1 Molecular Physiology Unit, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, and Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán Tlalpan 14000, Mexico City, Mexico; and 2 Department of Cellular and Molecular Physiology, Yale University Medical School, New Haven, Connecticut 06520
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The murine
apical bumetanide-sensitive
Na+-K+-2Cl cotransporter gene
(mBSC1) exhibits two spliced isoform products that differ at the
COOH-terminal domain. A long COOH-terminal isoform (L-mBSC1) encodes
the Na+-K+-2Cl
cotransporter, and
a short isoform (S-mBSC1) exerts a dominant-negative effect on L-mBSC1
cotransporter activity that is abrogated by cAMP. However, the
mechanism of this dominant-negative effect was not clear. In this
study, we used confocal microscopic analysis of an enhanced green
fluorescent protein (EGFP) fusion construct (L-mBSC1-EGFP) expressed to
characterize the surface expression of the L-BSC1 isoform in
Xenopus laevis oocytes. Functional expression was also
assessed in L-mBSC1-injected oocytes by measuring the bumetanide-sensitive 86Rb+ uptake. Oocytes
injected with L-mBSC1-EGFP cRNA developed a distinct plasma
membrane-associated fluorescence that colocalized with the fluorescent
membrane dye FM 4-64. The fluorescence intensity in L-mBSC1-EGFP
oocytes did not change after cAMP was added to the extracellular
medium. In contrast, L-mBSC1-EGFP fluorescence intensity was reduced in
a dose-dependent manner, with coexpression of S-mBSC1. The inhibitory
effect of S-mBSC1 was abrogated by cAMP. Finally, the exocytosis
inhibitor colchicine blocked the effect of cAMP on the
L-mBSC1-EGFP/S-mBSC1-coinjected oocytes. All changes in L-mBSC1
surface expression correlated with modification of bumetanide-sensitive
86Rb+ uptake. Our data suggest that the
dominant-negative effect of S-mBSC1 on L-mBSC1 transport function is
due to the effects of the cotransporter on trafficking.
kidney; thick ascending limb; Xenopus laevis; oocytes; green fluorescent protein; NKCC2
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SALT REABSORPTION
IN THE THICK ascending limb of Henle (TAL) involves NaCl entry
across apical membranes by the apical bumetanide-sensitive Na+-K+-2Cl cotransporter (BSC1,
NKCC2), apical recycling via ROMK K+ channels, and
basolateral Cl
efflux by CLCNKB channels
(38). Loss-of-function mutations in any of the genes
encoding these transport proteins in the human TAL cause Bartter's
syndrome (34-36), an autosomal recessive disease featuring hypokalemic metabolic alkalosis with hypercalciuria and low
arterial blood pressure (23).
A fundamental mechanism for enhancing salt transport in the TAL is the
generation of cAMP via activation of Gs-coupled
receptors by hormones such as vasopressin, calcitonin, parathyroid
hormone, glucagon, and catecholamine (13, 15, 16). The
effects of these hormones are crucial to the normal functioning of the
TAL in reabsorbing 10-15% of filtered NaCl, providing for normal
diluting and concentrating power and regulating divalent mineral
excretion. While vasopressin has been shown to directly activate the
apical Na+-K+-2Cl
cotransporter
in mouse TAL (25), the molecular mechanism of this
activation is not known.
An emerging field of regulation of several membrane transporters,
including the apical Na+-K+-2Cl
cotransporter, appears to involve alternative splicing that generates isoforms with regulatory roles (9). In this regard, the
murine renal specific Na+-K+-2Cl
cotransporter gene SLC12A1, known as BSC1 or NKCC2, gives
rise to six alternative spliced isoforms due to the combination of two
independent splicing mechanisms (18, 27). One splicing event produces A, B, and F isoforms that vary in a portion of the
predicted second transmembrane segment and the contiguous intracellular
loop connecting transmembrane domain 2 with
3 (18). The other splicing mechanism is an
alternative polyadenylation site that predicts two mBSC1 proteins that
are identical at the NH2-terminal and transmembrane
domains, as well as in the first 74 amino acid residues of the
COOH-terminal domain (27). These isoforms differ in the
sequence and length of the remaining COOH-terminal domain. The longer
isoform, L-mBSC1 (previously known as mBSC1-9), contains 1,095 amino acids residues, with the last 383 residues being unique. The
shorter isoform, S-mBSC1 (previously known as mBSC1-4), consists
of 770 amino acids residues, with the initial 74 amino acids of the
COOH terminus being identical to that of L-mBSC1 but followed by a
short, unique segment of 55 residues. The shorter COOH-terminal domain
of S-mBSC1 predicts distinct consensus PKA and PKC phosphorylation
sites. Because the two splicing events are independent of each other,
the combination of both mechanisms results in the production of six
different isoforms: three with a long COOH-terminal domain (L-mBSC1A,
B, or F) and three with a short COOH-terminal domain (S-mBSC1A, B, or
F). Both L-mBSC1 and S-mBSC1 are expressed in the apical membrane of
the TAL (27).
