cAMP-dependent activation of the renal-specific Na+-K+-2Clminus cotransporter is mediated by regulation of cotransporter trafficking

Patricia Meade1,2, Robert S. Hoover2, Consuelo Plata1, Norma Vázquez1, Norma A. Bobadilla1, Gerardo Gamba1, and Steven C. Hebert2

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Galpha s-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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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 beta -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% beta -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).

For immunodetection, we used a monoclonal antibody against GFP diluted 1:1,000 (Clontech, Palo Alto, CA). The membrane was blocked for 1 h in 10% milk (10 mM Tris, pH 9.0, 150 mM NaCl, 0.1% Tween 20; TBS-T) and exposed to anti-GFP antibody diluted in 1% milk powder/TBS-T overnight at 4°C. After a washing in TBS-T, the membrane was exposed to horseradish peroxidase-linked anti-mouse IgG secondary antibody (Amersham Life Science, Arlington Heights, IL) for 1 h at room temperature. After a washing in TBS-T, antigen-antibody complexes were detected by autoradiography-enhanced chemiluminescence (ECL Plus Western blot analysis system, Amersham Life Science).

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


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Fig. 1.   Top: representative confocal images of X. laevis oocytes injected with water (a) or 25 ng of long COOH-terminal isoform of murine apical bumetanide-sensitive Na+-K+-2Cl- cotransporter gene (mBSC1)-enhanced green fluorescent protein (EGFP) fusion construct (L-mBSC1-EGFP) cRNA with (b) or without the FM 4-64 fluorescence dye (c). d: superimpositon of b and c. Middle: confocal images of oocytes injected with 25 ng of short COOH-terminal isoform of mBSC1-EGFP fusion construct (S-mBSC1-EGFP; a) or with L-mBSC1-EGFP before (g) and after exposure to dibutyryl-cAMP+IBMX (h). f: Western blot using anti-GFP monoclonal antibody of proteins obtained from 6 oocytes injected with S-mBSC1-EGFP cRNA, S-mBSC1 cRNA, or water. Bottom: representative images of oocytes coinjected with 25 ng L-mBSC1-EGFP plus 25 ng of S-mBSC1 cRNA before (i) and after exposure to dibutyryl-cAMP+IBMX (j) and (k) or colchicine (l)as indicated.

To further exclude the possibility that we were detecting primarily EGFP fluorescence in vesicles below the plasma membrane, we also examined fluorescence from an EGPF-labeled protein that we had previously shown to be exclusively expressed just below the plasma membrane. S-mBSC1 protein does not reach the plasma membrane but remains in what appears to be a submembranal pool of vesicles in X. laevis oocytes incubated under isotonic conditions (31). S-mBSC1-EGFP-injected oocytes exhibited no EGFP surface fluorescence (Fig. 1e). The absence of surface fluorescence in the S-mBSC1-EGFP-injected oocytes was not due to the lack of protein expression because S-mBSC1-EGFP protein was detected by Western blot analysis using anti-GFP monoclonal antibody (Fig. 1f). In addition, S-mBSC1-EGFP protein retained the ability to interact with L-mBSC1 (see below).

To further demonstrate that L-mBSC1-EGFP fluorescence represents cotransporter expression in oocyte plasma membranes, we assessed the correlation between L-mBSC1-EGFP surface expression and L-mBSC1 functional expression (Fig. 2). Activity of the Na+-K+-2Cl- cotransporter assessed by measuring 86Rb+ uptake increased as a function of the amount of cRNA injected in oocytes, reaching a plateau at ~20 ng/oocyte. The surface expression of L-mBSC1-EGFP protein increased similarly as a function of the amount of cRNA injected. All of these results when taken together indicate that the surface EGFP fluorescence detected in our L-mBSC1-EGFP-injected oocytes was predominantly coming from cotransporter expression in plasma membranes.


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Fig. 2.   Correlation between the dose dependency of L-mBSC1-EGFP surface expression, assessed by laser-scanning confocal microscopy (), and the dose dependency of 86Rb+ uptake by L-BSC1 (open circle ) in Xenopus laevis oocytes. Each point represents the mean ± SE of 20 oocytes. AU, arbitrary units.

