ANG II and calmodulin/CaMKII regulate surface expression and

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ANG II and calmodulin/CaMKII regulate surface expression and
Am J Physiol Renal Physiol 293: F68–F77, 2007.
First published March 20, 2007; doi:10.1152/ajprenal.00454.2006.
ANG II and calmodulin/CaMKII regulate surface expression and functional
activity of NBCe1 via separate means
Clint Perry, Hong Le, and Irina I. Grichtchenko
Department of Physiology and Biophysics, University of Colorado and Denver Health Sciences Center, Aurora, Colorado
Submitted 13 November 2006; accepted in final form 13 March 2007
Perry C, Le H, Grichtchenko II. ANG II and calmodulin/CaMKII
regulate surface expression and functional activity of NBCe1 via separate
means. Am J Physiol Renal Physiol 293: F68–F77, 2007. First published
March 20, 2007; doi:10.1152/ajprenal.00454.2006.—We recently reported that ANG II inhibits NBCe1 current and surface expression in
Xenopus laevis oocytes (Perry C, Blaine J, Le H, and Grichtchenko II.
Am J Physiol Renal Physiol 290: F417–F427, 2006). Here, we
investigated mechanisms of ANG II-induced changes in NBCe1
surface expression. We showed that the PKC inhibitor GF109203X
blocks and EGTA reduces surface cotransporter loss in ANG IItreated oocytes, suggesting roles for PKC and Ca2⫹. Using the
endosomal marker FM 4-64 and enhanced green fluorescent protein
(EGFP)-tagged NBCe1, we showed that ANG II stimulates endocytosis of NBCe1. To eliminate the possibility that ANG II inhibits
NBCe1 recycling, we demonstrated that the recycling inhibitor monensin decreases surface expression, accumulates NBCe1-EGFP in
endosomes, and inhibits NBCe1 current. Monensin and ANG II
applied together produce greater inhibition of NBCe1 current than
either did alone. This additive effect of monensin and ANG II
suggests that ANG II stimulates internalization of NBCe1. We used
the calmodulin (CaM) antagonist W13, which controls recycling by
blocking the exit of the endocytosed cargo from early endosomes, to
determine the role of CaM in NBCe1 trafficking. We demonstrated
that W13 decreases surface expression of NBCe1, accumulates
NBCe1-EGFP in endosomal-like formations, and inhibits NBCe1
current. W13 and ANG II applied together produce greater inhibition
of NBCe1 current than either does alone, while W13 and monensin
applied together do not. The additive effect of ANG II and W13 and
lack of additive effect of monensin and W13 suggest that CaM is not
involved in ANG II stimulation of internalization but controls recycling of endocytosed NBCe1. The CaM-activated enzyme CaM kinase
II (CaMKII) applied with ANG II also gives an additive inhibitory
effect, suggesting a role for CaMKII in NBCe1 recycling.
W13; monensin; FM 4-64; trafficking; endocytosis
NBCe1 IS ABUNDANT IN MANY tissues (30). This membrane protein
plays a major role in the regulation of intracellular pH and in
absorption and secretion of HCO⫺
3 (6, 29, 34). The amount of
NBCe1 at the cell surface is a critical determinant of cotransporter function. Previously, we showed that ANG II inhibits
NBCe1 current (INBC) and decreases the level of surface
expression of NBCe1 in Xenopus laevis oocytes (28). Endocytosis is a common phenomenon that controls the level of
surface expression of many membrane proteins. X. laevis
oocytes have been successfully used to study endocytosis of
many membrane proteins, including the thiazide-sensitive
Na-Cl cotransporter (NCCT), ROMK channels, EAAT1 transporters, GABA(C) receptors, the epithelial Na⫹ channels
Address for reprint requests and other correspondence: I. I. Grichtchenko,
Univ. of Colorado and Denver Health Sciences Center, Dept. of Physiology
and Biophysics, Mail Stop 8307, PO Box 6511, Aurora, CO 80045 (e-mail:
[email protected]).
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(ENaCs), and the CFTR Cl channels (5, 7, 8, 12, 20, 33, 41,
42). However, before our study nothing was known of the
trafficking changes induced by ANG II which result in inhibition of NBCe1. Here, we utilized the properties of FM 4-64 and
monensin to test our hypothesis that ANG II induces endocytosis of NBCe1 in X. laevis oocytes. The impermeant, lipophilic styryl dye FM 4-64 partitions into the plasma membrane and is a well-established fluorescent endocytotic marker
(4, 19). The monovalent carboxylic ionophore monensin raises
pH within endosomes and inhibits recycling of internalized
proteins to the plasma membrane (3, 37, 40).
We reported earlier that ANG II inhibits INBC in a PKC- and
Ca2⫹-dependent manner (28). One objective of the current
study was to use the PKC inhibitor GF109203X and Ca2⫹
chelator EGTA to determine the roles of PKC and Ca in ANG
II-induced loss of NBCe1 from the cell surface.
It has been shown that the calcium-binding protein calmodulin (CaM) and CaM kinase II (CaMKII) are involved in the
regulation of functional activity of renal and intestinal NBCe1
(2, 10, 31, 32). It is also known that CaM controls the recycling
step of the endocytic pathway of several membrane proteins (9,
14). Furthermore, its antagonist, W13, blocks the exit of
internalized cargo from early endosomes, resulting in the
formation of large endosomes (1, 23, 39). However, before our
study nothing was known of the involvement of CaM or
CaMKII in the trafficking of NBCe1. Here, we utilized inhibitors W13 and KN93 to determine the roles of CaM and
CaMKII in the trafficking of NBCe1.
Through biotinylation, confocal fluorescent microscopy in
live cells, and two-electrode voltage clamp, we found that
ANG II and CaM/CaMKII regulate endocytosis of NBCe1 via
separate mechanisms: ANG II induces internalization in a
PKC- and Ca2⫹-dependent manner, while CaM/CaMKII controls recycling of NBCe1.
EXPERIMENTAL PROCEDURES
Materials. GF109203X, W-13, KN93, monensin, and [Asn1,Val5]ANG
II acetate salt were purchased from Sigma (St. Louis, MO). Living
Colors full-length monoclonal green fluorescent protein (GFP) antibody was purchased from BD Biosciences/Clontech (Palo Alto, CA).
Leibovitz’s L-15 medium and penicillin-streptomycin and the styryl
dye FM4-64, 5-(and-6)-carboxy-2⬘,7⬘-dichlorofluorescein diacetate
(CDCFDA) were purchased from Invitrogen (Carlsbad, CA). EZ-Link
Sulfo-NHS-Biotin and immobilized neutravidin biotin binding protein
were purchased from Pierce Biotechnology (Rockford, IL). All other
chemicals were purchased from Sigma.
Preparation of oocytes. Female X. laevis frogs (NASCO) were
anesthetized with 1.5 mg/ml tricaine. All experimental procedures
The costs of publication of this article were defrayed in part by the payment
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0363-6127/07 $8.00 Copyright © 2007 the American Physiological Society
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ANG II AND CAM/CAMKII DIFFERENTLY REGULATE NBCe1
were approved by the University of Colorado Health Sciences Center
Animal Care and Use Committee [protocol no. 65105106(05)1D].
