the magnitude, activation rate, or
I-V
relationship of
ICl,swell in
BALB/c-3T3 cells. Moreover,
ICl,swell under
control conditions (absence of PKA and PKC stimulation) is similar in
MDR1- and MDR1-3SA-expressing cells.
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Inhibition of ICl,swell by activation of PKC and PKA. Protein kinase stimulation was carried out by exposure to either 200 nM PMA (a membrane-permeant PKC activator) or 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP; a membrane-permeant PKA activator). These concentrations are sufficient to attain maximal stimulation (26).
Figure 5A shows the swelling-activated Cl
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PKA and PKC affect ICl,swell by different mechanisms. Recently, phosphorylation of MDR1 by PKC was shown to reduce the activation rate of ICl,swell following exposure to hypotonic solutions (7). However, other investigators have not found this effect (35). After examination of the time course of ICl,swell activation, we noticed that inhibition of the current by PKC activation was stronger at early times after reduction of the bath osmolality. This observation suggested that phosphorylation of MDR1 could alter the activation rate of ICl,swell but not its final magnitude. To test this possibility, we exposed BALB-MDR1 cells treated with either PMA or 8-BrcAMP to HYPO solution for a longer period. We stopped acquiring data 12-14 min after switching the bath to HYPO because membrane blebs appeared in the patched cells after that time, suggesting uncoupling of the membrane from the cytoskeleton. Figure 6A shows that phosphorylation of MDR1 by PKC slows down the activation of ICl,swell without altering its steady-state value. In contrast, activation of PKA reduced the steady-state level of ICl,swell in BALB-MDR1 cells without modifying the activation rate (Fig. 6B). As shown in Fig. 6C, the addition of both PMA and 8-BrcAMP generated a "mixed" effect, i.e., both activation rate and final magnitude of ICl,swell were reduced. In summary, PKA and PKC stimulation produced distinct effects on ICl,swell, and these effects were abolished by replacing Ser-661, Ser-667, and Ser-671 with Ala residues.
The delay of ICl,swell activation in BALB-MDR1 by PKC stimulation is not caused by cell shrinkage. One possible explanation for the delay in the activation of ICl,swell in MDR1-expressing cells treated with PMA is that MDR1 phosphorylation causes a decrease in cell volume upon stimulation of PKC. This putative reduction in cell volume could explain the delay in the onset of ICl,swell, i.e., exposure to HYPO during the first ~2 min would return the cell volume to the level before PMA exposure, without activation of ICl,swell. To test this possibility, changes in cell volume were monitored before and during exposure to 200 nM PMA using the fluorescent dye CMFDA as a marker for cell water volume. Pilot experiments showed that the decrease in fluorescence during the time course of the studies was due to photobleaching of the fluorophore (9 ± 1%, n = 30), with negligible probe efflux in both BALB/c-3T3 and BALB-MDR1 cells (data not shown). These observations indicate that the CMFDA-glutathione conjugate is not an MDR1 substrate. The conjugate could be a substrate for the multidrug resistance associated protein (MRP1), but BALB/c-3T3 cells are devoid of MRP1 (8). Fluorescence of the CMFDA-glutathione conjugate was monitored in cells exposed to either ISO or ISO plus PMA for 10 min. Fluorescence, normalized to the average value in ISO after correction for photobleaching, was 1.00 ± 0.01 in ISO (n = 22) and 0.99 ± 0.02 in ISO plus PMA (n = 8). These results show that PMA did not elevate cell fluorescence (as expected for cell shrinkage), thus ruling out a decrease in cell volume as the mechanism for the delay in ICl,swell activation. The validity of the fluorescent probe-based methods to assess changes in cell volume has been previously established (44) and was confirmed by measuring CMFDA fluorescence in control cells exposed to anisotonic solutions. Exposure to 11% HYPO reduced cell fluorescence by 12 ± 3% (n = 15), and exposure to 11% HYPER increased cell fluorescence by 11 ± 5% (n = 6).
Inhibition of ICl,swell
in BALB-MDR1 cells by PKA activation is not due to ATP release.
It has been suggested that ABC proteins can regulate other transporters
by providing a pathway for efflux of ATP, which can activate or inhibit
other transporters via activation of purinergic receptors (reviewed in
Ref. 11). In this context, phosphorylation of CFTR by PKA activates
outwardly rectifying Cl
channels via ATP efflux (39). Because external ATP reduces the
magnitude of
ICl,swell in
several cell lines (40), including BALB-MDR1 cells (Fig.
7A), it
is then possible that phosphorylation of MDR1 by PKA causes ATP release
and this extracellular ATP subsequently reduces the magnitude of
ICl,swell. To
test this possibility, BALB-MDR1 cells were exposed to 8-BrcAMP in the
continuous extracellular presence of hexokinase (0.5 U/ml; Sigma, St.
Louis, MO), glucose, and Mg2+.
