(Received for publication, September 25, 1995; and in revised form, November 20, 1995)
From the
Cl and cation conductances were characterized
in zymogen granules (ZG) isolated from the pancreas of wild-type mice
(+/+) or mice with a homozygous disruption of the multidrug
resistance P-glycoprotein gene mdr1a (-/-).
Cl
conductance of ZG was assayed in isotonic KCl
buffer by measuring osmotic lysis, which was induced by maximal
permeabilization of ZG membranes (ZGM) for K
with
valinomycin due to influx of K
through the artificial
pathway and of Cl
through endogenous channels. To
measure cation conductances, ZG (pH
6.0-6.5)
were suspended in buffered isotonic monovalent cation acetate solutions
(pH 7.0). The pH gradient was converted into an outside-directed
H
diffusion potential by maximally increasing
H
conductance of ZGM with carbonyl cyanide m-chlorophenylhydrazone. Osmotic lysis of ZG was induced by
H
diffusion potential-driven influx of monovalent
cations through endogenous channels and nonionic diffusion of the
counterion acetate. ZGM Cl
conductances were not
different in (-/-) and (+/+) mice (2.6 ±
0.3 h
versus 3.1 ± 0.2
h
(relative rate constant)). The nonhydrolyzable ATP
analog adenosine 5`-(
,
-methylene)triphosphate (AMP-PCP) (0.5
mM) activated the Cl
conductance both in
(+/+) and (-/-) mice. However, activation of
Cl
conductance by AMP-PCP was reduced in
(-/-) mice as compared with (+/+) mice (5.0
± 0.4 h
versus 7.6 ± 0.7
h
; p < 0.005). In contrast, ZGM
K
conductance was increased in (-/-) mice
as compared with (+/+) mice (14.2 ± 2.0 h
versus 8.5 ± 1.2 h
; p < 0.03). In the presence of 0.5 mM AMP-PCP, which
completely blocks K
conductance but leaves a
nonselective cation conductance unaffected, there was no difference
between (-/-) and (+/+) mice (5.3 ± 0.7
h
versus 3.2 ± 0.5
h
). In Western blots of ZGM from wild-type mice, a
polyclonal MDR1 specific antibody labeled a protein band of
80 kDa.
In mdr1a-deficient mice, the intensity of this band was
reduced to 39 ± 7% of the wild-type signal. This indicates that
a mdr1a gene product of
80 kDa enhances the
AMP-PCP-activated fraction of mouse ZGM Cl
conductance and reduces AMP-PCP-sensitive K
conductance.
Most members of the superfamily of ATP binding cassette (ABC) ()transporter proteins act as pumps carrying substrates as
diverse as large hydrophobic drugs, small anions, or peptides across
membranes at the cost of ATP hydrolysis. Two members of the ABC
transporter superfamily, however, have properties of Cl
channels and are also modulated by ATP: the cystic fibrosis
transmembrane conductance regulator (CFTR) and the multidrug resistance
P-glycoprotein (MDR1). CFTR is responsible for cAMP-activated
epithelial Cl
secretion or reabsorption (1) and functions as a Cl
channel that is
activated by protein kinase A- and protein kinase C-mediated
phosphorylation(2) , but activation also requires ATP binding
and hydrolysis(3) . P-glycoproteins can confer multidrug
resistance by actively extruding structurally unrelated, amphiphilic,
and hydrophobic drugs from the cells(4) . Recently, Higgins and
co-workers (5, 6, 11) found that MDR1
overexpression in different cell lines correlates with the appearance
of a volume-regulated Cl
current. The MDR1 associated
Cl
current was induced by swelling (5) and
required allosteric interaction with ATP or nonhydrolyzable ATP analogs
for activation(6) . However, the role of MDR1 as a
swelling-activated Cl
channel has been
challenged(7, 8, 9, 10) . Recent
evidence suggests that MDR1 is not the Cl
channel,
but a regulator of a latent channel protein(11) . Thus, MDR1
appears to be multifunctional, associated with both transporter and
channel regulator activities.