The ovarian lobes were surgically removed, dissected, and then
treated with 2 mg/ml collagenase type IA in Ca2⫹-free ND96-HEPES
solution (see below). Oocytes were incubated at 18°C in OR3 medium, a 1:2 dilution in dH2O of Leibovitz’s L-15 medium, supplemented with 50 U/ml of penicillin-streptomycin, 10 mM of HEPES,
and titrated to pH 7.5.
Expression in X. laevis oocytes. The cDNAs encoding human
hkNBCe1 and rat AT1B (the kind gift of Dr. Lakshmidevi Pulakat,
Bowling Green State University, Bowling Green, OH) were each
subcloned into the pGH19 expression vector. A chimera of the
enhanced GFP (EGFP)-tagged hkNBC1 cDNA was subcloned into
pGH19 (the kind gift of Dr. Leila V. Virkki, Institute of Physiology,
University of Zurich, Zurich, Switzerland). DNAs were transcribed in
vitro using the mMessageMachine kit (Ambion, Austin, TX) to
generate synthesized capped mRNAs. Oocytes were injected with 50
nl of 0.5 ng/nl hkNBC1 mRNA, 25 nl of 1 ng/nl hkNBC1 mRNA plus
25 nl of 1 ng/nl AT1B mRNA, or 50 nl of dH2O.
Solutions. Nominally bicarbonate-free ND96-HEPES solution contained 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, and
5 mM HEPES, pH 7.50. Bicarbonate-containing solutions were prepared by replacing 33 mM NaCl with 33 mM NaHCO3 and equilibrating with 5% CO2-balanced oxygen. Ca-free ND96-HEPES solutions were prepared by adding 0.5 mM EGTA. The osmolality of all
solutions was ⬃200 mosmol/kgH2O.
Drug treatments. W13 was made as a 1,000⫻ stock in sterile
dH2O, KN93 was made as a 1,000⫻ stock solutions in DMSO, and
monensin was made as a 10,000⫻ stock solution in methanol. All
three were diluted with ND96-HEPES solution to the final concentrations before use. As a control, we used 0.1% DMSO and 0.01%
methanol. ANG II was made as a 1,000⫻ stock in sterile dH2O and
diluted with ND-96-HEPES solution to the final concentrations before
use. Where indicated, 50 ␮M monensin was applied overnight to
oocytes kept at 18°C. Where indicated, 50 nl of 50 mM EGTA were
injected into oocytes before the experiments.
Two-electrode oocyte voltage clamp. As described previously (28),
we injected synthesized mRNAs encoding NBCe1-EGFP and AT1
into oocytes and 3 days later performed electrophysiological experiments. Oocytes were voltage clamped at room temperature using a
two-electrode oocyte clamp (Warner Instrument, New Haven, CT)
and microelectrodes made by pulling borosilicate glass capillary
tubing (Warner Instruments) on a microelectrode puller. The cells
were impaled with microelectrodes filled with 3 M KCl (resistance ⫽
0.3–1.0 M⍀). We clamped oocytes to a ⫺50 mV holding potential
and measured INBC in response to a 60-s depolarization, from ⫺50
mV to 0 mV. The currents were filtered at 20 Hz (4-pole Bessel filter)
and digitized. An oocyte was placed in a chamber with a 4 ml/min
constant superfusion. Bath solutions were delivered with syringe
pumps (Harvard Apparatus, South Natick, MA), and solutions were
switched with pneumatically operated valves (Clippard Instrument
Laboratory, Cincinnati, OH).
Biotinylation of surface proteins. As described previously (28),
oocytes injected with hkNBCe1-EGFP mRNA, or dH2O, or coinjected with hkNBCe1-EGFP and AT1B mRNAs and after 3 days were
incubated for 20 min with 1 ␮M ANG II alone, 1 ␮M ANG II with
100 nM GF, or 1 ␮M ANG II was added to oocytes injected with 50
nl of 50 mM EGTA. In separate experiments oocytes were incubated
for 50 min with 100 ␮M W13 or 50 ␮M KN93. In separate experiments, oocytes were treated overnight with 50 ␮M monensin. Next,
oocytes were incubated in the presence or absence of EZ-Link
Sulfo-NHS-Biotin for 1 h at 4°C, and the biotinylated proteins were
recovered from the membrane fractions with immobilized neutravidin
biotin binding protein by precipitation overnight at 4°C. Proteins were
boiled in Laemmli sample buffer and subjected to SDS-PAGE (21).
The hkNBCe1-EGFP bands were detected by Western blot analysis
with monoclonal anti-GFP antibodies using KODAK Image Station
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440CF. The intensity of the hkNBC1 bands was measured using
ImageJ software (http://rsb.info.nih.gov/ij/). As a control, we used
biotinylated oocytes for immunoprecipitation without avidin and nonbiotinylated oocytes immunoprecipitated with avidin.
Confocal microscopy in live oocytes. Fluorescent images of
“green” NBCe1-EGFP and “red” styryl dye FM 4-64 were captured in
a confocal XY section of the plasma membrane by excitation at 488
nm and emission at 510 nm (GFP) and by excitation at 545 nm and
emission at 740 nm (FM 4-64). Brightfields were taken in the same
confocal XY sections of the plasma membrane in series with fluorescent images. For brightfields, we used an argon laser to illuminate the
oocyte and captured the image using a channel without an emission
filter. We used a LSM510 microscope (Carl Zeiss Laser Scanning
System, available at Light Microscopy Facility at University of Colorado and Denver Health Sciences Center; see http://www.uchsc.edu/
lightmicroscopy/) with a ⫻25 water-immersion objective (NA 0.8
mm) and argon (“green” EGFP) and HeNa1 (“red” FM 4-64) lasers
for excitation. Fluorescence was acquired with Carl Zeiss LSM Image
software.
Data acquisition. INBC data were recorded digitally on a personal
computer using an analog-to-digital converter (ADC-30, CONTEC
Microelectronics, San Jose, CA) and sampling at a rate of 1 Hz.
Statistics and data analysis. All averages are reported as means ⫾
SD, along with the number of observations (n). For ratios, averages
are presented as log-normal means. The statistical significance of
log-normal data was determined using an unpaired Student t-test (18).
Differences were considered significant at a level of P ⬍ 0.05.
Fig. 1. ANG II-induced loss of surface NBCe1 is blocked by PKC inhibitor
GF109203X and reduced by EGTA in Xenopus laevis oocytes. A: results are
typical of 6 surface biotinylation studies using oocytes coexpressing NBC1enhanced green fluorescent protein (EGFP) and AT1. Biotinylated NBCe1
bands represent surface expression of NBCe1 detected by Western blot
analysis using anti-GFP monoclonal antibodies in a dilution of 1:1,000 (see
EXPERIMENTAL PROCEDURES). Oocytes were untreated (lane 1), treated with 1
␮M ANG II for 20 min (lane 2), treated for 20 min with a mixture of 1 ␮M
ANG II and 100 nM GF109203X (GF; lane 3), and treated with 1 ␮M ANG
II applied for 20 min to oocytes injected with 50 nl of 50 mM EGTA before
ANG II application (lane 4). B: bars represent means ⫾ SD of normalized
intensity of biotinylated NBCe1 bands acquired expressed as the ratio of
intensity of biotinylated NBCe1 band from drug-treated oocytes to those from
untreated control oocytes in n experiments as in A. The intensity of the
biotinylated NBC1 band in oocytes treated with ANG II (bar 2) was 39.3 ⫾
11.5 (n ⫽ 6) of untreated cells (bar 1). *P ⬍ 0.005 vs. untreated cells by
Student’s t-test. The intensities of the biotinylated NBCe1 band in oocytes
treated with ANG II and GF (bar 3) and EGTA (bar 4) were 102.8 ⫾ 25.9 (n ⫽
6) and 63.0 ⫾ 13.5% (n ⫽ 6), respectively, of untreated cells (bar 1). *P ⬍
0.01 vs. ANG II-treated cells by Student’s t-test (bar 2).