Under these conditions, hexokinase catalyzes the phosphorylation of
glucose by ATP, thus scavenging any secreted ATP. In CFTR-expressing cells, this experimental maneuver has been shown to prevent the activation of outwardly rectifying
Cl
channels via purinergic
receptors (39). Figure 7B shows
ICl,swell measured at +80 mV in BALB-MDR1 cells both under control conditions and
in the presence of 8-BrcAMP or 8-BrcAMP plus hexokinase. Addition of
external hexokinase did not block the PKA-induced effect. This proves
that external ATP is not involved in the inhibition of ICl,swell by PKA
in BALB-MDR1 cells. Addition of external hexokinase alone did not
affect ICl,swell
in BALB-MDR1 cells.
ICl,swell
measured at +80 mV, 6 min after exposure to HYPO bath, was 83 ± 25 pA/pF (n = 5) in control conditions
and 122 ± 10 pA/pF (n = 5) in the presence of hexokinase.
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DISCUSSION |
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Current evidence indicates that expression of MDR1 regulates or
modifies swelling-activated
Cl channels (7, 24, 35, 45,
46). The main results supporting such a role for MDR1 are
1) the modulation of the volume
sensitivity and/or the activation rate of swelling-activated
Cl
channels by MDR1
expression (7, 32, 35, 45) and 2)
the inhibition of
ICl,swell or a
decrease in its activation rate caused by PKC activation in MDR1 (and
mdr1a)-expressing cells (7, 24). The PKC effect was abolished by
mutating all the consensus PKC phosphorylation sites in the mini-linker
domain of MDR1 to Ala (24), indicating that phosphorylation of MDR1 is
part of the mechanisms by which PKC activation affects
ICl,swell. The present results provide additional evidence that phosphorylation of
MDR1, not MDR1 expression per se, alters the activity of
swelling-activated Cl
channels.
It has been shown by several investigators that expression of MDR1 alters the activation rate of ICl,swell by either increasing it (7, 32, 35, 45) or decreasing it (33) and that this effect is reversed by PKC activation (7, 45). However, MDR1 expression does not always alter the activation rate of ICl,swell (Ref. 15; see Fig. 4). It is possible that the rate at which ICl,swell is activated in MDR1-expressing cells, vis à vis the parental, non-MDR1-expressing cells, is dependent on experimental factors that include the way cells are prepared for the experimental procedure (23) and exposure of MDR1-expressing cells to chemotherapeutic agents (32, 33).
In our hands, activation of PKC by PMA affects the activation rate of
ICl,swell in an
MDR1-expressing cell line, not its final magnitude, thus confirming
observations by Bond et al. (7). We also demonstrated that
phosphorylation of residues Ser-661, Ser-667, and Ser-671 in the MDR1
mini-linker domain is sufficient for the effect of PKC stimulation.
Moreover, because our cell lines were never exposed to chemotherapeutic
drugs, our results rule out a nonspecific effect of PKC related to cell
exposure to those agents (33). However, the effects of MDR1
phosphorylation on swelling-activated
Cl channels are
controversial because other investigators did not find any effects of
PKC stimulation on
ICl,swell in
MDR1-expressing cells (35). Among the possible explanations for this
discrepancy are the following. 1)
The data shown by Miwa et al. (35) were obtained at steady state, when
no effect is expected (see Fig. 4).
2) The MDR1-dependent effects of PKC
stimulation on swelling-activated Cl
channels may well vary
from cell to cell, depending on the presence and location of specific
PKC isoenzymes as well as on the specific molecule underlying
ICl,swell. In
this context, although ClC-3 has been tentatively identified as a
swelling-activated Cl
channel (13), it seems that
ICl,swell is
underlain by different channels in different cells (46), and it is
possible that MDR1 may influence only some of these channels. The
observations on the association of
Kir channels with SUR support
this hypothesis. SUR1 has been shown to modify the function of some
(e.g., Kir 6.1, Kir 6.2) but not all (e.g.,
Kir 2.1, Kir 4.3)
Kir channels (1).
The present results demonstrate for the first time that stimulation of
PKA affects swelling-activated
Cl channels selectively in
MDR1-expressing cells. Exposure to a membrane-permeable analog of cAMP
reduced the magnitude of
ICl,swell elicited by hyposmotic swelling. This effect was observed only in the
cells expressing MDR1 and was abolished by substitution of Ser-661,
Ser-667, and Ser-671 of MDR1 with Ala. The results in the parental
cells devoid of MDR1 are in agreement with the notion that the PKA
pathway does not regulate swelling-activated Cl
channels (36). In
contrast, it has been shown that activation of myocardial
ICl,swell is
blocked by PKA-mediated phosphorylation (21). It has been proposed (36)
that such a mechanism could form part of a negative feedback of
ICl,swell
activation because some cells accumulate cAMP during swelling (48). The
possibility that MDR1 expressed in the myocardium (17) is responsible
for the modulation of
ICl,swell remains
to be explored.
