Phosphorylation of P-glycoprotein by PKA and PKC
modulates swelling-activated Cl2 currents
CARLOS G. VANOYE,1 ARIEL F. CASTRO,1 THIERRY POURCHER,2
LUIS REUSS,1 AND GUILLERMO A. ALTENBERG1
1Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas
77555-0641; and 2Laboratoire Jean Maetz, Commissariat a l’Energie Atomique-Centre
National de la Recherche Scientifique, 06230 Villefranche Sur Mer, France
Vanoye, Carlos G., Ariel F. Castro, Thierry Pourcher,
Luis Reuss, and Guillermo A. Altenberg. Phosphorylation
of P-glycoprotein by PKA and PKC modulates swelling-
activated Cl2 currents. Am. J. Physiol. 276 (Cell Physiol. 45):
C370–C378, 1999.—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 resis-
tance protein P-glycoprotein (MDR1). We measured whole
cell swelling-activated Cl2 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-bromoad-
enosine 38,58-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 mu-
tant. In summary, it is likely that phosphorylation of MDR1
by PKA and by PKC alters swelling-activated Cl2 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 58-triphosphate-
binding cassette proteins; cystic fibrosis transmembrane con-
ductance regulator; sulfonylurea receptor; chloride channels
THE ATP-BINDING CASSETTE (ABC) superfamily of mem-
brane 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 mem-
bers of the ABC superfamily expressed in mammalian
cells have two membrane-spanning regions, each fol-
lowed 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 do-
main; see Ref. 19) joins the two membrane spanning
region-nucleotide binding domain tandems. The CFTR
The costs of publication of this article were defrayed in part by the
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C370 0363-6143/99 $5.00 Copyright r 1999 the American Physiological Society
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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 regula-
tor 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
Na1 channel activity (49).
Some members of the ABC family have been pro-
posed 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 Cl2 channels
and epithelial Na1 channels (39, 41). Activation of
protein kinase A (PKA) has been shown to activate
outwardly rectifying Cl2 channels and inhibit epithe-
lial Na1 channels only in the presence of CFTR (14, 41).
SUR seems to be a subunit of ATP-sensitive K1 chan-
nels [KATP; formed by SUR and inwardly rectifying K1
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 Cl2 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 phosphoryla-
tion 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 phosphory-
lation has been suggested (42).
The aims of this study were to determine whether 1)
activation of PKC alters ICl,swell in MDR1-expressing
 REGULATION OF CL2 CHANNELS BY P-GLYCOPROTEIN
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.
MATERIALS AND METHODS
General procedures. Mouse fibroblast cells (BALB/c-3T3; a
gift from Dr. E. B. Mechetner) were grown in DMEM supple-
mented 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 b-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 observa-
tions). Functional expression of MDR1 in the plasma mem-
brane 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 Ca21-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% hypos-
motic (HYPO)] were designed to have Cl2 as the main
conductive ion. Their chemical compositions (in mM) were:
ISO, 140 N-methyl-D-glucammonium chloride (NMDG-Cl),
1.3 CaCl2, 0.5 MgCl2, 10 HEPES, and 7.8 glucose, pH ,7.4,
,275 mosmol/kg; HYPO, same as ISO, except that NMDG-Cl
was reduced to 105 mM (,215 mosmol/kg); pipette solution,
140 NMDG-Cl, 1.2 MgCl2, 10 HEPES, 2 ATP, and 1 EDTA, pH
,7.0, ,270 mosmol/kg. To prevent cell swelling and activa-
tion of ICl,swell in ISO, the pipette solution was diluted 5–10%
with distilled water. The glass pipettes were pulled with a
multistage P-87 Flaming-Brown micropipette puller (Sutter
Instruments, San Rafael, CA) and fire polished. Pipette tip
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C371
resistances were 3–5 MV (nearly symmetrical NMDG-Cl
solutions in the pipette and bath).