Using heterologous expression in Xenopus laevis oocytes, we
have previously shown that the L-mBSC1 isoform is a
bumetanide-sensitive Na+-K+-2Cl
cotransporter (32, 33), whereas the S-mBSC1 isoform is a hypotonicity-activated, K+-independent,
bumetanide-sensitive Na+-Cl
cotransporter
that is inhibited by cAMP (31). Under isotonic conditions,
however, S-mBSC1 is not trafficked to the plasma membrane when
expressed alone but exerts a dominant-negative effect on the
Na+-K+-2Cl
cotransporter
function. This latter effect can be abrogated with PKA activation by
cAMP (33). Thus S-mBSC1 and L-mBSC1 interaction could be
critical for activation of the
Na+-K+-2Cl
cotransporter by
hormones such as vasopressin. This type of interaction between
alternatively spliced isoforms has been observed in several renal
cotransporters, but the mechanisms are not known (9). Competition between intracellular vesicles or heterodimers containing different isoforms has been suggested as possible mechanisms of interaction; however, no study has addressed this issue for membrane transporters.
In the present study, we investigated the mechanism of interaction
between the long and short isoforms of the
Na+-K+-2Cl cotransporter. To this
end, we assessed the effects of the S-mBSC1 isoform on both the surface
and functional expression of the L-mBSC1 isoform in X. laevis oocytes. Our results show that S-mBSC1 reduces the surface
expression, and hence the activity, of the L-mBSC1 Na+-K+-2Cl
cotransporter isoform
by a mechanism that involves PKA phosphorylation processes and the
exocytosis machinery.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
X. laevis oocyte preparation and injection.
Adult female X. laevis frogs were purchased from Nasco (Fort
Atkinson, MI). Frogs were maintained at the animal facility under constant control of room temperature and humidity at 16°C and 65%,
respectively. Oocytes were harvested from tricaine
(0.17%)-anesthetized frogs and incubated for 1 h with vigorous
shaking in Ca2+-free ND-96 [(in mM) 96 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES/Tris, pH 7.4] in the presence of 2 mg/ml of collagenase B. Oocytes were washed three times in regular
ND-96 [(in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES/Tris, pH 7.4], manually defolliculated, and incubated overnight at 16°C in incubation medium (ND-96
supplemented with 2.5 mM sodium pyruvate and 5 mg/100 ml of
gentamicin). Oocytes (6) were injected with 50 nl of water
or a solution containing L-mBSC1 cRNA at 0.5 µg cRNA/µl (25 ng
cRNA/oocyte). In coinjection experiments, oocytes were injected with
the same volume and amount of L-mBSC1 plus varying amounts of S-mBSC1
cRNA. After injection, oocytes were incubated at 16°C during 4-5
days, and the medium was changed every day. Oocytes were incubated
overnight in Cl-free ND-96 [(in mM) 96 sodium
isethionate, 2 potassium gluconate, 1.8 calcium gluconate, 1.0 magnesium gluconate, 5 HEPES, and 2.5 sodium pyruvate as well as 5 mg/100 ml gentamicin, pH 7.4 (11)] before
86Rb+ uptake experiments were performed.
mBSC1 cDNA isoforms.
The cDNA encoding the long and short COOH-terminal spliced isoforms of
the apical Na+-K+-2Cl
cotransporter, L-mBSC1 and S-mBSC1, respectively, was inserted in the
plasmid pSPORT1 (Invitrogen, Carlsbad, CA) as described (27). For preparation of a cRNA template, each cDNA
isoform was linearized at the 3'-end using XbaI (New England
Biolabs, Beverly, MA) restriction enzyme, and cRNA was in vitro
transcribed, using the T7 RNA polymerase mMESSAGE kit (Ambion, Austin,
TX). Transcript integrity was confirmed on agarose gels, and
concentration was determined by absorbance at 260 nm (DU 640, Beckman,
Fullerton, CA) and by densitometry of the corresponding bands in
agarose gels. cRNA was stored frozen in aliquots at
80°C until use.
Assessment of the
Na+-K+-2Cl
cotransporter function.
The function of the Na+-K+-2Cl
cotransporter was assessed by measuring tracer
86Rb+ uptake (New England Nuclear) in groups of
10-15 oocytes. Four days after water or cRNA injection,
86Rb+ uptake was measured with the following
protocol: a 30-min incubation period in mild hypotonic (~160
mosmol/kgH2O) K+- and Cl
-free
medium [(in mM) 68 sodium gluconate, 4.6 calcium gluconate, 1.0 magnesium gluconate, and 5 HEPES/Tris, pH 7.4], with 1 ouabain; this
was followed by a 60-min uptake period in a mild hypotonic (~160
mosmol/kgH2O) uptake medium [(in mM) 62 NaCl, 10 KCl, 1.8 CaCl2, 1 MgCl-2, 5 HEPES/Tris, pH 7.4], with 1 ouabain and 2.0 µCi/ml of 86Rb+. To study the
effect of PKA activation on
Na+-K+-2Cl
cotransporter
function, during the present study the 86Rb+
uptake in groups of oocytes was analyzed in the absence or presence of
10
3 M concentration of the cell membrane-permeable
dibutyryl-cAMP (Roche, Mannheim, Germany) plus 10
6 M
concentration of the phosphodiesterase inhibitor IBMX (Sigma, St.