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.


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Fig. 3.   Effect of dibutyryl-cAMP and IBMX on the function and surface expression of L-BSC1 or L-BSC1-EGFP in groups of X. laevis oocytes analyzed in the absence (open bars) or presence (hatched bars) of 1 mM cAMP+1 µM IBMX. A: laser-scanning confocal microscopy of 80 oocytes from 5 different frogs injected with L-mBSC1-EGFP cRNA before and 30 min after dibutyryl-cAMP+IBMX exposure. Representative images are shown in Fig. 1, g and h. B: bumetanide-sensitive 86Rb+ uptake in 2 groups of oocytes injected with 25 ng of L-BSC1 cRNA. Bumetanide was added to the preincubation and uptake medium at 10-4 M. Each bar represents the mean ± SE of ~100 oocytes from 5 different frogs.

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.

To begin to assess the mechanism by which S-mBSC1 regulates L-mBSC1 in a cAMP-dependent manner, we examined the effects of the S-mBSC1 isoform on the surface expression of the L-mBSC1 isoform. Thus we assessed the fluorescence intensity in oocytes injected with L-mBSC1-EGFP cRNA alone or with S-mBSC1 cRNA. We also measured 86Rb+ uptake in the same batch of X. laevis oocytes injected with L-mBSC1 cRNA alone or together with S-mBSC1 cRNA. As shown in Fig. 4A, the fluorescence intensity of oocytes injected with L-mBSC1-EGFP alone was significantly higher (73.9 ± 5.3 AU) than intensity observed in oocytes coinjected with L-mBSC1-EGFP and S-mBSC1 (42.9 ± 3.5 AU, P < 0.001). A representative example showing the confocal images from a L-mBSC1-EGFP oocyte and a L-mBSC1-EGFP+S-mBSC1 oocyte is presented in Fig. 1, g and i, respectively. Consistent with the reduced surface expression of L-mBSC1-EGFP induced by S-mBSC1 (Fig. 4), functional analysis confirmed a significant reduction in bumetanide-sensitive 86Rb+ uptake in L-mBSC+S-mBSC1-injected oocytes (7.76 ± 0.5 nmol · oocyte-1 · h-1) compared with values obtained in L-mBSC1 oocytes (15.8 ± 0.7 nmol · oocyte-1 · h-1, P < 0.001). The reduction in surface expression of L-mBSC1-EGFP and in L-mBSC1 bumetanide-sensitive 86Rb+ transport showed similar relationships to the amount of coinjected S-mBSC1. As shown in Fig. 5A, when oocytes injected with 25 ng of L-mBSC1-EGFP cRNA were additionally injected with increasing concentrations of S-mBSC1 cRNA, from 6 to 50 ng/oocyte, a significant dose-dependent decrease in fluorescence intensity was observed. We observed a dose-dependent inhibition of 86Rb+ uptake within the same range of S-mBSC1 coinjections (Fig. 5B). Thus the lower the L-mBSC1-to-S-mBSC1 ratio, the lower the surface expression and activity of the Na+-K+-2Cl- cotransporter.


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Fig. 4.   Reduction of the Na+-K+-2Cl- cotransporter surface and functional expression induced by S-mBSC1 cRNA in X. laevis oocytes injected with 25 ng of L-mBSC1-EGFP or L-BSC1 cRNA alone (open bars) or together with 25 ng of S-mBSC1 cRNA (filled bars). A: laser-scanning confocal microscopy fluorescence intensity. Representative images are depicted in Fig. 1, g and i.**P < 0.001 vs. L-mBSC1-EGFP-injected group. B: bumetanide-sensitive 86Rb+ uptake in nmol · oocyte-1 · h-1. Bumetanide was added to the preincubation and uptake mediums at 10-4 M concentration. Each bar depicts the mean ± SE of 100 oocytes from 5 different frogs. *P < 0.001 vs. L-mBSC1.