After obtaining a 5-GV seal between the pipette and the
cell membrane, the patch was ruptured by applying negative
pressure or negative pressure plus a large voltage pulse (1.2 V
for 0.5–3 ms). Currents were measured in the whole cell
configuration by the patch-clamp technique (22) using an
Axopatch 200A amplifier (Axon Instruments, Foster City,
CA), sampled at 20 kHz, and filtered at 2 or 5 kHz. The
holding voltage was 0 mV. The Cl2 currents were measured
from 280 to 180 mV at 20-mV steps, 40 ms after the start of
the voltage pulse, as indicated. Pulse generation and data
collection and analyses were performed using pCLAMP 6
(Axon Instruments). The measured currents are expressed
relative to apparent membrane capacitance. The access resis-
tance and apparent membrane capacitance were estimated as
described by Lindau and Neher (30). Access resistance values
ranged from 7 to 10 MV, and the apparent membrane
capacitances were 31 6 1 pF for BALB/c-3T3 cells (n 5 35),
30 6 1 pF for BALB-MDR1 cells (n 5 58), and 29 6 2 pF for
BALB-MDR1-3SA cells (n 5 42).
Cell 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-chloromethylfluo-
rescein 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 superfus-
ate 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 cali-
brated 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 6 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
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 experi-
ments 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 rhoda-
C372 REGULATION OF CL2 CHANNELS BY P-GLYCOPROTEIN

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.
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 38,58-cyclic mono-
phosphate (8-BrcAMP; a membrane-permeant PKA
activator). These concentrations are sufficient to attain
maximal stimulation (26).
Figure 5A shows the swelling-activated Cl2 cur-
rents measured at 180 and 280 mV in BALB/c-3T3,
Fig. 1. Functional expression of multidrug resistance protein (MDR1; BALB-MDR1, and BALB-MDR1-3SA cells under con-
P-glycoprotein) in BALB-MDR1 and BALB-MDR1-3SA cells. Cells trol conditions and after PKC activation. The cells were
were preloaded with rhodamine 123, and decay in intracellular exposed to ISO plus PMA for 8–10 min before the whole
rhodamine 123 fluorescence (FR123 ) was measured following superfu- cell configuration was obtained. About 2 min after the
sion with rhodamine 123-free solution. a.u., Arbitrary units. After
removal of rhodamine 123, intracellular fluorescence follows a single- breaking of the seal, the bath solution was changed to
exponential decay as a function of time. Mouse cells transfected with HYPO plus PMA for ,6 min. Activation of PKC by
human MDR1 cDNA, either MDR1 (BALB-MDR1) or MDR1-3SA exposure to PMA reduced ICl,swell only in the MDR1-
(BALB-MDR1-3SA), display significant rhodamine 123 efflux, whereas expressing BALB-MDR1 cells, i.e., PMA did not affect
efflux in parental cells (no detectable MDR1 expression) was .10-fold
slower. Each trace is representative of 6 similar experiments.
ICl,swell in either BALB/c-3T3 or BALB-MDR1-3SA cells.
These results indicate that MDR1 expression is re-
mine 123 unidirectional efflux, whereas the rhodamine quired for the inhibitory effect of PKC on ICl,swell. In
123 efflux from the parental cells was ,10-fold slower addition, one or more of Ser-661, Ser-667, and Ser-671
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.
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. Representa-
tive records of Cl2 currents under basal conditions
(ISO) and after activation by cell swelling (HYPO) are
shown in Fig. 2. Whole cell Cl2 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 Cl2 currents with similar outward
rectification. The ratio of the absolute currents mea-
sured at 40 ms, at 180 and 280 mV, used to assess the
degree of rectification, was not significantly different
Fig. 2. Whole cell Cl2 currents in parental cells and MDR1-
among all cell lines and was on average 1.25 6 0.02 transfected cells. Representative traces of Cl2 currents (expressed
(n 5 35). The three cell lines showed reversibility of relative to apparent membrane capacitance) measured in isotonic
ICl,swell when the bath solution was changed back to ISO (ISO; 2 min after breaking patch) and hypotonic (HYPO; 6 min after
(data not shown). Figure 4 shows the swelling-acti- lowering bath osmolality) bath solutions. Currents were measured in
whole cell configuration by patch-clamp technique. Holding voltage
vated currents measured, as a function of time, in was 0 mV, and whole cell currents were measured 40 ms after onset of
parental and in MDR1- and MDR1-3SA-expressing voltage pulses of amplitude ranging from 280 to 180 mV at 20-mV
cells. As seen in Fig. 4, expression of either wild-type or steps. Data are from 6–12 experiments.