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ANG II AND CAM/CAMKII DIFFERENTLY REGULATE NBCe1
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Fig. 3. Monensin decreases surface expression and voltage-clamp current of NBCe1 by inhibiting recycling of NBCe1 in X. laevis oocytes. A: experiment typical
of 5 surface biotinylation studies using oocytes expressing NBC1-EGFP. Biotinylated NBCe1 bands represent the level of surface expression of NBCe1 detected
by Western blot analysis using anti-GFP monoclonal antibodies in a dilution of 1:1,000 (see EXPERIMENTAL PROCEDURES). Oocytes were untreated or treated with
50 ␮M monensin overnight. B: values are means ⫾ SD of normalized intensity of biotinylated NBCe1 bands acquired as the ratio of intensity of biotinylated
NBCe1 band from monensin-treated oocytes to untreated control oocytes in 5 experiments as in A. The intensity of the biotinylated NBCe1 band in oocytes
treated with monensin was 47.9 ⫾ 7.7% (n ⫽ 5) of that of untreated cells. *P ⬍ 0.005 by Student’s t-test. C: representative single equatorial optical slice of
confocal images of a live oocyte treated with 50 ␮M monensin overnight. Images show fluorescence of NBCe1-EGFP localized within the membrane of the
oocyte and in the endosomal-like formations. Inset: white arrow in high-magnification image points at endosome-like formation below plasma membrane. Scale
bars ⫽ 5 ␮m. D: as described earlier (28), oocytes were superfused with 5% CO2/33 mM HCO3 solution, voltage clamped to ⫺50 mV for 10 min, and depolarized
from ⫺50 to 0 mV to record amplitude of NBCe1 current. We corrected our data for a small background current of ⬃25 nA recorded in HCO⫺
3 -free solution,
in the presence of the NBC1 inhibitor DIDS (200 nM), or in water-injected oocytes. Values are means ⫾ SD of amplitude of voltage-clamp NBCe1 current from
17 untreated oocytes (2.3 ⫾ 0.2 ␮A) and 17 oocytes treated overnight with 50 ␮M monensin (1.4 ⫾ 0.3 ␮A). *P ⬍ 0.005 by Student’s t-test.
RESULTS
and Ref. 28). We found that the
intensity of the biotinylated NBCe1 band from oocytes treated
for 20 min with 1 ␮M ANG II (Fig. 1A, lane 2) was significantly reduced compared with that of untreated cells (Fig. 1A,
lane 1). The intensity of the biotinylated NBCe1 band from
oocytes treated for 20 min with 100 nM GF applied together
with 1 ␮M ANG II (Fig. 1A, lane 3) was similar to that of
untreated cells (Fig. 1A, lane 1). The intensity of the biotinylated NBCe1 band from oocytes injected with 50 nl of 50 mM
EGTA before treatment with 1 ␮M ANG II (Fig. 1A, lane 4)
was less than that of untreated cells (Fig. 1A, lane 1) but greater
than that of ANG II-treated cells (Fig. 1A, lane 2). Bars in Fig.
1B show that the normalized intensity of the biotinylated
EXPERIMENTAL PROCEDURES
GF109203X blocks and EGTA decreases ANG II-induced
loss of NBCe1 cotransporters from the cell surface in X. laevis
oocytes. In our previous paper, we showed that ANG IIinduced inhibition of INBC is PKC and Ca2⫹ dependent (28).
We also reported that ANG II induces a significant decrease in
the surface expression of NBCe1 (28). To investigate whether
PKC and intracellular Ca2⫹ also play a role in the ANG
II-induced decrease in NBCe1 surface expression, we utilized
the PKC-specific inhibitor GF109203X and EGTA. Using
surface biotinylation followed by Western blot analysis, we
determined levels of surface expression of cotransporters in
oocytes coexpressing hkNBCe1-EGFP with rat AT1B (see
Fig. 2. Confocal images of NBCe1 endocytosis. A: representative single equatorial optical slice of confocal images of a live untreated oocyte expressing
NBCe1-EGFP and loaded with 2 ␮M red FM 4-64 dye. The first panel on left shows brightfield image of outer edge of the oocyte. The second panel from the
left shows “red” fluorescence of FM 4-64 styryl dye localized in the plasma membrane of oocyte. The third panel from the left shows “green” fluorescence of
NBCe1-EGFP localized within the membrane of the oocyte. Far right panel shows merged image of red and green with yellow, indicating colocalization of FM
4-64 and NBCe1-EGFP within the oocyte membrane. B: representative single equatorial optical slice of confocal images of a live oocyte treated for 20 min with
1 ␮M ANG II and 2 ␮M FM 4-64 at room temperature. White arrows in merged field point to endosomal formations costained with FM 4-64 and NBCe1-EGFP.
Yellow indicates colocalization of FM 4-64 (red) and NBCe1-EGFP (green). C, left and middle: representative high-magnification image of endosomal formations
in live oocyte treated for 20 min with 1 ␮M ANG II. Outlined regions (white dotted circles) show colocalization of FM 4-64 and NBCe1-GFP within endosomal
formations budding into the cytosol of the oocyte; right, fluorescence intensity representations of left image showing highest intensities of red and green localized
within outlined regions. Scale on far right color-codes low to high intensity of fluorescence. Scale bars ⫽ 5 ␮m. Confocal images were acquired with a Zeiss
LSM510 microscope.
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ANG II AND CAM/CAMKII DIFFERENTLY REGULATE NBCe1
Fig. 4. Monensin and ANG II produce a greater inhibition of NBCe1
current than either does alone in voltage-clamped X. laevis oocytes. Voltageclamp currents were obtained from oocytes coexpressing NBCe1 and AT1.
Oocytes were superfused with 5% CO2/33 mM HCO3 solution, voltage
clamped to ⫺50 mV for 10 min, and depolarized from ⫺50 to 0 mV to record
either control currents before treatment or test currents following treatment
(See EXPERIMENTAL PROCEDURES). Values are means ⫾ SD of relative NBCe1
peak currents acquired as the ratio of test to control peak currents in n
experiments [as described earlier (28) and see EXPERIMENTAL PROCEDURES].
Oocytes were untreated (bar A); treated with 1 ␮M ANG II for 20 min (bar
B; *P ⬍ 0.005 vs. untreated cells by Student’s t-test); treated with 50 ␮M
monensin overnight (bar C ; *P ⬍ 0.005 vs. untreated cells by Student’s
t-test); and treated with 50 ␮M monensin overnight followed by 1 ␮M
ANG II for 20 min (bar D; *P ⬍ 0.005 vs. ANG II-treated cells by
Student’s t-test).