Interestingly, our results show that the MDR1-related effects of PKA and PKC on ICl,swell are different. PKA stimulation reduces steady-state ICl,swell, whereas PKC stimulation reduces the rate of activation of the currents by swelling, without affecting their steady-state level. Moreover, the effects of PKA and PKC stimulation are different and additive (reduction of speed of response plus decrease in steady-state currents). The latter result indicates that the two effects are independent. It has been shown that MDR1 is phosphorylated by PKC at Ser-661, Ser-667, and Ser-671 (probably at the same residues in vitro and in vivo; Refs. 9, 19), whereas PKA phosphorylation occurs at Ser-667, Ser-671, and Ser-683 (only in vitro data are available; see Ref. 19). Because the effects of stimulation of PKA and PKC are different and additive and require phosphorylation of Ser-661, Ser-667, and/or Ser-671, it is likely that phosphorylation of Ser-661 is involved in the response to PKC stimulation and that Ser-683 is involved in the response to PKA stimulation. However, additional mutagenesis studies are required to confirm this hypothesis.
Other ABC proteins, besides MDR1, regulate ion channels. CFTR is a
Cl channel that regulates
outwardly rectifying Cl
channels, epithelial Na+ channels,
and KATP channels (14, 34, 41),
whereas SUR regulates certain Kir
channels (1). There is little information on the mechanism by which ABC
proteins alter the activity of ion channels. ATP secretion has been
implicated in the regulation of outwardly rectifying
Cl
channels by CFTR (39),
but ATP secretion dependent on CFTR is controversial (29, 37), and it
cannot explain our results. External ATP blocks
ICl,swell, and
ATP scavenging by hexokinase had no effect on the
ICl,swell
reduction by PKA stimulation. Direct interaction between CFTR and the
-subunit of the epithelial Na+
channel has been suggested to mediate the inhibition of
Na+ channels by phosphorylation of
CFTR by PKA (28). The relationship between SUR and
Kir also supports protein-protein
interaction as the mechanism for regulation by ABC proteins. It has
been established that the effects of sulfonylureas (inhibition) and
Mg-ADP (stimulation) on Kir
channels are mediated by SUR. SUR probably mediates the effects of
sulfonylureas and Mg-ADP on Kir
channels via protein-protein interaction, because SUR and
Kir channels form functional
oligomers (KATP channels), likely
consisting of four Kir and four
SUR molecules (1). The precise mechanism of alteration in
swelling-activated Cl
channels by phosphorylation of MDR1 is unknown. MDR1-associated transport of substrates with stimulatory or inhibitory effects on
Cl
channels is unlikely
because the magnitude of
ICl,swell is
independent of MDR1 expression (see Ref. 46). Efflux of ATP can also be ruled out from our studies. On the basis of the available information on the effects of CFTR and SUR on ion channels, we favor the hypothesis that the regulation of swelling-activated
Cl
channels mediated by
MDR1 phosphorylation is via protein-protein interaction.
Our conclusions are as follows. 1)
MDR1 phosphorylation alters the function of swelling-activated
Cl channels. This
modulation could depend on the cell type, e.g., differences in
expression and compartmentalization of PKC isoenzymes and different
Cl
channels underlying
ICl,swell.
2) The effects of PKA and PKC on
swelling-activated Cl
channels by phosphorylation of MDR1 require phosphorylation of one or
more of Ser-661, Ser-667, and Ser-671. These residues are located in
the MDR1 mini-linker domain (10), homologous to the R domain of CFTR
(the R domain is a target for modulation of CFTR activity by
phosphorylation; see Ref. 11). 3)
The effects of PKA and PKC are different and additive. Stimulation of
PKC reduces the rate of the increase in
ICl,swell
following exposure to hyposmotic solution, whereas stimulation of PKA
reduces the magnitude of ICl,swell,
without affecting the speed of the response.
4) The simplest explanation for our
results is that phosphorylation of Ser-661 is involved in the response
to PKC stimulation, whereas Ser-683 is involved in the response to PKA
stimulation. This conclusion is based on the observations that Ser-667
and Ser-671 are phosphorylated by both PKA and PKC, that PKA also
phosphorylates Ser-683, and that PKC also phosphorylates Ser-661.
The recent observation that both SUR and CFTR increase the sensitivity
of KATP to sulfonylureas suggests
that several members of the ABC superfamily can associate with the same
channels (e.g., KATP channels).
Therefore, understanding how MDR1 phosphorylation affects
swelling-activated Cl
channels may provide useful information on the mechanism(s) by which
ABC proteins modulate ion channels.
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ACKNOWLEDGEMENTS |
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We thank Drs. S. A. Weinman and J.-T. Zhang for comments on a preliminary version of this paper and K. Spilker for technical assistance. BALB/c-3T3 cells were generously provided by Dr. E. Mechetner. The vector pLK444 was a gift of Dr. P. Melera.
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FOOTNOTES |
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This work was supported by a grant from Searle Research and Development, a grant-in-aid from the American Heart Association (Texas Affiliate), and National Institutes of Health Grants CA-72783 and DK-08865.
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.
Address for reprint requests: G. A. Altenberg, Dept. of Physiology and Biophysics, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0641.
Received 21 September 1998; accepted in final form 2 November 1998.
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