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 REGULATION OF CL2 CHANNELS BY P-GLYCOPROTEIN C373

Fig. 3. Expression of either MDR1 or MDR1-3SA does not alter swelling-activated Cl2 currents (ICl,swell ) in
BALB/c-3T3 cells. Current-voltage (I-V) relationships for whole cell currents measured at voltages ranging from
280 to 180 mV at 20-mV steps, 40 ms from start of voltage pulse in ISO (2 min after breaking patch) and HYPO (6
min after lowering bath osmolality) are shown. Data are means 6 SE of currents (expressed relative to estimated
cell membrane capacitance; n 5 4–12) plotted as function of holding voltage.

are required for this inhibition. Because Ser-667 and
Ser-671 are also substrates for PKA, we tested whether
PKA activation reproduces the PKC effect. Figure 5B
shows ICl,swell measured at 180 and 280 mV in BALB/c-
3T3, BALB-MDR1, and BALB-MDR1-3SA cells under
control conditions and after PKA activation, by a proto-
col similar to that used to study the effect of PKC
stimulation but with 8-BrcAMP instead of PMA. The
results show that activation of PKA inhibits ICl,swell in
BALB/c-3T3 cells expressing MDR1 but not in the
parental or mutant MDR1-expressing cells. Hence,
inhibition of ICl,swell in BALB/c-3T3 cells by either PKA
or PKC activation requires MDR1 expression, and
Ser-661, Ser-667, and/or Ser-671 are required for this
effect. In addition, because PKA and PKC have Ser-667
and Ser-671 as common substrates, one or both resi-
dues are essential for the inhibition of ICl,swell by MDR1
phosphorylation.
Fig. 4. Activation rate of ICl,swell in BALB/c-3T3 cells is not affected by
expression of either MDR1 or MDR1-3SA. Whole cell currents
measured at 180 mV, 40 ms after voltage pulse, are plotted as a
function of time after lowering of bath osmolality. Bath solution was
changed to HYPO at time 0. Data are means 6 SE of currents
(expressed relative to estimated cell membrane capacitance; n 5
10–25).
The effects of both PKA and PKC stimulation were
always present but were somewhat variable in magni-
tude when experiments performed months apart were
compared (e.g., compare the decreases in the rate of
ICl,swell activation by PMA at 6 min in Figs. 5 and 6). For
this reason, experimental and control studies were
always contemporaneous.
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 expo-
sure to hypotonic solutions (7). However, other investi-
gators have not found this effect (35). After examina-
tion 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 phosphory-
lation 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 uncou-
pling 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 re-
duced 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 activa-
tion 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 resi-
dues.

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C374 REGULATION OF CL2 CHANNELS BY P-GLYCOPROTEIN
Fig. 5. Activation of protein kinase C (PKC) by phorbol 12-myristate
13-acetate (PMA) and activation of protein kinase A (PKA) by
8-bromoadenosine 38,58-cyclic monophosphate (8-BrcAMP) inhibit
ICl,swell only in cells expressing wild-type MDR1. A: cells were exposed
to ISO 1 200 nM PMA for 6–10 min before whole cell configuration
was obtained and continuously thereafter. ICl,swell values were ob-
tained 6 min after lowering of bath osmolality. B: cells were exposed
to ISO 1 1 mM 8-BrcAMP for 6–10 min before whole cell configura-
tion was obtained and continuously thereafter. ICl,swell values at 280
and 180 mV are shown for both control cells and 8-BrcAMP-exposed
cells. Data are means 6 SE (n 5 4–13) of currents measured at 280
mV (downward bars) and 180 mV (upward bars), 40 ms after start of
voltage pulse, and expressed relative to estimated cell membrane
capacitance. * P , 0.05 compared with control.