Pancreatic acinar cells secrete NaCl,
fluid, and digestive enzymes upon stimulation by secretagogues. In
permeabilized pancreatic acini, we have demonstrated that enzyme
secretion evoked by the Ca and cAMP signaling
pathways depends on the ionic environment of the secretory granules,
since it required the presence of isosmotic Cl
and
K
in the medium and was abolished by application of
Cl
and K
channel blockers (12) . We therefore postulated that hormonally regulated
Cl
and K
selective channels are
present in the membrane of ZG. Upon fusion of ZG with the luminal
plasma membrane, the increased influx of salt and water through the
granule Cl
and K
channels would
promote enzyme secretion(13) . Subsequently, we have
demonstrated the presence of regulated ion conductance pathways in rat
pancreatic ZG membranes. A K
conductance is inhibited
by ATP and nonhydrolyzable ATP analogs and blocked by K
channel blockers, e.g. quinine and glibenclamide, in a
similar manner as ATP-sensitive K
channels(14) . A nonselective cation conductance is not
affected by adenine nucleotides and blocked by micromolar
concentrations of Ba
and also by flufenamic
acid(15) . A Cl
conductance is activated by
ATP and nonhydrolyzable ATP analogs and is blocked by
DIDS(16, 17) .
Recently, we have demonstrated that
two monoclonal antibodies against cytosolic epitopes of MDR1,
JSB-1(18) , and C219(19) , prevent activation of the
Cl conductance of ZG by the nonhydrolyzable ATP
analog AMP-PCP(15) . In immunoblots of rat ZG membranes, a
protein band of
65 kDa was labeled with both antibodies, but no
full sized mdr1 protein of 170-180 kDa was detected (15) . We proposed that the
65-kDa protein is the
Cl
channel of rat ZG membrane or a regulator of that
channel and hypothetized that the
65-kDa protein could represent a
truncated or alternatively spliced form of mdr1 or a separate member of
the ABC transporter superfamily with structural homology to mdr1.
In
the present study, we have investigated the effect of homologous
disruption of the mdr1a()gene on AMP-PCP-regulated
mouse ZG anion and cation conductances and carried out Western blots of
ZG membranes from wild-type and mdr1a-deficient mice using a
polyclonal MDR1-specific antibody. The results demonstrate that
disruption of the mdr1a gene is associated with a partial
reduction of ZG Cl
conductance in the presence of
AMP-PCP and with an increase of AMP-PCP inhibitable K
conductance. A MDR1-specific polyclonal antibody labels a band of
80 kDa in mouse ZG membranes, which is reduced by
50% in mdr1a-deficient mice.
Purified ZG membranes were obtained as
described previously(22) , with slight modifications. ZG were
diluted about 20-fold in an ice-cold hypotonic ``lysis
buffer'' containing 0.1 mM MgSO, 5 mM EDTA, 10 mM HEPES, adjusted to pH 7.0 with Tris, plus a
protease inhibitor mixture (10 µg/ml leupeptin, 1 mM benzamidine, 0.2 mM Pefabloc® SC, and 50 µg/ml
trypsin inhibitor), and lysis was allowed to proceed on ice for 30 min.
The clear suspension was centrifuged at 100,000
g for
60 min. The pellet was resuspended in lysis buffer containing 1 M guanidinium HCl to remove membrane-associated proteins, and
membranes were centrifuged once more at 100,000
g for
60 min. ZG membranes were stored in liquid nitrogen. Protein
concentration was assayed as described by Bradford (23) using
bovine serum albumin as a standard.
Since bulk salt influx into the intragranular space and the resulting granule lysis are limited by the flux of ions through the endogenous conductance pathway, but not by the flux of counterions through the shunt pathway, the slope of the decrease in absorbance with time will represent an estimate of the rate of ions transported through the endogenous conductance pathway.