Louis, MO). Although the hypotonic conditions used in our experiments
inhibit the endogenous Na+-K+-2Cl
cotransporter that is expressed in X. laevis oocytes
(10), uptakes were also measured in water-injected oocytes
(data not shown), and the mean values for water groups were subtracted
from corresponding L-mBSC1 groups to assess total
86Rb+ uptake due to L-mBSC1. Ouabain (Sigma)
was added to prevent 86Rb+ entry via
Na+-K+-ATPase. Uptakes were performed at
32°C, and at the end of the uptake period oocytes were washed five
times in ice-cold uptake solution without isotope to remove tracer in
extracellular fluid. Oocytes were then dissolved in 10% SDS. Tracer
activity was determined for each oocyte by
-scintillation counting.
Assessment of mBSC1 expression in oocyte plasma membranes. The surface expression of the L-mBSC1 isoform in the oocyte plasma membrane was measured by fluorescence using an enhanced green fluorescent protein (EGFP)-mBSC1 fusion construct. To obtain the pSPORT1/S-mBSC1-EGFP construct, the fragment containing the EGFP sequence was removed from pSPORT1/EGFP-L-mBSC1 and ligated into pSPORT1-S-mBSC1. L-mBSC1-EGFP cRNA was in vitro transcribed and microinjected into X. laevis oocytes in a volume of 50 nl at 0.5 µg cRNA/µl (25 ng cRNA/oocyte). In coinjection experiments, oocytes were injected with the same volume and amount of L-mBSC1-EGFP plus varying amounts of S-mBSC1 cRNA. After injection, oocytes were incubated at 16°C for 4-5 days. During this time, the incubation medium was changed every day. Individual oocytes were monitored for EGFP fluorescence (excitation = 488 n;. emittance = 505 nm), using a Zeiss confocal laser-scanning microscope LSM510 (×10 objective lens, excitation with 488-nm line of a multiline argon ion laser; Carl Zeiss). Fluorescent emissions were passed through a 505-nm band-pass filter. Water and wild-type L-mBSC1-injected oocytes were used as controls. Background autofluorescence of water-injected oocytes was minimized by adjusting brightness and contrast settings at a constant pinhole size. These settings were then used to assess fluorescence of L-mBSC1-EGFP. To study the effect of PKA activation on the surface expression of L-mBSC1-EGFP, the fluorescence intensity was assessed in oocytes before and 30 min after exposure to dibutyryl-cAMP+IBMX. All confocal microscopic experiments were performed using conditions identical to those for the functional experiments (i.e., the same hypotonic solutions, incubation times, and drug concentrations).
For membrane colocalization, oocytes were bathed at 4°C with 2 µM FM 46-4 [N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl)-hexatrienyl) pyridinium dibromide] (Molecular Probes, Eugene, OR), a fluorescent membrane marker. The low temperature was used to minimize endocytosis of FM 4-64 so as to ensure that fluorescence was primarily coming from dye localization in the plasma membrane. For determination of fluorescence secondary to FM 4-64 membrane labeling, the excitation was at 543 nm and the emissions were passed through a 650-nm band pass filter. To use this dye for colocalization experiments with EGFP-tagged membrane proteins, the fluorescence emission of FM 4-64 detected at ~670 nm becomes undetectable at wavelengths below 580 nm (data from Molecular Probes). We found in preliminary experiments (data not shown) that FM 4-64 fluorescence becomes undetectable when measured at 505 nm, the emission wavelength for EGFP fluorescence. Fluorescence of both EGFP and FM 4-64 was quantified at equatorial focal sections of oocytes using SigmaScan Pro (Jandel Scientific, San Rafael, CA) image-analysis software.Western blotting.
Oocyte homogenates were obtained 4 days after injection with water,
S-mBSC1, or S-mBSC1-EGFP cRNA. Groups of 30-50 oocytes were
homogenized in 2 µl/oocyte of homogenization buffer [(in mM) 250 sucrose, 0.5 EDTA, and 5 Tris/HCl, pH 6.9, plus protease inhibitors],
centrifuged twice at 100 g for 10 min at 4°C, and the
supernatant was recollected. Oocyte protein (6 oocytes/lane, 12 µl)
was heated in sample buffer containing 6% SDS, 15% glycerol, 0.3%
bromophenol blue, 150 mM Tris, pH 7.6, and 2% -mercaptoethanol, resolved by Laemmli SDS-PAGE (7.5%), and transferred in 10 × Tris/CAPS to a polyvinylidene difluoride membrane by semidry
electroblotting. Prestained molecular mass markers were used (Bio-Rad,
Hercules, CA).