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
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volume upon stimulation of PKC. This putative reduc-
tion 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 6 1%, n 5
30), with negligible probe efflux in both BALB/c-3T3
and BALB-MDR1 cells (data not shown). These observa-
tions indicate that the CMFDA-glutathione conjugate
is not an MDR1 substrate. The conjugate could be a
Fig. 6. PKA and PKC have different effects on ICl,swell in MDR1-
expressing cells. A: activation of PKC reduced rate of activation of
ICl,swell but not its steady-state value. B: activation of PKA reduced
steady-state current levels. C: exposure of BALB-MDR1 cells to both
agents simultaneously generated a ‘‘mixed’’ effect, i.e., onset of
current was delayed and final current was reduced. Experimental
protocol was similar to that used for Fig. 3, but cells were maintained
in HYPO solution for 8–14 min (A and C) or 8–12 min (B). Data are
means 6 SE (n 5 4–10) of currents measured at 180 mV, 40 ms after
start of voltage pulse, and are normalized to ICl,swell measured at
8 min under control conditions. Bath osmolality was reduced at
time 0. * P , 0.05 for difference between control and treatment at
same time point.
 REGULATION OF CL2 CHANNELS BY P-GLYCOPROTEIN C375

substrate for the multidrug resistance associated pro-

tein (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 photobleach-
ing, was 1.00 6 0.01 in ISO (n 5 22) and 0.99 6 0.02 in
ISO plus PMA (n 5 8). These results show that PMA did
not elevate cell fluorescence (as expected for cell shrink-
age), 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 aniso-
tonic solutions. Exposure to 11% HYPO reduced cell
fluorescence by 12 6 3% (n 5 15), and exposure to 11%
HYPER increased cell fluorescence by 11 6 5% (n 5 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 trans-
porters 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 Cl2 channels via ATP efflux (39).
Because external ATP reduces the magnitude of ICl,swell Fig. 7. Inhibition of ICl,swell in MDR1-expressing cells by PKA activa-
in several cell lines (40), including BALB-MDR1 cells tion is not due to ATP release. A: addition of external ATP (2 mM)
(Fig. 7A), it is then possible that phosphorylation of inhibits ICl,swell in BALB-MDR1 cells (28 6 4%, n 5 4). Data are
means 6 SE of currents measured at 180 mV, 40 ms after start of
MDR1 by PKA causes ATP release and this extracellu- voltage pulse, and are expressed as percentage of current measured
lar ATP subsequently reduces the magnitude of ICl,swell. before ATP addition. * P , 0.05 compared with control. B: activation
To test this possibility, BALB-MDR1 cells were exposed of ICl,swell in BALB-MDR1 cells under control conditions (n 5 4), after
to 8-BrcAMP in the continuous extracellular presence 1 mM 8-BrcAMP alone (n 5 4), or after 1 mM 8-BrcAMP 1 0.5 U/ml
of hexokinase (0.5 U/ml; Sigma, St. Louis, MO), glu- hexokinase (HK; n 5 3). Hexokinase does not alter PKA-induced
effect, suggesting that inhibition of ICl,swell is not due to ATP release.
cose, and Mg21. Under these conditions, hexokinase Data are means 6 SE of currents measured at 180 mV, 40 ms after
catalyzes the phosphorylation of glucose by ATP, thus start of voltage pulse, and are normalized to ICl,swell measured at
scavenging any secreted ATP. In CFTR-expressing cells, 8 min under control conditions. Bath osmolality was reduced at
this experimental maneuver has been shown to prevent time 0.
the activation of outwardly rectifying Cl2 channels via
purinergic receptors (39). Figure 7B shows ICl,swell mea-
sured at 180 mV in BALB-MDR1 cells both under activation rate caused by PKC activation in MDR1 (and
control conditions and in the presence of 8-BrcAMP or mdr1a)-expressing cells (7, 24). The PKC effect was
8-BrcAMP plus hexokinase. Addition of external hexo- abolished by mutating all the consensus PKC phosphor-
kinase did not block the PKA-induced effect. This ylation sites in the mini-linker domain of MDR1 to Ala
proves that external ATP is not involved in the inhibi- (24), indicating that phosphorylation of MDR1 is part of
tion of ICl,swell by PKA in BALB-MDR1 cells. Addition of the mechanisms by which PKC activation affects ICl,swell.
external hexokinase alone did not affect ICl,swell in The present results provide additional evidence that
BALB-MDR1 cells. ICl,swell measured at 180 mV, 6 min phosphorylation of MDR1, not MDR1 expression per se,
after exposure to HYPO bath, was 83 6 25 pA/pF (n 5 alters the activity of swelling-activated Cl2 channels.