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Fig. 5.   Effect of increasing concentrations of S-mBSC1 cRNA on surface expression of L-mBSC1-EGFP and functional expression of L-mBSC1 in X. laevis oocytes. A: fluorescence intensity in oocytes injected with 25 ng of L-mBSC1-EGFP cRNA alone or together with increasing amounts of S-BSC1 cRNA as indicated. **P < 0.001 vs. L-mBSC1-EGFP-injected group. B: bumetanide-sensitive 86Rb+ uptake was assessed in groups of oocytes that were injected with 25 ng of L-mBSC1 cRNA alone or together with increasing amounts of S-BSC1 cRNA as stated. Bumetanide was added to the preincubation and uptake mediums at 10-4 M concentration. Each bar depicts the mean ± SE of 50 oocytes from 3 different frogs. *P < 0.001 vs. L-mBSC1.

Given the reduction in Na+-K+-2Cl- cotransporter surface expression and function induced by S-mBSC1 isoform in X. laevis oocytes, we measured the effect of dibutyryl-cAMP+IBMX on the fluorescence intensity and 86Rb+ uptake in coinjected oocytes. As shown in Fig. 6A, the reduction in the surface expression of L-mBSC1-EGFP in S-mBSC1 coinjected oocytes was partially reversed by the addition of dibutyryl-cAMP+IBMX (30.3 ± 4.8 without vs. 49 ± 7.1 AU with dibutyryl-cAMP+IBMX, P < 0.001). A representative example of an oocyte coinjected with L-mBSC1-EGFP and S-mBSC1 isoforms before and 30 min after exposure to dibutyryl-cAMP+IBMX is shown in Fig. 1, i and j, respectively. A clear increase in fluorescence intensity is observed in Fig. 1j compared with Fig. 1i. As depicted in Fig. 6B, similar results were obtained when functional expression was assessed (7.76 ± 0.5 in the absence vs. 10.9 ± 0.7 nmol · oocyte-1 · h-1 in the presence of dibutyryl-cAMP+IBMX, P < 0.001). In addition, Fig. 6C shows that the S-mBSC1-EGFP construct is also able to reduce the function of L-mBSC1 and that this reduction can also be abrogated by dibutyryl-cAMP+IBMX. Thus in X. laevis oocytes, the surface expression, and hence the function, of the Na+-K+-2Cl- cotransporter can be modulated by dibutyryl-cAMP only when L-mBSC1 and S-mBSC1 isoforms are coexpressed but not when the L-mBSC1 isoform is expressed alone (Fig. 3). Furthermore, similar 86Rb+ responses to dibutyryl-cAMP+IBMX are observed whether the EGFP reporter is on S-mBSC1 or L-mBSC1. Moreover, experiments were performed in which L-mBSC1 and S-mBSC1-EGFP were coinjected and EGFP fluorescence intensity was assessed in the absence and presence of cAMP. No S-mBSC1-EGFP fluorescence was detected 30 min after cAMP [H2O injected 0.18 ± 0.1 AU (n = 10); L-mBSC1+S-mBSC1 coinjected, 0.28 ± 0.06 AU (-cAMP; n = 18) and 0.23 ± 0.04 AU (+cAMP; n = 19); P = NS compared with H2O injected], suggesting that S-mBSC1 may be rapidly removed from the membrane after coinsertion with L-mBSC1 after cAMP.