NBCe1 band from oocytes treated with ANG II alone was
39.3 ⫾ 11.5% (n ⫽ 6); ANG II together with 100 nM GF was
102.8 ⫾ 25.9% (n ⫽ 6); and injected with EGTA and treated
with ANG II was 63.0 ⫾ 13.5% (n ⫽ 6) of that of untreated
cells. Our data suggest that PKC plays a major role in the ANG
II-induced decrease in the surface expression of NBCe1. These
data also indicate a partial involvement of intracellular Ca2⫹.
Monitoring endocytosis of NBCe1-EGFP with FM 4-64
fluorescent dye. Here, we investigated the involvement of
endocytosis in the ANG II-induced decrease in surface expression of NBCe1. It is known that X. laevis oocytes have
machinery for endocytosis, which occurs via plasma membrane vesicles that bud into the cytosol (7, 13, 25, 26, 33).
Therefore, to provide evidence for ANG II-stimulated endocytosis of NBCe1, we needed to demonstrate that endosomes
which form in ANG II-treated oocytes are stained with EGFPtagged NBCe1. For this we used confocal fluorescent microscopy in live oocytes coexpressing NBCe1-EGFP with AT1B
(See EXPERIMENTAL PROCEDURES). We utilized styryl fluorescent
dye FM 4-64, which partitions within the lipid bilayer of the
plasma membrane and reversibly stains membranes of endosomes which have been endocytosed (4, 27). We used the
brightfield view to position oocytes and visualize the external
and internal division (Fig. 2A, far left). Loading oocytes with 2
␮M FM 4-64 resulted in strong red dye labeling of the plasma
membrane of untreated oocytes (Fig. 2A, second panel from the
left). NBCe1-EGFP can be seen localized within the plasma
membrane of the oocyte (Fig. 2A, third panel from left). Figure
2A, far right is a merged image of FM 4-64 and NBCe1-GFP
showing that both are localized within the plasma membrane.
Next, we treated oocytes for 20 min with 1 ␮M ANG II,
transferred oocytes to dye- and ANG II-free media, and detected colocalization of NBCe1-EGFP and FM 4-64 within
vesicular formations that bud into the cytosol (Fig. 2B, white
arrows). High-magnification views of these endosomal formations allow one to see the high intensity and colocalization of FM 4-64 and NBCe1-EGFP fluorescence (Fig. 2C,
dashed white circles). These data indicate that ANG II
induces endocytosis of NBCe1 cotransporters from the cell
surface of X. laevis oocytes.
Fig. 5. CaM inhibitor W13 decreases surface expression of NBCe1 and causes endosome-like formations stained with NBCe1EGFP in X. laevis oocytes. A: typical surface biotinylation study using oocytes
coexpressing NBCe1-EGFP and AT1. Biotinylated NBCe1 bands represent the level
of surface expression of NBCe1 detected by
Western blot analysis using anti-GFP monoclonal antibodies in a dilution of 1:1,000 (see
EXPERIMENTAL PROCEDURES). Oocytes were
untreated or treated with 100 ␮M W13 for 50
min. B: bars represent means ⫾ SD of normalized intensity of biotinylated NBCe1
bands acquired as the ratio of intensity of
biotinylated NBCe1 bands from W13-treated
oocytes to those from untreated control oocytes in 5 experiments. The intensity of the
biotinylated NBCe1 bands in oocytes treated
with W13 was 34.7 ⫾ 25.7% (n ⫽ 5) of that
of untreated cells. *P ⬍ 0.005 by Student’s
t-test. C: representative single equatorial optical slice of confocal images of a live oocyte
treated for 50 min with 100 ␮M W13. Images
show fluorescence of NBCe1-EGFP. White
arrows in high-magnification image point to
endosome-like formations. Scale bars ⫽ 5 ␮m.
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ANG II AND CAM/CAMKII DIFFERENTLY REGULATE NBCe1
Recycling inhibitor monensin significantly reduces the level
of surface expression and INBC. Endocytosis can be carried out
by increasing internalization of NBCe1 from the plasma membrane or by decreasing recycling of the endocytosed cotransporters back to the plasma membrane (36). Here, we used a
recycling inhibitor, the monovalent carboxylic ionophore monensin, to determine whether recycling of the transporter is
involved in the ANG II-induced endocytosis of NBCe1. We
first performed a surface biotinylation study followed by Western
blot analysis to determine whether monensin affects surface expression of NBCe1 in X. laevis oocytes (see EXPERIMENTAL
PROCEDURES and Ref. 28). Oocytes coexpressing NBCe1-EGFP
with AT1B were untreated and treated with 50 ␮M monensin
overnight. A typical experiment shown in Fig. 3A illustrates a
significant decrease in the intensity of the surface NBCe1 band
in monensin-treated cells. We found that the normalized
intensity of the biotinylated NBCe1 band in monensintreated oocytes was 47.9 ⫾ 7.7% (n ⫽ 5) of that of untreated
control cells (Fig. 3B). To support our biotinylation data, we
used confocal fluorescent microscopy in live oocytes to
visualize monensin’s effect on NBCe1 surface expression
(see EXPERIMENTAL PROCEDURES). Equatorial optical slice images of oocytes revealed that treatment with 50 ␮M monensin
overnight caused endosomal-like formations stained with
NBCe1-EGFP (Fig. 3C). In the high-magnification view in Fig.
3C, the white arrow points to a large endosomal formation that
buds into the cytosol. Thus our surface biotinylation and
confocal microscopy studies revealed that the recycling inhibitor monensin produces a significant loss of NBCe1 cotransporters from the cell surface.
Next, we determined the effect of monensin on NBCe1
functional activity, acquired as INBC in voltage-clamped oocytes
coexpressing NBCe1-EGFP with AT1B (see EXPERIMENTAL
PROCEDURES and Ref. 28). We found that mean INBC recorded
from oocytes treated overnight with 50 ␮M monensin was
1.4 ⫾ 0.3 ␮A (n ⫽ 17) compared with 2.3 ⫾ 0.2 ␮A (n ⫽ 17)
for untreated control oocytes (Fig. 3D). Thus we detected that
INBC was significantly inhibited in oocytes treated with monensin overnight, with the remaining current being 59.7 ⫾
12.0% (n ⫽ 17) (Fig. 4, bar C) of that from untreated oocytes.
These data show that monensin produces a considerable decrease in the functional activity of NBCe1, probably through
significant loss of the cotransporters from the cell surface by
inhibiting the recycling of NBCe1 cotransporters back to
plasma membrane.
Applied together, recycling inhibitor monensin and ANG II
produce a cumulative inhibitory effect on INBC. We showed
above that ANG II induces endocytosis of NBCe1, but we did
not clarify whether ANG II induces internalization or inhibits
recycling of endocytosed NBCe1 back to the plasma membrane. Monensin, as shown in the above experiments, inhibits
recycling of endocytosed NBCe1. To examine whether ANG II
and monensin inhibit activity of NBC1 through the same or
separate means, we applied ANG II to monensin-treated oocytes. Overnight treatment with 50 ␮M monensin approximately halves INBC. Twenty-minute treatment with 1 ␮M ANG
II also approximately halves INBC. The remaining currents
were 59.7 ⫾ 12.0 (n ⫽ 17) and 52.4 ⫾ 5.9% (n ⫽ 7),
respectively, of the control current before treatment (bars C
and B, respectively, in Fig. 4). In oocytes treated overnight
with 50 ␮M monensin followed by a 20-min application of 1
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␮M ANG II, INBC was drastically inhibited, with the remaining
current being 25.0 ⫾ 8.7% (n ⫽ 15) of the control (Fig. 4, bar
D). This cumulative inhibitory effect indicates that ANG II and
monensin decrease functional activity of NBC1 through separate means. Our data suggest that monensin inhibits NBCe1 by
blocking the recycling of NBCe1 to the plasma membrane,
while ANG II inhibits NBCe1 by inducing internalization of
NBCe1.