Anion conductance was measured by resuspending ZG in
HEPES-buffered (20 mM, pH 7.0) iso-osmotic (150 mM)
potassium salts of the anions tested plus 1 mM EGTA, 0.1
mM MgSO, and the addition of 5 µM valinomycin, which selectively and maximally permeabilizes ZG
membranes to the major cation K
(13) . To
measure K
or nonselective cation conductances, ZG were
suspended in 150 mM monovalent cation/acetate solutions
containing 1 mM EDTA, 0.1 mM EGTA and buffered with
50 mM imidazole (pH 7.0, adjusted with acetic acid). Since the
intragranular pH is about 6.5(14) , an inside-to-outside
directed H
concentration gradient of
0.5 pH units
between intragranular space and incubation solution was generated.
Cation influx was initiated by addition of 16 µM electrogenic protonophore CCCP, which maximally permeabilizes the
granular membrane to H
and converts the H
concentration gradient into an inside negative H
diffusion potential. The inside negative membrane potential, in
turn, energizes cation influx through endogenous cation permeabilities.
Anion influx occurs through the uncharged molecule acetic acid, which
permeates through the lipid membrane by nonionic diffusion and
dissociates to provide the intragranular space continuously with
protons for protonation of imidazole as well as for proton efflux from
the acidic interior(14) . Under these conditions, cation influx
through endogenous cation permeabilities is rate-limiting.
Half-time of granular lysis was estimated from the slope of the decrease in absorbance with time between ionophore addition and either experimental half-time or the entire observation period if the half-time was not reached. The absorbance of the suspension was measured at a wavelength of 540 nm in a spectrophotometer at 37 °C. The slope of the absorbance change with time was calculated by linear regression of the digitized data. Lysis rates were expressed as half-times of granular lysis or its reciprocal value, i.e. the inverse half-time of lysis, which was considered proportional to the rate constant of lysis. Unless otherwise indicated, data were expressed as means ± S.E. of different preparations. Statistical analysis was carried out with the Statgraphics program using unpaired Student's t test. Results with levels of p < 0.05 were considered significant.
To characterize anion conductance of wild-type
(+/+) and mdr1a-deficient (-/-) mouse ZG,
granules were incubated in different isotonic K salts
in the absence (Fig. 1A) or presence (Fig. 1B) of valinomycin (5 µM), which
renders the ZG membrane (ZGM) maximally permeable to
K
. In the presence of valinomycin, granule lysis
occurred within seconds, both in (+/+) and (-/-)
mice, as the result of K
influx through the
valinomycin pore and of diffusion of the lipophilic anion
SCN
through the lipid membrane. In the presence of
the large anion gluconate, granules from (+/+) and
(-/-) mice remained equally stable before (Fig. 1A) and after the addition of valinomycin (Fig. 1B) throughout the duration of the experiment.
This indicates that the osmotic properties and mechanical stability of
the granules, as well as the permeability of the granule membranes to
lipophilic and large hydrophilic anions are similar in wild-type and mdr1a-deficient mice.
Figure 1:
A, osmotic lysis of
pancreatic ZG from wild-type (+/+) or mdr1a-deficient (-/-) mice suspended in
iso-osmotic K salts. ZG were suspended in a solution
containing 150 mM potassium salts and incubated at 37 °C
in a cuvette. B, anion conductance of wild-type
(+/+) or mdr1a-deficient (-/-) mouse
pancreatic ZG. The experimental buffer system was identical to that
described in A. Conductance was recorded after addition of the
K
selective ionophore valinomycin (5 µM)
at the arrow. C, effect of the nonhydrolyzable ATP analog
AMP-PCP on Cl
conductance of wild-type
(+/+) and mdr1a-deficient (-/-) mouse
pancreatic ZG.
Whereas ZG incubated in isotonic KCl
did not lyse in the absence of ionophore, the addition of the
electrogenic K ionophore valinomycin enhanced lysis (Fig. 1B), which indicates that Cl
permeates through an anion-selective conductance pathway. Its
permeability sequence was I
> Br
> Cl
for both (-/-) and
(+/+) mice (not shown), which is similar to that found in rat
pancreatic (13) and parotid ZG(27) . However, the
conductance for Cl
(as well as for Br
and I
; not shown) was slightly reduced in
(-/-) mice, as compared with (+/+) mice (Fig. 1B). We also investigated Cl
conductance in (+/+) and (-/-) mice in the
absence and presence of 0.5 mM of the nonhydrolyzable ATP
analog AMP-PCP (Fig. 1C). AMP-PCP (0.5 mM),
which increases rat pancreatic ZG Cl
conductance(15, 17) , enhanced lysis both in
(+/+) and (-/-) mice. The increase in
Cl
conductance was, however, more pronounced in
(+/+) mice than in (-/-) mice.