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Fig. 6.   Effect of dibutyryl-cAMP and IBMX on S-mBSC1-induced reduction of surface expression and in S-mBSC1 or S-mBSC1-EGFP-induced reduction in functional expression of the Na+-K+-2Cl- cotransporter. A: fluorescence intensity in oocytes injected with 25 ng of L-mBSC1-EGFP cRNA alone (open bar) or together with 25 ng of S-mBSC1 in the absence (filled bar) or presence (hatched bar) of 1 mM cAMP and 1 µM IBMX. The filled and hatched bars depict the fluorescence intensity in the same group of oocytes before and 30 min after exposure to dibutyryl-cAMP+IBMX. Representative images are shown in Fig. 1, i and j. B: bumetanide-sensitive 86Rb+ uptake in oocytes injected with 25 ng of L-mBSC1 cRNA alone (open bar) or together with 25 ng of S-mBSC1 cRNA in the absence (filled bar) and presence (hatched bar) of dibutyryl-cAMP+IBMX. C: bumetanide-sensitive 86Rb+ uptake in oocytes injected with 25 ng of L-mBSC1 cRNA alone (open bar) and together with 25 ng of S-mBSC1-EGFP cRNA in the absence (filled bar) and presence (hatched bar) of dibutyryl-cAMP+IBMX. In both B and C, bumetanide, cAMP, and IBMX were added to the preincubation and uptake media at 10-4, 10-3, and 10-6 M concentration, respectively. Each bar represents the mean ± SE of 40 oocytes from 3 different frogs.

The results in Fig. 6 suggest that the mechanism by which the S-mBSC1 isoform reduces the function of the Na+-K+-2Cl- cotransporter includes a reduction in the surface expression of L-mBSC1, which is partially abrogated after PKA activation with dibutyryl-cAMP+IBMX. The modulation of L-mBSC1 surface expression could result from alterations in trafficking to (exocytosis) or from (endocytosis) the plasma membrane. To study this possible mechanism, we assessed the effects of colchicine, an inhibitor of microtubules (26), on the surface and functional expression of the Na+-K+-2Cl- cotransporter in oocytes coinjected with L-mBSC1-EGFP or L-mBSC1 cRNA, together with S-mBSC1 cRNA. As shown in Fig. 7A, the fluorescence intensity in L-mBSC1-EGFP+S-mBSC1-coinjected oocytes was lower than oocytes alone and the dibutyryl-cAMP+IBMX increment was prevented by prior exposure to colchicine. Colchicine alone had no effect on the fluorescence intensity either in the L-mBSC1-EGFP-injected group (data not shown) or in the L-mBSC1-EGFP+S-mBSC1-coinjected group. Representative images of an oocyte exposed to colchicine alone or to colchicine and dibutyryl-cAMP+IBMX are shown in Fig. 1, k and l, respectively. No change in fluorescence intensity is observed. Similar results were observed when 86Rb+ uptake was assessed. The 86Rb+ uptake observed in L-mBSC1+S-mBSC1-injected oocytes (6.2 ± 0.6 nmol · oocyte-1 · h-1) was not affected in the presence of colchicine (5.9 ± 0.8 nmol · oocyte-1 · h-1) or by dibutyryl-cAMP+IBMX in the presence of colchicine (6.3 ± 0.8 nmol · oocyte-1 · h-1), suggesting that blocking the exocytosis mechanisms with colchicine prevents the L-mBSC1 response to dibutyryl-cAMP+IBMX (Fig. 7B).


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Fig. 7.   Colchicine prevents the effect of the dibutyryl-cAMP and IBMX on surface expression and transport in L-mBSC1 and S-mBSC1 coinjected oocytes. A: fluorescence intensity in oocytes injected with 25 ng of L-mBSC1-EGFP cRNA alone (open bar) or together with 25 ng of S-mBSC1 (filled bars) with or without colchicine and dibutyryl-cAMP+IBMX as indicated. The filled bars depict the fluorescence intensity in the same group of oocytes before and 30 min after exposure to colchicine and 30 min after exposure to colchicine and dibutyryl-cAMP+IBMX. Representative images are shown in Fig. 1, k and l. **P < 0.001 vs. L-mBSC1-EGFP-injected group. B: bumetanide-sensitive 86Rb+ uptake in oocytes injected with 25 ng of L-mBSC1 cRNA alone (open bar) and together with 25 ng of S-mBSC1 cRNA (filled bars) with or without colchicine and/or dibutyryl-cAMP+IBMX as indicated. Colchicine was added to the preincubation medium at 20 µM, 15 min before the addition of cAMP+IBMX. Each bar represents the mean ± SE of 25 oocytes from 3 different frogs. *P < 0.001 vs. L-mBSC1.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


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