Statistical analysis. The significance of the differences between groups was tested by one-way ANOVA with multiple comparisons using Bonferroni correction. The significance within the same group was analyzed using the paired t-test. The results are presented as means ± SE.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Surface expression of L-mBSC1-EGFP protein in X. laevis oocytes.
X. laevis oocytes injected with L-mBSC1-EGFP cRNA exhibited
a ring of fluorescence at their surfaces, as determined by confocal fluorescence microscopy (Fig.
1b). Control oocytes injected
with water showed no significant fluorescence at the emission and
excitation wavelengths for EGFP (Fig. 1a). To determine
whether the L-mBSC1-EGFP-specific fluorescence was mainly at the oocyte
plasma membrane, we loaded oocytes with FM 4-64 under conditions that
would suppress endocytosis of the dye, i.e., 4°C (22).
FM 4-64 is a lipophilic fluorophore that has been used to measure
surface expression of other membrane transporters (19,
20), including the rat thiazide-sensitive Na+-Cl cotransporter in X. laevis
oocytes (17), because it possesses the optimal properties
for a fluorescent membrane marker. Figure 1c shows FM 4-64 fluorescence at 4°C obtained in the same L-mBSC1-EGFP-injected oocyte
shown in Fig. 1b. The red FM 4-64-specific fluorescence was
detected in a surface ring pattern similar to that for
L-mBSC1-EGFP-specific fluorescence (Fig. 1b). As shown in
Fig. 1d, the superimposition of the two images (Fig. 1,
b and c) gives a yellow signal, indicating colocalization of FM 4-64 and L-mBSC1-EGFP protein. We observed >99%
surface colocalization of L-mBSC1-EGFP and FM 4-64 fluorescence in all
tested oocytes, suggesting that the L-mBSC1-EGFP fluorescence measured
at equatorial confocal sections in oocytes was indicative of expression
on plasma membrane.
|
|
Absence of cAMP on functional and surface expression of L-mBSC1 in
X. laevis oocytes.
We have shown previously (33) that microinjection of
X. laevis oocytes with L-mBSC1 cRNA results in a significant
increase in 22Na+ uptake that is not affected
by PKA activation. Because
Na+-K+-2Cl cotransport in native
TAL cells is enhanced by cAMP and PKA (25), this previous
experiment suggested that some factor or protein was missing in
L-mBSC1-injected oocytes that would allow the activation of this
cotransporter by cAMP. To determine the effect of dibutyryl-cAMP+IBMX on the surface expression of the mouse
Na+-K+-2Cl
cotransporter in
X. laevis oocytes, we analyzed 20 oocytes injected with
L-mBSC1-EGFP cRNA before and 30 min after addition of
dibutyryl-cAMP+IBMX. As shown in Fig.
3A, no change in fluorescence
intensity was observed after PKA activation. Oocytes exhibited a mean
of 37.5 ± 3.1 arbitraty units (AU) before and 35.8 ± 5.8 AU
after addition of dibutyryl-cAMP+IBMX. A representative example of the
fluorescence observed in a single L-mBSC1-EGFP cRNA-injected oocyte
before and after addition of dibutyryl-cAMP+IBMX is shown in Fig. 1,
g and h, respectively. Note that fluorescence
intensity is similar in both pictures. Similarly, dibutyryl-cAMP+IBMX
failed to increase bumetanide-sensitive 86Rb+
uptake in L-mBSC1-injected oocytes [15.8 ± 0.7 vs. 17.4 ± 1.4 nmol · oocyte
1 · h
1,
respectively, P = not significant (NS)], consistent
with our previous results (32). Thus the plasma membrane
and functional expression of
Na+-K+-2Cl
cotransport induced by
heterologous expression of the L-mBSC1 isoform were not affected by PKA
activation with dibutyryl-cAMP+IBMX.
|
Coexpression of the long and short mBSC1 isoform and regulation by
cAMP.
In native TAL cells, L-mBSC1 and S-mBSC1 isoforms are coexpressed in
the apical membrane (27). We have also previously shown that S-mBSC1 exerts a dominant-negative-like effect on the
bumetanide-sensitive Na+-K+-2Cl
cotransport activity of L-mBSC1 expressed in oocytes and that the
inhibitory effect of S-mBSC1 was largely reversed by addition of
cAMP+IBMX (33). Given that S-mBSC1 is exclusively
expressed in a submembranal pool when expressed alone
(31), we hypothesized that the dominant-negative effect of
S-mBSC1 on L-mBSC1 function may involve modulation in trafficking of
L-mBSC1 to or from the plasma membrane.
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present work describes the effect of the COOH-terminal
truncated, alternatively spliced isoform of the murine
SLC12A1 gene S-mBSC1 on the surface and functional
expression of the Na+-K+-2Cl
cotransporter (L-mBSC1). Using the X. laevis oocyte's
heterologous expression system, we show by confocal laser image
analysis that the S-mBSC1 isoform exerts a dominant-negative effect on
the surface expression of the longer isoform L-mBSC1, which results in
a reduction of the cotransporter activity (Figs. 1, 4, and 5). The
dominant-negative effect on surface expression, and hence functional
activity, was partially abrogated by PKA activation with
dibutyryl-cAMP+IBMX (Fig. 6). Finally, we demonstrate that the effect
of dibutyryl-cAMP+IBMX was prevented by the exocytosis inhibitor
colchicine (Fig. 7).