Calmodulin inhibitor W13 significantly reduces the level of
surface expression of NBCe1. To help support our results
found with monensin, we decided to use another inhibitor of
recycling, W13. W13 is known to inhibit the recycling of
various transporters in other cells (39) and is also a highly
specific CaM antagonist (15). Therefore, we aimed to support
our results separating the mechanisms of ANG II and monensin
as well as determine the role of CaM in the trafficking of
NBCe1. We first set out to determine the effect of W13, as
a recycling inhibitor, on the surface expression of NBCe1
in X. laevis oocytes. The experiment shown in Fig. 5A represents a typical surface biotinylation experiment in oocytes
coexpressing NBCe1-EGFP and AT1 (See EXPERIMENTAL
PROCEDURES). A typical Western blot in Fig. 5A shows that a
50-min treatment with 100 ␮M W13 significantly reduces the
level of NBCe1 protein at the cell surface. Figure 5B shows
that the normalized intensity of the biotinylated NBCe1 bands
from oocytes treated with W13 was 34.7 ⫾ 25.7% (n ⫽ 5) of
that of untreated cells. These data indicate that inhibition of
CaM produces a significant loss of surface NBCe1 protein,
probably through the inhibition of recycling NBCe1 to the
Fig. 6. Effect of calmodulin (CaM) antagonist W13 on NBCe1 current in
voltage-clamped X. laevis oocytes. Voltage-clamp currents were obtained
from oocytes coexpressing NBCe1 and AT1. Oocytes were superfused with
5% CO2/33 mM HCO3 solution, voltage clamped to ⫺50 mV for 10 min, and
depolarized from ⫺50 to 0 mV to record either control currents before
treatment or test currents following treatment (see EXPERIMENTAL PROCEDURES).
Values are means ⫾ SD of relative NBCe1 peak currents acquired as the ratio
of test to control peak currents in n experiments (as described above). Oocytes
were untreated (bar A), treated with 1 ␮M ANG II for 20 min (bar B; *P ⬍
0.005 vs. untreated cells by Student’s t-test); treated with 100 ␮M W13 for 50
min (bar C; *P ⬍ 0.005 vs. untreated cells by Student’s t-test); treated with
W13 (100 ␮M for 30 min) followed by 100 ␮M W13 plus 1 ␮M ANG II for
20 min [bar D; *P ⬍ 0.005 vs. cells treated with ANG II and W13 by Student’s
t-test (separate analysis)]; treated with 50 ␮m monensin overnight (bar
E; *P ⬍ 0.005 vs. untreated cells by Student’s t-test); and treated with 50 ␮M
monensin overnight followed by 100 ␮M W13 plus monensin for 50 min (bar
F; *P ⬍ 0.005 vs. untreated cells by Student’s t-test).
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ANG II AND CAM/CAMKII DIFFERENTLY REGULATE NBCe1
plasma membrane. We used confocal fluorescent microscopy
in live cells to visualize W13’s effect on NBCe1 surface
expression. Equatorial optical slice images revealed that a
50-min treatment with 100 ␮M W13 produced endosomal-like
formations stained with NBCe1-EGFP and clearly visible at
high magnification (Fig. 5C, white arrows). Note that all
oocytes also expressed AT1 for control and comparison purposes.
Applied together, W13 with ANG II, but not W13 with
monensin, produce a cumulative inhibitory effect on INBC. We
determined the effect of W13 on INBC. We pretreated oocytes
for 50 min with 100 ␮M W13. We found that in voltageclamped oocytes, the remaining INBC was 35.7 ⫾ 6.5% (n ⫽
11) of control current before treatment (Fig. 6, bar C). Note
that in oocytes treated with ANG II alone, the remaining INBC
was 52.4 ⫾ 5.9% (n ⫽ 7) of control (Fig. 6, bar B). Next, we
pretreated oocytes for 30 min with 100 ␮M W13 followed by
a 20-min treatment with a mixture of 1 ␮M ANG II and 100
␮M W13. The remaining INBC was 18.7 ⫾ 4.9% (n ⫽ 6) of
control current before treatment (Fig. 6, bar D). Thus we found
that W13 and ANG II applied together produce a much
stronger inhibition of INBC than either alone. This cumulative
effect suggests that W13 and ANG II inhibit NBCe1 through
separate means. Our data and the literature suggest that W13
inhibits NBCe1 by blocking the recycling of NBCe1 to the
plasma membrane, while ANG II inhibits NBCe1 by stimulat-
ing the internalization of NBCe1. We then provided additional
evidence that W13 works as a recycling inhibitor in X. laevis
oocytes, as it does in other cells. We applied 50 ␮M monensin
overnight and then 100 ␮M W13 for 50 min. The remaining
INBC was 59.4 ⫾ 8.2% (n ⫽ 6) of control (Fig. 6, bar F). Note
that monensin treatment resulted in a remaining current of
59.7 ⫾ 12.0% (n ⫽ 17) of control (Fig. 6, bar E). W13 and
monensin applied together produced an inhibition approximately equal to monensin alone, while W13 alone causes
greater inhibition. We speculate that this is due to the action of
monensin and the timing of applications. Monensin was applied overnight before W13 application. Once monensin causes
its effect on endosomes and recycling is blocked, CaM’s
role in controlling NBCe1 recycling may be rendered useless.
In summary, lack of a cumulative effect suggests that monensin and W13 affect NBCe1 through similar means. Both
monensin and W13 inhibit recycling of NBCe1. Our data
suggest that CaM plays a role in the recycling of NBCe1 to the
plasma membrane.
CaMKII inhibitor KN93 significantly decreases surface expression and inhibits INBC and when applied together with
ANG II produces a cumulative inhibitory effect on INBC. The
above experiments suggest that CaM is involved in recycling
of NBCe1 in X. laevis oocytes. Since CaMKII effects intestinal
NBCe1 (2) and CaMKII is a CaM-activated enzyme, we
decided to determine the role of CaMKII in NBCe1 surface
Fig. 7. CaM kinase II (CaMKII) antagonist KN93, which inhibits surface expression and current of NBCe1, produces an additive effect with ANG II. A: typical
surface biotinylation study using oocytes expressing NBCe1-EGFP and AT1. Biotinylated NBCe1 bands represent the level of surface expression of NBCe1
detected by Western blot analysis using anti-GFP monoclonal antibodies in a dilution of 1:1,000 (see EXPERIMENTAL PROCEDURES). Oocytes were untreated or
treated for 50 min with 50 ␮M KN93. B: values are means ⫾ SD of normalized intensity of biotinylated NBCe1 bands acquired as the ratio of intensity of
biotinylated NBCe1 band from KN93-treated oocytes to untreated control oocytes in 4 experiments. The intensity of the biotinylated NBCe1 band in oocytes
treated with KN93 was 39.8 ⫾ 24.6% (n ⫽ 4) that of untreated cells. *P ⬍ 0.005 by Student’s t-test. C: voltage-clamp currents were obtained from oocytes
coexpressing NBCe1 and AT1. Oocytes were superfused with 5% CO2/33 mM HCO3 solution, voltage clamped to ⫺50 mV for 10 min, and depolarized from
⫺50 to 0 mV to record either control currents before treatment or test currents following treatment (see EXPERIMENTAL PROCEDURES). Values are means ⫾ SD
of relative NBCe1 peak currents acquired as the ratio of test to control peak currents in n experiments (see above). Oocytes were untreated (bar A), treated for
20 min with 1 ␮m ANG II (bar B; *P ⬍ 0.005 vs. untreated cells by Student’s t-test); treated with 50 ␮M KN93 for 50 min (bar C; *P ⬍ 0.005 vs. untreated
cells by Student’s t-test); and treated by KN93 (50 ␮M for 30 min) followed by 50 ␮M KN93 plus 1 ␮M ANG II for 20 min [bar D; *P ⬍ 0.005 vs. cells treated
with ANG II and KN93 (separate analysis)].