To investigate
whether the differences in Cl conductance between
(+/+) and (-/-) mice were significant, mean rate
constants of ZG lysis from 10-14 experiments were calculated and
analyzed by Student's t test for two independent
samples. As can be seen from the left panel of Fig. 3A, mean Cl
conductance
(expressed as mean rate constant of ZG lysis) in (+/+) mice
was 3.1 ± 0.2 h
and 2.6 ± 0.3
h
(mean ± S.E.) in (-/-) mice.
This difference was not significant. In the presence of AMP-PCP (0.5
mM), Cl
conductance increased to 7.6
± 0.7 h
in (+/+) mice and to 5.0
± 0.4 h
in (-/-) mice. This
difference was significant (p < 0.005; n =
10). AMP-PCP-sensitive Cl
conductance, i.e. the component of the Cl
conductance that is
activated by 0.5 mM AMP-PCP, was reduced by
50% in mdr1a-deficient mice, as compared with controls (2.8 ±
0.5 h
versus 5.1 ± 0.7
h
; p < 0.02; n = 9). ZG
Cl
conductance in (-/-) mice was not
abolished, which indicated that ZG Cl
conductance is
affected by disruption of the mdr1a gene, but that
mdr1a-independent Cl
transport also contributes to ZG
Cl
conductance.
Figure 3:
Synopsis of of Cl (A) and K
conductance (B)
measurements in pancreatic ZG of wild-type (+/+) and mdr1a-deficient (-/-) mice with or without
AMP-PCP. Solid bars represent conductance values for wild-type
(+/+), and open bars values for mdr1a-deficient mice (-/-). The difference between
conditions with and without AMP-PCP corresponds to AMP-PCP activated
Cl
and AMP-PCP sensitive K
conductances, respectively. Data are means ± S.E. of
7-14 experiments. p values were calculated using
Student's t test for unpaired comparison of condition
without (+/+) or with mdr1a disruption
(-/-). n.s., not
significant.
When granules were incubated in
KSCN buffer, granule lysis occurred in the absence of valinomycin and
was faster in (-/-) mice than in (+/+) mice (Fig. 1A). For lysis to occur, influx of K and SCN
was necessary. Since the difference in
lysis between (-/-) and (+/+) mice was no longer
present after addition of valinomycin (Fig. 1B),
endogenous K
conductance pathways, but not
SCN
diffusion across the ZGM, must have been
responsible for the faster lysis in mdr1a-deficient mice. Rat
pancreatic ZG contain a K
- and
Rb
-selective cation conductance and a nonselective
cation conductance in their membrane(14, 15) .
Therefore, mouse ZG were tested for K
-selective and
nonselective cation conductances (Fig. 2), as described under
``Methods.'' As shown in Fig. 2A,
K
conductance was increased in mdr1a-deficient mice (-/-,CCCP) as compared with
mice expressing the mdr1a gene (+/+, CCCP). 0.5
mM AMP-PCP, which blocks rat pancreatic K
conductance(14) , also reduced K
conductance in both (+/+) and (-/-) mice.
Even in the presence of 0.5 mM AMP-PCP, K
selective conductance was higher in (-/-) mice than
in (+/+) mice. The increase of K
conductance
in mdr1a-deficient mice appeared to be specific, since the
nonselective cation conductance of ZGM, determined using Na
as permeating cation, was not affected by mdr1a gene
disruption (Fig. 2B).