5) in control conditions and 122 6 10 pA/pF (n 5 5) in It has been shown by several investigators that
the presence of hexokinase. expression of MDR1 alters the activation rate of ICl,swell
by either increasing it (7, 32, 35, 45) or decreasing it
DISCUSSION (33) and that this effect is reversed by PKC activation
(7, 45). However, MDR1 expression does not always
Current evidence indicates that expression of MDR1 alter the activation rate of ICl,swell (Ref. 15; see Fig. 4). It
regulates or modifies swelling-activated Cl2 channels is possible that the rate at which ICl,swell is activated in
(7, 24, 35, 45, 46). The main results supporting such a MDR1-expressing cells, vis à vis the parental, non-
role for MDR1 are 1) the modulation of the volume MDR1-expressing cells, is dependent on experimental
sensitivity and/or the activation rate of swelling- factors that include the way cells are prepared for the
activated Cl2 channels by MDR1 expression (7, 32, 35, experimental procedure (23) and exposure of MDR1-
45) and 2) the inhibition of ICl,swell or a decrease in its expressing cells to chemotherapeutic agents (32, 33).

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C376 REGULATION OF CL2 CHANNELS BY P-GLYCOPROTEIN
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 observa-
tions 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 Cl2 channels are controversial because other
investigators did not find any effects of PKC stimula-
tion 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 stimula-
tion on swelling-activated Cl2 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 Cl2 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 Cl2
channels selectively in MDR1-expressing cells. Expo-
sure to a membrane-permeable analog of cAMP re-
duced 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 Cl2 channels (36). In
contrast, it has been shown that activation of myocar-
dial ICl,swell is blocked by PKA-mediated phosphoryla-
tion (21). It has been proposed (36) that such a mecha-
nism 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 cur-
rents). 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-
Downloaded from journals.physiology.org/journal/ajpcell (037.239.094.088) on June 13, 2021.
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 stimula-
tion 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 Cl2 channel that regulates out-
wardly rectifying Cl2 channels, epithelial Na1 chan-
nels, and KATP channels (14, 34, 41), whereas SUR
regulates certain Kir channels (1). There is little infor-
mation on the mechanism by which ABC proteins alter
the activity of ion channels. ATP secretion has been
implicated in the regulation of outwardly rectifying Cl2
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 be-
tween CFTR and the a-subunit of the epithelial Na1
channel has been suggested to mediate the inhibition of
Na1 channels by phosphorylation of CFTR by PKA (28).
The relationship between SUR and Kir also supports
protein-protein interaction as the mechanism for regu-
lation by ABC proteins. It has been established that the
effects of sulfonylureas (inhibition) and Mg-ADP (stimu-
lation) on Kir channels are mediated by SUR. SUR
probably mediates the effects of sulfonylureas and
Mg-ADP on Kir channels via protein-protein interac-
tion, 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 Cl2 channels by phos-
phorylation of MDR1 is unknown. MDR1-associated
transport of substrates with stimulatory or inhibitory
effects on Cl2 channels is unlikely because the magni-
tude 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
Cl2 channels mediated by MDR1 phosphorylation is via
protein-protein interaction.
Our conclusions are as follows. 1) MDR1 phosphory-
lation alters the function of swelling-activated Cl2
channels. This modulation could depend on the cell
type, e.g., differences in expression and compartmental-
ization of PKC isoenzymes and different Cl2 channels
underlying ICl,swell. 2) The effects of PKA and PKC on
swelling-activated Cl2 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
 REGULATION OF CL2 CHANNELS BY P-GLYCOPROTEIN
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 mag-
nitude 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 in-
volved in the response to PKA stimulation. This conclu-
sion 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 sug-
gests 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 Cl2 channels may provide
useful information on the mechanism(s) by which ABC
proteins modulate ion channels.
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.
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.
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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