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ANG II AND CAM/CAMKII DIFFERENTLY REGULATE NBCe1
expression and function. The experiment shown in Fig. 7A
represents a typical surface biotinylation study in oocytes
coexpressing NBCe1-EGFP and AT1. The Western blot in Fig.
7A shows that a 50-min treatment with 50 ␮M KN93 significantly reduced the level of NBCe1 protein at the cell surface.
Figure 7B shows that the normalized intensity of the biotinylated NBCe1 band from oocytes treated with KN93 was 39.8 ⫾
24.6% (n ⫽ 4) of that of control untreated cells. These data
suggest that CaMKII plays a role in controlling surface expression of NBCe1. We determined the effect of KN93 on INBC in
voltage-clamped oocytes. We found that a 50-min treatment
with 50 ␮M KN93 caused inhibition of NBCe1 functional
activity, with the remaining INBC being 55.0 ⫾ 7.5% (n ⫽ 9)
(Fig. 7C, bar C) of the control current before treatment. Next,
we pretreated oocytes for 30 min with 50 ␮M KN93, followed
by a 20-min treatment with a mixture of 1 ␮M ANG II and 50
␮M KN93. Figure 7C, bar C, shows that the remaining INBC in
oocytes treated with a mixture of KN93 and ANG II was
18.1 ⫾ 6.1% (n ⫽ 8, bar D) of the control current before
treatment. Note that in oocytes treated with ANG II alone, the
remaining INBC was 52.4 ⫾ 5.9% (n ⫽ 7) of control (Fig. 7C,
bar B). These data suggest that inhibition of CaMKII produces
a significant decrease in the functional activity of NBCe1
cotransporters. The cumulative effect of ANG II and KN93
together suggests that they work through separate means to
inhibit NBCe1. Considering this, and that CaM controls recycling of NBCe1 and that CaMKII is a CaM-activated enzyme,
it is very likely that CaMKII plays a role in the recycling of
NBCe1 back to the plasma membrane.
DISCUSSION
We reported earlier that 1 ␮M ANG II inhibits current and
decreases surface expression of NBCe1 (28). We also reported
that ANG II-inhibition of INBC is mediated by PKC and
intracellular Ca2⫹ (28). Binding of ANG II to AT1 receptors
F75
causes a cascade of events which eventually activates PKC and
increases intracellular Ca2⫹ concentration (11). The present
report shows that PKC plays a major role and intracellular
Ca2⫹ plays some role in the ANG II-induced loss of NBCe1
from the cell surface. This conclusion is based on the demonstration that GF109203X, which specifically inhibits all PKC
isoforms, applied together with ANG II completely prevents
the loss of NBCe1 from the cell surface (Fig. 1A, lane 3, Fig.
1B, bar 3). Furthermore, chelating intracellular Ca2⫹ with
EGTA in oocytes treated with ANG II reduces the amount of
NBCe1 lost from ANG II application alone (Fig. 1A, lane 4,
Fig. 1B, bar 4).
Endocytosis is a common mechanism responsible for the
loss of surface expression of several membrane proteins (35).
Our findings indicate that endocytosis also regulates surface
expression of NBCe1 in ANG II-treated oocytes. To show this,
we utilized FM 4-64, an impermeant styryl dye, which fluoresces exclusively in a lipid environment of either plasma or
endosomal membranes (4). We used FM 4-64 as a plasmalemma marker in nontreated oocytes when dye was present in
the media and as an endocytotic marker when dye was washed
from the media. By confocal microscopy in live cells, we
showed that NBCe1 localized at the plasma membrane of
oocytes and colocalized with FM 4-64 in endosomal formations after ANG II treatment (Fig. 2).
We demonstrated that ANG II-induced endocytosis is not a
“global” event involving all membrane proteins in X. laevis
oocytes. For this we used another membrane transporter, an
excitatory amino acid transporter, EAAT3, coexpressed with
AT1B in X. laevis oocytes (the EAAT3-EGFP construct was a
kind gift of Drs. Thomas S. Otis and Meera Pratap, University
of California, Los Angeles, CA). Using confocal microscopy,
no loss of surface expression of EAAT3-EGFP was observed
after a 20-min 1 ␮M ANG II treatment, with normalized
fluorescent intensity of total plasma membrane region being
Fig. 8. Putative model of the membrane trafficking
of NBCe1 in X. laevis oocytes. In internalization
from the plasma membrane, ANG II binds to the AT1
receptor, activating PKC and raising intracellular
Ca2⫹. ANG II-activated PKC and Ca2⫹ induce endocytosis of NBCe1. Endocytosis can be carried out
by increasing internalization of NBCe1 or by decreasing recycling of endocytosed cotransporters
(36). ANG II induces the internalization of NBCe1
and does not change constitutive recycling of the
cotransporter, causing loss of NBC1 at the surface
and inhibition of the cotransporter activity. In
oocytes treated with GF109203X or EGTA, the ANG
II-induced internalization of NBCe1 was prevented.
In recycling back to the plasma membrane, CaM
plays an important role in the recycling pathway but
not in endocytosis (16, 17, 22, 24). CaM and CaMKII
control recycling of NBCe1. CaM/CaMKII antagonists W13 and KN93 inhibit NBCe1 recycling, causing loss of the surface expression and activity of the
cotransporter. The recycling inhibitor monensin also
reduces recycling of NBCe1 back to the plasma
membrane, causing loss of surface expression and
activity of the cotransporter.
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ANG II AND CAM/CAMKII DIFFERENTLY REGULATE NBCe1
101 ⫾ 13% (n ⫽ 17) of that of untreated oocytes (data not
shown). In parallel experiments, similar treatment resulted
in a significant loss of surface expression of NBCe1-EGFP,
with normalized fluorescent intensity of total plasma membrane region being 56.2 ⫾ 12.8% (n ⫽ 7) of that of
untreated oocytes (data not shown). These data suggest that
ANG II-induced endocytosis of NBCe1 is not global and
may be specific for the NBCe1 cotransporter under conditions of our experiments.