Figure 2:
A, Effect of AMP-PCP on K selective conductance of pancreatic ZG from wild-type
(+/+) and mdr1a-deficient (-/-) mice. ZG
were suspended in buffered 150 mM potassium acetate with or
without 0.5 mM AMP-PCP. At the arrow, the
protonophore CCCP (16 µM) was added, where indicated. B, nonselective cation conductance of wild-type
(+/+) and mdr1a deficient (-/-) mouse
pancreatic ZG. ZG were suspended in 150 mM sodium acetate.
Otherwise, the experimental conditions were identical as described in A.
A summary of the results from
7-11 different experiments is shown in Fig. 3B.
K conductance was increased in (-/-) mice
roughly by a factor of 2 (from 8.5 ± 1.2 h
to
14.2 ± 2.0 h
; left panel of Fig. 3B). This difference was statistically significant (p < 0.03; n = 11). AMP-PCP (0.5
mM) reduced K
conductance in (+/+)
mice to 3.2 ± 0.5 h
and to 5.3 ± 0.7
h
in (-/-) mice (Fig. 3B). This AMP-PCP insensitive portion of ZG
K
conductance, which corresponds to the nonselective
cation conductance(15) , was not different in (+/+)
and (-/-) mice (see also Fig. 2B). In
contrast, the AMP-PCP-sensitive K
conductance was
significantly increased in mdr1a knockout mice (6.5 ± 1.0 h
), compared with wild-type animals (3.5
± 0.6 h
; p < 0.03; n = 7).
From these results we conclude that there is a
relationship between mdr1a expression and the activity of ZG
Cl and K
conductance pathways. We
therefore correlated ion conductances of wild-type (+/+) and mdr1a deficient (-/-) mice with Western blot
analyses of mouse ZGM. We used an affinity-purified rabbit polyclonal
antibody against amino acids 389-406 of human MDR1 (anti-pgp 389)
to probe electrophoretically separated membrane proteins from ZGM of
wild-type or mdr1-deficient mice. The antibody was used
because of low background, high affinity, and specificity toward MDR1
when compared with C219 and JSB-1 antibodies. As shown in the first
lane of Fig. 4A, the antibody (dilution 1:2,000)
labeled mouse mdr1a, which is the only mdr1 isoform expressed in mouse
intestinal brush-border membranes(21) . Preincubation of the
antibody with 1 µM peptide antigen abolished antibody
labeling of the 160-180 kDa band associated with mdr1a,
suggesting specificity of antibody labeling (Fig. 4B).
In mouse ZGM, the antibody labeled a major band of
80 kDa (Fig. 4A), a molecular mass that is higher than that
found in rat ZGM and could result from differences in glycosylation,
the use of different alternative splice sites or may represent a
species-specific isoform. Labeling was abolished by competition
experiments with 1 µM peptide epitope (Fig. 4B). In mdr1a-deficient mice, no
160-180 kDa band was detected in intestinal brush-border
membranes (second lane of Fig. 4A). In ZGM from
mdr1a-deficient mice, the
80 kDa band was still present, but
the signal was much weaker than in the wild-type mice (Fig. 4A, fourth lane). This band was also
abolished by preincubation of the antibody with antigenic peptide (Fig. 4B). Signals from four different experiments were
scanned, and the intensity of the signals was quantified. Mean
intensity of the
80 kDa band in ZGM from mdr1a-deficient
mice was calculated to 39 ± 7% of the wild-type signal (mean
± S.D.). This suggested that a fraction of the protein
associated with the
80 kDa band of ZGM is a mdr1a gene
product. Since anti-pgp 389 antibody also labels mdr1b (Western blots
of mouse adrenal gland, a tissue that exclusively expresses
mdr1b(28) ; not shown), it is likely that the weak
80 kDa
band detected in ZGM from mdr1a-deficient mice (Fig. 4A) represents an mdr1b gene product.
Western blots of ZGM with a polyclonal antiserum to gp300, a sulfated
glycoprotein mainly localized to the zymogen granule membrane of mouse
pancreas(26) , showed signals of comparable intensity in
wild-type and mdr1a-deficient mice (Fig. 4C),
which indicates that the reduction in intensity of the
80 kDa band
in mdr1a-deficient mice is specific. Western blot analyses of
ZGM with the MDR1-specific antibody therefore suggests that both mdr1a and mdr1b gene products are coexpressed in
wild-type mouse pancreatic acinar cells and contribute equally to the
80 kDa band labeled by anti-pgp 389 antibody.