It is well established that generation of cAMP by hormones, such as
vasopressin, activates transepithelial salt transport in the murine TAL
(1, 12, 15). However, the mechanism of this activation was
unknown. We previously showed that PKA activation with cAMP did not
enhance the uptake of 22Na+ by L-mBSC1
expressed in X. laevis oocytes, suggesting that other factors/proteins not present in oocytes are required to reconstitute the observed cAMP activation of apical
Na+-K+-2Cl cotransport in murine
TAL (33). We had previously suggested that the critical
additional protein required for cAMP-dependent modulation of the
functional cotransporter isoform, L-mBSC1, was the short COOH-terminal
mBSC1 splice variant. This was based on the following observations.
First, both isoforms are expressed in the mouse TAL. The L-mBSC1
isoform was expressed almost equally in both medullary (MTAL) and
cortical TAL (CTAL) segments, but expression of the S-mBSC1 isoform was
highest in the MTAL and diminished significantly along the CTAL toward
the cortical surface (27). This gradient of S-mBSC1
expression parallels the medullary-to-cortical magnitude of
hormone-induced cAMP accumulation in the TAL and may account for the
observation that vasopressin enhances salt reabsorption predominantly
in MTAL rather than CTAL (14). In addition, we had also
previously reported that the short COOH-terminal isoform, S-mBSC1,
exerts a dominant-negative-like effect on ion transport by the L-mBSC1
Na+-K+-2Cl
cotransporter
(33). This was not due to competition for translation in
S-mBSC1+L-mBSC1-coinjected oocytes because unrelated cRNAs such as
renin or the Shaker K+ channel did not
significantly affect transport by L-mBSC1. Thus S-mBSC1 would reduce
the functional expression of L-mBSC1 in native TAL cells. Importantly,
we showed that cAMP reversed the negative effect of S-mBSC1 on L-mBSC1
function (33). The latter could provide a mechanism for
cAMP-mediated activation of
Na+-K+-2Cl
cotransport in native
TAL cells.
There is precedence for a dominant-negative type of effect of alternatively spliced isoforms of several genes, including several renal cotransporters (9). For example, the Na+-phosphate cotransporter gene can express truncated isoforms with dominant-negative effects on the cotransporter function (37). One interesting example of dominant-negative regulation occurs in the human KvLQT1 K+ channel that is associated with the congenital long QT syndrome. One of the two alternatively spliced KvLQT1 variants forms the functional K+ channel. The other, a COOH-terminal truncated isoform, does not possess channel function but exerts a strong dominant-negative effect on channel function (4). It has been shown that transgenic mice overexpressing the truncated isoform develop several interesting cardiac arrhythmias (5) and that, in humans with the recessive form of the long QT syndrome (Jervell and Lange-Nielsen syndrome), mutations in the dominant-negative isoform correlate with the phenotype of the cardiac arrhythmia (24). The mechanisms by which truncated isoforms produce their negative effects are still poorly understood.
The competition between intracellular vesicles containing different isoforms or formation of heterodimers between isoforms has been suggested as possible mechanisms. In the glucocorticoid receptor, the formation of nonfunctional heterodimers seems to be at least part of the mechanism by which the dominant-negative isoform reduces the function of the receptor (29). However, to our knowledge no study has addressed this issue in membrane transporters.
The interaction between the long and short isoforms of mBSC1 could be due to competition between S-mBSC1 and L-mBSC1 containing vesicles. L-mBSC1 has been detected in subapical vesicles (28). Thus trafficking of L-mBSC1 vesicles to the apical membrane could play a role in cotransporter regulation. We observed no S-mBSC1-EGFP fluorescence in L-mBSC1+S-mBSC1-EGFP-coinjected oocytes after cAMP treatment, suggesting that S-mBSC1-EGFP protein may be rapidly removed from the membrane after coinsertion with L-mBSC1. In addition, we have shown that S-mBSC1 antibody staining was predominantly in a subapical distribution in mouse kidney (27). Therefore, it is possible that the alternatively spliced S-mBSC1 isoform affects the interaction of the transporter proteins with the cytoskeleton (7) and/or the vesicular trafficking machinery. If so, then coassociation of S-mBSC1 and L-mBSC1 isoforms may result in "trapping" of the L-mBSC1 complex within subapical vesicles.
To begin to understand the interaction between the short and long
isoforms of the murine Na+-K+-2Cl
cotransporter, we assessed the effect of the short mBSC1 isoform on the
surface expression of an EGFP-tagged long mBSC1 isoform using the
X. laevis heterologous expression system. Several lines of
evidence suggest that the EGFP fluorescence observed in our present
study quantified cotransporter localization in the oocyte plasma
membrane. First, EGFP has been used to assess surface expression of
many membrane proteins expressed in oocytes (2, 3, 8, 21)
as well as cultured cells (30). In oocytes, intracellular EGFP fluorescence is not detected with confocal microscopy because the
laser does not penetrate deeply enough to visualize the intracellular EGFP pool at equatorial sections in these large cells. Even if there
were some intracellular light penetration, the distribution of the EGFP
signal in these large cells is so diffuse that it would likely fall
below the level of detection (3). Second, we show in Fig.