The loss of surface NBCe1 could be carried out through
increasing internalization of or by inhibiting the recycling of
constitutively endocytosed cotransporters (35). To investigate
the mechanism of ANG II-stimulated endocytosis of NBCe1,
we required a means of blocking recycling of NBCe1 separate
from ANG II. To do this, we used the recycling inhibitor
monensin (3, 37, 38, 40). Monensin caused a significant loss of
NBCe1 cotransporters from the cell surface (Fig. 3, A and B)
and induced the appearance of large endosome-like vesicles
stained with NBCe1-EGFP (Fig. 3C, white arrow). Monensin
also inhibited INBC in voltage-clamped oocytes (Figs. 3D and
4, bar C). When applied together, ANG II and monensin
resulted in a cumulative inhibitory effect on INBC (Fig. 4, bar
D). This suggests that monensin and ANG II work through
separate means to inhibit NBCe1. Thus we showed that ANG
II most likely induces internalization of NBCe1 from the
plasma membrane. However, our data are not able to differentiate between ANG II causing additional internalization and
that causing an increase in the rate of constitutive internalization without affecting the rate of recycling back to the membrane. It will be of great interest to determine in mammalian
renal cells the rates of constitutive and stimulated endocytosis
of NBCe1 and to identify what mechanisms are involved
(clathrin, caveolae, etc.).
It has been shown that recycling of assorted endocytosed
transporters back to the membrane is dependent on CaM (1, 9,
14, 23). It has been reported by others that CaM plays a
significant role in the regulation of functional activity of
NBCe1 in various cells (2, 10, 31, 32). However, nothing was
known before our study of CaM’s role in the control of NBCe1
surface expression, nor CaM’s effects of NBCe1 in X. laevis
oocytes.
The present report shows that CaM plays a role in the
recycling of endocytosed NBCe1 back to the plasma membrane. This conclusion is based on the demonstration that
inhibiting CaM with W13 resulted in removal of the cotransporter from the cell surface (Fig. 5, A and B). W13 induces the
appearance of endosome-like vesicles stained with NBCe1EGFP (Fig. 5C, white arrows). W13 also significantly inhibited
INBC in voltage-clamped oocytes (Fig. 6, bar C). Moreover,
when applied together ANG II and W13 resulted in a cumulative inhibitory effect on INBC (Fig. 6, bar D). In contrast,
when monensin and W13 were applied together there was no
cumulative effect (Fig. 6, bar F).
The CaM-dependent enzyme CaMKII has been reported to
affect NBCe1 function (2). Therefore, we aimed to define the
role of CaMKII in the regulation of NBCe1 trafficking. We
showed that inhibiting CaMKII with KN93 results in a significant loss of NBCe1 from the cell surface (Fig. 7, A and B) and
in inhibition of INBC (Fig. 7C, bar C). When applied together,
ANG II and KN93 produce a cumulative effect on INBC (Fig.
7C, bar D). This suggests that these two work through separate
AJP-Renal Physiol • VOL
means to inhibit NBCe1. Knowing that CaMKII is a CaMactivated enzyme and by showing that CaM is involved in the
recycling of NBCe1 back to the plasma membrane, it is likely
that CaM works through CaMKII to regulate the recycling
process of NBCe1.
Our results show that the inhibitory effect of W13 on NBCe1
is faster and more robust than monensin’s effect (Figs. 3B, 5B,
and Fig. 6, bars C and E). The observed differences may be
attributed to possibly different mechanisms behind W13- and
monensin-inhibition of NBCe1 recycling. Since these mechanisms are still not well understood, we can only speculate
about the reasons behind the observed differences in action of
these two compounds. It has been shown that calmodulin
antagonists and monensin can raise endosomal pH, which
causes a major disruption of the recycling pathway (1, 3, 37,
40). However, some have suggested that monensin is ineffective in mildly acidic early endosomes, and thus recycling may
be incompletely blocked by monensin (22). Additionally, it has
been suggested that CaM is involved in various portions of the
recycling pathway, which also may explain the stronger and
faster effect of W13 (16, 17, 22, 24).
A model proposed from our data is summarized in Fig. 8.
Our findings suggest that 1) acute application of high concentration of ANG II induces endocytosis of renal NBCe1 expressed in X. laevis oocytes; 2) ANG II-induced endocytosis of
NBCe1 is mediated mainly by PKC and in part by intracellular
Ca2⫹; 3) CaM and CaMKII control surface expression and
functional activity of NBCe1 in the absence of induced endocytosis; and 4) ANG II and CaM/CaMKII regulate NBCe1 via
separate mechanisms: ANG II induces internalization of
NBCe1, while CaM/CaMKII controls recycling of the endocytosed transporters back to the cell surface.
ACKNOWLEDGMENTS
We thank Dr. Leila V. Virkki (Institute of Physiology, University of Zurich,
Zurich, Switzerland) for providing the NBCe1-EGFP cDNA construct;
Dr. Lakshmidevi Pulakat (Bowling Green State University, Bowling Green,
OH) for providing the AT1B cDNA construct; and Dr. Thomas S. Otis and Dr.
Meera Pratap (University of California, Los Angeles, CA) for providing the
EAAT3-EGFP cDNA construct.
GRANTS
This work was supported by Howard Hughes Medical Institute Grant
HHMI 76200-550102 to I. I. Grichtchenko.
REFERENCES
1. Apodaca G, Enrich C, Mostov KE. The calmodulin antagonist, W-13,
alters transcytosis, recycling, and the morphology of the endocytic pathway in Madin-Darby canine kidney cells. J Biol Chem 269: 19005–19013,
1994.
2. Bachmann O, Reichelt D, Tuo B, Manns MP, Seidler UE. Carbachol
increases Na⫹/HCO⫺
3 cotransport activity in murine colonic crypts in
an M3-, Ca2⫹/calmodulin-, and PKC-dependent manner. Am J Physiol
Gastrointest Liver Physiol 291: G650 –G657, 2006.
3. Basu SK, Goldstein JL, Anderson RG, Brown MS. Monensin interrupts
the recycling of low density lipoprotein receptors in human fibroblasts.
Cell 24: 493–502, 1981.
4. Betz WJ, Mao F, Smith CB. Imaging exocytosis and endocytosis. Curr
Opin Neurobiol 6: 365–371, 1996.
5. Boehmer C, Henke G, Schniepp R, Palmada M, Rothstein JD, Broer
S, Lang F. Regulation of the glutamate transporter EAAT1 by the
ubiquitin ligase Nedd4-2 and the serum and glucocorticoid-inducible
kinase isoforms SGK1/3 and protein kinase B. J Neurochem 86: 1181–
1188, 2003.
293 • JULY 2007 •
www.ajprenal.org
ANG II AND CAM/CAMKII DIFFERENTLY REGULATE NBCe1
6. Boron WF. Sodium-coupled bicarbonate transporters. JOP 2: 176 –181,
2001.
7. Bradbury NA, Clark JA, Watkins SC, Widnell CC, Smith HS, Bridges
RJ. Characterization of the internalization pathways for the cystic fibrosis
transmembrane conductance regulator. Am J Physiol Lung Cell Mol
Physiol 276: L659 –L668, 1999.
8. Cope G, Murthy M, Golbang AP, Hamad A, Liu CH, Cuthbert AW,
O’Shaughnessy KM. WNK1 affects surface expression of the ROMK
potassium channel independent of WNK4. J Am Soc Nephrol 17: 1867–
1874, 2006.
9. de Figueiredo P, Brown WJ. A role for calmodulin in organelle membrane tubulation. Mol Biol Cell 6: 871– 887, 1995.