Figure 4: Western blot analysis of intestinal brush-border (Intest.M) and ZGM from wild-type (+/+) and mdr1a-deficient (-/-) mice with the MDR1 specific polyclonal antibody anti-pgp 389 (A, B), or with a polyclonal antiserum against gp 300 (C). A, proteins (10 µg) were probed with anti-pgp 389 antibody (1:2,000 dilution); B, the diluted antibody solution was preincubated for 30 min with 1 µM peptide epitope sequence. C, ZGM proteins (10 µg) were probed with a polyclonal antiserum to gp300 (1.10,000 dilution; left) (26) , or with a preimmune serum (right).
The experiments
with ZG isolated from mdr1a-deficient mice demonstrate that
disruption of the mdr1a gene partially reduces the ZG
Cl conductance, which is activated by AMP-PCP, and
increases AMP-PCP inhibitable K
conductance. As
summarized in the model in Fig. 5, we propose that a mdr1a gene product in ZGM is an adenine nucleotide binding protein that
regulates both Cl
and K
conductances
in a coupled but inverse manner. Further support for our hypothesis is
also derived from the reciprocal effects of pharmacological channel
modulators, such as AMP-PCP, the sulfonylurea derivative glibenclamide,
and quinine, which activate rat ZG Cl
and inhibit
K
conductance(29) . This hypothesis is
highlighted by the recent cloning of a pancreatic islet high affinity
sulfonylurea receptor, which modulates ATP-sensitive K
channels and turns out to be a novel member of the ABC
superfamily of transporters(30) . In view of our finding that
the increased ZG K
conductance in mdr1a (-/-) remains fully AMP-PCP inhibitable (Fig. 2A and 3B), the K
channel in ZGM, similar to the ubiquitously expressed Kir6.1
K
channel(46) , is also likely to interact
more directly with AMP-PCP, in addition to its putative interaction
with the mdr1a gene product. Furthermore, our results likewise
do not allow to discriminate between a model in which AMP-PCP interacts
solely with P-glycoprotein, with the Cl
channel, or
with both the regulator and the channel (see Fig. 5).
Figure 5: Model of the electrogenic ion pathways of pancreatic zymogen granule membranes and their regulation by a mdr1 P-glycoprotein gene product. For further details, see text.
The
results of this study support the
(disputed(7, 8, 9, 10) ) view that
there is a regulatory relationship between MDR1 P-glycoprotein
gene products and Cl channels, as proposed by Hardy et al.(11) for ATP-dependent volume-regulated
Cl
currents and by Luckie et al.(31) for swelling-activated Cl
and
K
currents. As reported earlier for the
P-glycoprotein-regulated volume-sensitive Cl
current
by Gill et al.(6) , Cl
channel
activation (as well as K
channel inhibition) by the mdr1a gene product in ZGM apparently proceeds in the presence
of the nonhydrolyzable ATP analog AMP-PCP, implying that P-glycoprotein
interaction with the ion channels does not require ATP hydrolysis, in
clear contrast to its activity as a drug pump.
There is increasing
evidence that ABC transporters may regulate ion channel function as
well. Both MDR1 and CFTR have structural homology (32) , share
common functions as Cl(2, 5) and
ATP-channels(33, 34) , and exhibit a multidrug
resistance phenotype (35) . There is no doubt that CFTR
operates as a Cl
channel(36) . However, it is
interesting to note in the context of our conclusions that cystic
fibrosis is associated with a decreased activity of an outwardly
rectifying Cl
channel(37, 38, 39, 40) and
an increased open probability of amiloride-sensitive Na
channels(41, 42) . These examples and our
results emphasize the possibility that MDR1 and CFTR gene
products, including potential splicing
variants(43, 44, 45) , may be responsible for
the regulation of different ion channels.