1 that L-mBSC1-EGFP fluorescence is colocalized with the membrane
marker FM 4-64 under conditions that would reduce FM 4-64 endocytosis
and potential labeling of a subplasma membrane pool. Third, we show
that the S-mBSC1-EGFP isoform, which under isotonic conditions remains
in the submembranal compartment of the oocyte (31), was
not detected by confocal analysis. Finally, we show in Fig. 2 a
direct correlation between EGFP fluorescence and the magnitude of
bumetanide-sensitive 86Rb+ uptake. When taken
together, these results demonstrate that the L-mBSC1-EGFP fluorescence
detected in the present study was predominantly at the oocyte plasma
membrane. While our results in oocytes suggest one mechanism for
regulation of BSC1 function by cAMP, verification of this mechanism in
TAL cells would be informative; however, no one has been able to stably
express BSC1 in a mammalian cell line.
Our results indicate that the S-mBSC1 isoform regulates cotransporter
function by inhibiting L-mBSC1 trafficking to the plasma membrane. The
following observations support this conclusion. The alternatively
spliced S-mBSC1 isoform reduces, in a dose-dependent manner, both
86Rb+ uptake by, and plasma membrane expression
of, the L-mBSC1 Na+-K+-2Cl
cotransporter. We also show that in the presence of the S-mBSC1 isoform, but not in its absence, the surface expression of L-mBSC1-EGFP is increased by PKA activation with dibutyryl-cAMP+IBMX. This increase
in plasma membrane expression is also associated with an increase in
86Rb+ uptake by the cotransporter. Finally, the
dibutyryl-cAMP+IBMX-induced increase in surface and functional
expression was blocked by colchicine, an inhibitor of the exocytosis
machinery. Thus the presence of the S-mBSC1 isoform precludes the
L-mBSC1 complex from migrating to the plasma membrane, and this
inhibitory effect is abrogated by cAMP. To our knowledge, this is the
first study to address the mechanisms of interaction between
alternatively spliced isoforms of membrane cotransporters. Further
studies will be required to determine whether S-mBSC1 and L-mBSC1 form
heterodimers and the role of PKA phosphorylation processes on this
interaction and the trafficking mechanism.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Jorge Sosa-Melgarejo for help in using the laser-scanning confocal microscope and to members of the Molecular Physiology Unit for suggestions and assistance.
![]() |
FOOTNOTES |
---|
This work was supported by Mexican Council of Science and Technology (CONACYT) Research Grants 97629m and 36124m, Howard Hughes Medical Institute Grant 75197-553601 (to G. Gamba), Robert Wood Johnson Grant 038545 (to R. S. Hoover), and National Institutes of Health Grant DK-36803 to (S. C. Hebert and G. Gamba). P. Meade was supported by scholarship grants from CONACYT and from the Dirección General del Personal Académico of the National University of Mexico.
Present address of R. S. Hoover: Dept. of Medicine, The Univ. of Chicago, 5841 S. Maryland Ave., MC-5100, Chicago, IL 60637.
Address for reprint requests and other correspondence: S. C. Hebert, Yale Univ. School of Medicine, 333 Cedar St., SHM B147, PO Box 208026, New Haven, CT 06520-8026(E-mail: steven.hebert{at}yale.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 25, 2003;10.1152/ajprenal.00421.2002
Received 10 December 2002; accepted in final form 4 February 2003.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bailly, C.
Transducing pathways involved in the control of NaCl reabsorption in the thick ascending limb of Henle's loop.
Kidney Int
53, Suppl 65:
S-29-S-35,
1998.
2.
Chan, KW,
Sui JL,
Vivaudou M,
and
Logothetis DE.
Specific regions of heteromeric subunits involved in enhancement of G protein-gated K+ channel activity.
J Biol Chem
272:
6548-6555,
1997
3.
Chapell, R,
Bueno OF,
Alvarez-Hernandez X,
Robinson LC,
and
Leidenheimer NJ.
Activation of protein kinase C induces gamma-aminobutyric acid type A receptor internalization in Xenopus oocytes.
J Biol Chem
273:
32595-32601,
1998
4.
Demolombe, S,
Baró I,
Péréon Y,
Bliek J,
Mohammad-Panah R,
Pollard H,
Morid S,
Mannens M,
Wilde A,
Barhanin J,
Charpentier F,
and
Escande D.
A dominant negative isoform of the long QT syndrome 1 gene product.
J Biol Chem
273:
6837-6843,
1998
5.
Demolombe, S,
Lande G,
Charpentier F,
van Roon MA,
van den Hoff MJ,
Toumaniantz G,
Baro I,
Guihard G,
Le Berre N,
Corbier A,
de Bakker J,
Opthof T,
Wilde A,
Moorman AF,
and
Escande D.