10. Espiritu DJ, Bernardo AA, Robey RB, Arruda JA. A central role for
Pyk2-Src interaction in coupling diverse stimuli to increased epithelial
NBC activity. Am J Physiol Renal Physiol 283: F663–F670, 2002.
11. Forster IC, Traebert M, Jankowski M, Stange G, Biber J, Murer H.
Protein kinase C activators induce membrane retrieval of type II Na⫹phosphate cotransporters expressed in Xenopus oocytes. J Physiol 517:
327–340, 1999.
12. Golbang AP, Cope G, Hamad A, Murthy M, Liu CH, Cuthbert AW,
O’shaughnessy KM. Regulation of the expression of the Na/Cl cotransporter (NCCT) by WNK4 and WNK1: evidence that accelerated dynamindependent endocytosis is not involved. Am J Physiol Renal Physiol 291:
F1369 –F1376, 2006.
13. Haucke V. Phosphoinositide regulation of clathrin-mediated endocytosis.
Biochem Soc Trans 33: 1285–1289, 2005.
14. Hidaka H, Takara T. Modulation of Ca2⫹-dependent regulatory systems
by calmodulin antagonists and other agents. In: Calmodulin Antagonists
and Cellular Physiology, edited by Hidaka H and Hartshorne DJ. New
York: Academic, 1985, p. 13–23.
15. Hidaka H, Tanaka T. Naphthalenesulfonamides as calmodulin antagonists. Methods Enzymol 102: 185–194, 1983.
16. Huber LA, Fialka I, Paiha K, Hunziker W, Sacks DB, Bahler M, Way
M, Gagescu R, Gruenberg J. Both calmodulin and the unconventional
myosin Myr4 regulate membrane trafficking along the recycling pathway
of MDCK cells. Traffic 1: 494 –503, 2000.
17. Hunziker W. The calmodulin antagonist W-7 affects transcytosis, lysosomal transport, and recycling but not endocytosis. J Biol Chem 269:
29003–29009, 1994.
18. Kleinbaum DG, Kupper LL, Muller KE. Applied Regression Analysis
and Other Multivariable Methods. Belmont, CA: Duxbury, 1988.
19. Klingauf J, Kavalali ET, Tsien RW. Kinetics and regulation of fast
endocytosis at hippocampal synapses. Nature 394: 581–585, 1998.
20. Kusama T, Hatama K, Saito K, Kizawa Y, Murakami H. Activation of
protein kinase C induces internalization of the GABA(C) receptors expressed in Xenopus oocytes. Jpn J Physiol 50: 429 – 435, 2000.
21. Laemmli UK. Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature 227: 2680 –2685, 1970.
22. Llado A, Tebar F, Calvo M, Moreto J, Sorkin A, Enrich C. Protein
kinaseCdelta-calmodulin crosstalk regulates epidermal growth factor receptor exit from early endosomes. Mol Biol Cell 15: 4877– 4891, 2004.
23. Llorente A, Garred O, Holm PK, Eker P, Jacobsen J, van Deurs B,
Sandvig K. Effect of calmodulin antagonists on endocytosis and intracellular transport of ricin in polarized MDCK cells. Exp Cell Res 227:
298 –308, 1996.
AJP-Renal Physiol • VOL
F77
24. Mayorga LS, Beron W, Sarrouf MN, Colombo MI, Creutz C, Stahl
PD. Calcium-dependent fusion among endosomes. J Biol Chem 269:
30927–30934, 1994.
25. Mukhopadhyay A, Barbieri AM, Funato K, Roberts R, Stahl PD.
Sequential actions of Rab5 and Rab7 regulate endocytosis in the Xenopus
oocyte. J Cell Biol 136: 1227–1237, 1997.
26. Opresko L, Wiley HS, Wallace RA. Differential postendocytotic compartmentation in Xenopus oocytes is mediated by a specifically bound
ligand. Cell 22: 47–57, 1980.
27. Penalva MA. Tracing the endocytic pathway of Aspergillus nidulans with
FM4-64. Fungal Genet Biol 42: 963–975, 2005.
28. Perry C, Blaine J, Le H, Grichtchenko II. PMA- and ANG II-induced
PKC regulation of the renal Na⫹-HCO⫺
3 cotransporter (hkNBCe1). Am J
Physiol Renal Physiol 290: F417–F427, 2006.
2⫺
29. Pushkin A, Kurtz I. SLC4 base (HCO⫺
3 , CO3 ) transporters: classification, function, structure, genetic diseases, and knockout models. Am J
Physiol Renal Physiol 290: F580 –F599, 2006.
30. Romero MF, Fulton CM, Boron WF. The SLC4 family of HCO⫺
3
transporters. Pflügers Arch 447: 495–509, 2004.
31. Ruiz OS, Arruda JAL. Regulation of the renal Na-HCO3 cotransporter
by cAMP and Ca-dependent protein kinases. Am J Physiol Renal Fluid
Electrolyte Physiol 262: F560 –F565, 1992.
32. Ruiz OS, Qiu YY, Cardoso LR, Arruda JA. Regulation of the renal
Na-HCO3 cotransporter. VII. Mechanism of the cholinergic stimulation.
Kidney Int 51: 1069 –1077, 1997.
33. Shimkets RA, Lifton RP, Canessa CM. The activity of the epithelial
sodium channel is regulated by clathrin-mediated endocytosis. J Biol
Chem 272: 25537–25541, 1997.
34. Soleimani M. Functional and molecular properties of Na⫹:HCO⫺
3 cotransporters (NBC). Minerva Urol Nefrol 55: 131–140, 2003.
35. Sorkin A, von Zastrow M. Signal transduction and endocytosis: close
encounters of many kinds. Nat Rev Mol Cell Biol 3: 600 – 614, 2002.
36. Sorkina T, Hoover BR, Zahniser NR, Sorkin A. Constitutive and
protein kinase C-induced internalization of the dopamine transporter is
mediated by a clathrin-dependent mechanism. Traffic 6: 157–170, 2005.
37. Stein BS, Bensch KG, Sussman HH. Complete inhibition of transferrin
recycling by monensin in K562 cells. J Biol Chem 259: 14762–14772,
1984.
38. Tartakoff AM. Perturbation of vesicular traffic with the carboxylic
ionophore monensin. Cell 32: 1026 –1028, 1983.
39. Tebar F, Villalonga P, Sorkina T, Agell N, Sorkin A, Enrich C.
Calmodulin regulates intracellular trafficking of epidermal growth factor
receptor and the MAPK signaling pathway. Mol Biol Cell 13: 2057–2068,
2002.
40. Whittaker J, Hammond VA, Taylor R, Alberti KG. Effects of monensin on insulin interactions with isolated hepatocytes. Evidence for inhibition of receptor recycling and insulin degradation. Biochem J 234: 463–
468, 1986.
41. Yang CL, Angell J, Mitchell R, Ellison DH. WNK kinases regulate
thiazide-sensitive Na-Cl cotransport. J Clin Invest 111: 1039 –1045, 2003.
42. Zeng WZ, Babich V, Ortega B, Quigley R, White SJ, Welling PA,
Huang CL. Evidence for endocytosis of ROMK potassium channel via
clathrin-coated vesicles. Am J Physiol Renal Physiol 283: F630 –F639,
2002.
293 • JULY 2007 •
www.ajprenal.org

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