Transgenic mice overexpressing human KvLQT1 dominant-negative isoform. Part I: phenotypic characterisation.
Cardiovasc Res
50:
314-327,
2001[ISI][Medline].
6.
Dumont, JN.
Oogenesis in Xenopus laevis (Daudin). Stages of oocyte development in laboratory maintained animals.
J Morph
136:
153-180,
1970.
7.
Ehlers, MD,
Fung ET,
O'Brien RJ,
and
Huganir RL.
Splice variant-specific interaction of the NMDA receptor subunit NR1 with neuronal intermediate filaments.
J Neurosci
18:
720-730,
1998
8.
Flagg, TP,
Tate M,
Merot J,
and
Welling PA.
A mutation linked with Bartter's syndrome locks Kir 1.1a (ROMK1) channels in a closed state.
J Gen Physiol
114:
685-700,
1999
9.
Gamba, G.
Alternative splicing and diversity of renal transporters.
Am J Physiol Renal Physiol
281:
F781-F794,
2001
10.
Gamba, G,
Miyanoshita A,
Lombardi M,
Lytton J,
Lee WS,
Hediger MA,
and
Hebert SC.
Molecular cloning, primary structure and characterization of two members of the mammalian electroneutral sodium-(potassium)-chloride cotransporter family expressed in kidney.
J Biol Chem
296:
17713-17722,
1994.
11.
Gamba, G,
Saltzberg SN,
Lombardi M,
Miyanoshita A,
Lytton J,
Hediger MA,
Brenner BM,
and
Hebert SC.
Primary structure and functional expression of a cDNA encoding the thiazide-sensitive, electroneutral sodium-chloride cotransporter.
Proc Natl Acad Sci USA
90:
2749-2753,
1993[Abstract].
12.
Greger, R.
Physiology of renal sodium transport.
Am J Med Sci
319:
51-62,
2000[ISI][Medline].
13.
Hebert, SC,
and
Andreoli TE.
Control of NaCl transport in the thick ascending limb.
Am J Physiol Renal Fluid Electrolyte Physiol
246:
F745-F756,
1984
14.
Hebert, SC,
Culpepper RM,
and
Andreoli TE.
NaCl transport in mouse medullary thick ascending limbs. I. Functional nephron heterogeneity and ADH-stimulated NaCl cotransport.
Am J Physiol Renal Fluid Electrolyte Physiol
241:
F412-F431,
1981
15.
Hebert, SC,
Culpepper RM,
and
Andreoli TE.
NaCl transport in mouse medullary thick ascending limbs. II. ADH enhancement of transcellular NaCl cotrasport; origin of transepithelial volatge.
Am J Physiol Renal Fluid Electrolyte Physiol
241:
F432-F442,
1981
16.
Hebert, SC,
Culpepper RM,
and
Andreoli TE.
NaCl transport in mouse medullary thick ascending limbs. III. Modulation of ADH efect by peritubular osmolality.
Am J Physiol Renal Fluid Electrolyte Physiol
241:
F443-F451,
1981
17.
Hoover, RS,
Poch E,
Monroy A,
Vazquez N,
Nishio T,
Gamba G,
and
Hebert SC.
N-glycosylation at two sites critically alters thiazide binding and activity of the rat thiazide-sensitive Na+-Cl cotansporter.
J Am Soc Nephrol
14:
271-282,
2003
18.
Igarashi, P,
Vanden Heuver GB,
Payne JA,
and
Forbush B, III.
Cloning, embryonic expression, and alternative splicing of a murine kidney-specific Na-K-Cl cotransporter.
Am J Physiol Renal Fluid Electrolyte Physiol
269:
F406-F418,
1995.
19.
Janecki, AJ,
Janecki M,
Akhter S,
and
Donowitz M.
Basic fibroblast growth factor stimulates surface expression and activity of Na+/H+ exchanger NHE3 via mechanism involving phosphatidylinositol 3-kinase.
J Biol Chem
275:
8133-8142,
2000
20.
Janecki, AJ,
Janecki M,
Akhter S,
and
Donowitz M.
Quantitation of plasma membrane expression of a fusion protein of Na/H exchanger NHE3 and green fluorescence protein (GFP) in living PS120 fibroblasts.
J Histochem Cytochem
48:
1479-1492,
2000
21.
Ji, HL,
Chalfant ML,
Jovov B,
Lockhart JP,
Parker SB,
Fuller CM,
Stanton BA,
and
Benos DJ.
The cytosolic termini of the beta- and gamma-ENaC subunits are involved in the functional interactions between cystic fibrosis transmembrane conductance regulator and epithelial sodium channel.
J Biol Chem
275:
27947-27956,
2000
22.
Kuismanen, E,
and
Saraste J.
Low temperature-induced transport blocks as tools to manipulate membrane traffic.
Methods Cell Biol
32:
257-274,
1989[ISI][Medline].
23.
Kurtz, I.
Molecular pathogenesis of Bartter's and Gitelman's syndromes.
Kidney Int
54:
1396-1410,
1998[ISI][Medline].
24.
Mohammad-Panah, R,
Demolombe S,
Neyroud N,
Guicheney P,
Kyndt F,
van den HM,
Baro I,
and
Escande D.
Mutations in a dominant-negative isoform correlate with phenotype in inherited cardiac arrhythmias.
Am J Hum Genet
64:
1015-1023,
1999[ISI][Medline].
25.
Molony, DA,
Reeves WB,
Hebert SC,
and
Andreoli TE.
ADH increases apical Na+,K+,2Cl entry in mouse medullary thick ascending limbs of Henle.
Am J Physiol Renal Fluid Electrolyte Physiol
252:
F177-F187,
1987
26.
Moral, Z,
Dong K,
Wei Y,
Sterling H,
Deng H,
Ali S,
Gu R,
Huang XY,
Hebert SC,
Giebisch G,
and
Wang WH.
Regulation of ROMK1 channels by protein-tyrosine kinase and -tyrosine phosphatase.
J Biol Chem
276:
7156-7163,
2001
27.
Mount, DB,
Baekgard A,
Hall AE,
Plata C,
Xu J,
Beier DR,
Gamba G,
and
Hebert SC.
Isoforms of the Na-K-2Cl transporter in murine TAL. I. Molecular characterization and intrarenal localization.
Am J Physiol Renal Physiol
276:
F347-F358,
1999
28.
Nielsen, S,
Maunsbach AB,
Ecelbarger CA,
and
Knepper MA.
Ultrastructural localization of Na-K-2Cl cotransporter in thick ascending limb and macula densa of rat kidney.
Am J Physiol Renal Physiol
275:
F885-F893,
1998
29.
Oakley, RH,
Jewell CM,
Yudt MR,
Bofetiado DM,
and
Cidlowski JA.
The dominant negative activity of the human glucocorticoid receptor beta isoform. Specificity and mechanisms of action.
J Biol Chem
274:
27857-27866,
1999
30.
Petrecca, K,
Atanasiu R,
Akhavan A,
and
Shrier A.
N-linked glycosylation sites determine HERG channel surface membrane expression.
J Physiol
515:
41-48,
1999
31.
Plata, C,
Meade P,
Hall AE,
Welch RC,
Vazquez N,
Hebert SC,
and
Gamba G.
Alternatively spliced isoform of the apical Na-K-Cl cotransporter gene encodes a furosemide sensitive Na-Cl cotransporter.
Am J Physiol Renal Physiol
280:
F574-F582,
2001
32.
Plata, C,
Meade P,
Vazquez N,
Hebert SC,
and
Gamba G.
Functional properties of the apical Na+-K+-2Cl cotransporter isoforms.
J Biol Chem
277:
11004-11012,
2002
33.
Plata, C,
Mount DB,
Rubio V,
Hebert SC,
and
Gamba G.
Isoforms of the Na-K-2Cl cotransporter in murine TAL. II. Functional characterization and activation by cAMP.
Am J Physiol Renal Physiol
276:
F359-F366,
1999
34.
Simon, DB,
Bindra RS,
Mansfield TA,
Nelson-Williamns C,
Mendonca E,
Stone R,
Schurman S,
Nayir A,
Alpay H,
Bakkaloglu A,
Rodriguez-Soriano J,
Morales JM,
Sanjad SA,
Taylor CM,
Pilz D,
Brem A,
Trachtman H,
Griswold W,
Richard GA,
John E,
and
Lifton RP.
Mutations in the chloride channel gene, CLCNKB, cause Bartter's syndrome type III.
Nature Genet
17:
171-178,
1997[ISI][Medline].
35.
Simon, DB,
Karet FE,
Hamdan JM,
Di Pietro A,
Sanjad SA,
and
Lifton RP.
Bartter's syndrome, hypokalaemic alkalosis with hypercalciuria, is caused by mutations in the Na-K-2Cl cotransporter NKCC2.
Nature Genet
13:
183-188,
1996[ISI][Medline].
36.
Simon, DB,
Karet FE,
Rodriguez-Soriano J,
Hamdan JH,
DiPietro A,
Trachtman H,
Sanjad SA,
and
Lifton RP.
Genetic heterogeneity of Bartter's syndrome revealed by mutations in the K+ channel, ROMK.
Nature Genet
14:
152-156,
1996[ISI][Medline].
37.
Tatsumi, S,
Miyamoto K,
Kouda T,
Motonaga K,
Katai K,
Ohkido I,
Morita K,
Segawa H,
Tani Y,
Yamamoto H,
Taketani Y,
and
Takeda E.
Identification of three isoforms for the Na+-dependent phosphate cotransporter (NaPi-2) in rat kidney.
J Biol Chem
273:
28568-28575,
1998
38.
Winters, CJ,
Reeves WB,
and
Andreoli TE.
A survey of transport properties of the thick ascending limb.
Semin Nephrol
11:
236-247,
1991[ISI][Medline].