Isoforms of the Na-K-2Cl cotransporter in murine TAL II. Functional characterization and activation by cAMP

Consuelo Plata1, David B. Mount2, Verena Rubio1, Steven C. Hebert2, and Gerardo Gamba1

1 Molecular Physiology Unit, Department of Nephrology and Mineral Metabolism, Instituto Nacional de la Nutrición Salvador Zubirán and Department of Medicine, Instituto de Investigaciones Biomédicas, National University of Mexico, Mexico City CP 14000, Mexico; and 2 Division of Nephrology, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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The functional properties of alternatively spliced isoforms of the mouse apical Na+-K+-2Cl- cotransporter (mBSC1) were examined, using expression in Xenopus oocytes and measurement of 22Na+ or 86Rb+ uptake. A total of six isoforms, generated by the combinatorial association of three 5' exon cassettes (A, B, and F) with two alternative 3' ends, are expressed in mouse thick ascending limb (TAL) [see companion article, D. B. Mount, A. Baekgaard, A. E. Hall, C. Plata, J. Xu, D. R. Beier, G. Gamba, and S. C. Hebert. Am. J. Physiol. 276 (Renal Physiol. 45): F347-F358, 1999]. The two 3' ends predict COOH-terminal cytoplasmic domains of 129 amino acids (the C4 COOH terminus) and 457 amino acids (the C9 terminus). The three C9 isoforms (mBSC1-A9/F9/B9) all express Na+-K+-2Cl- cotransport activity, whereas C4 isoforms are nonfunctional in Xenopus oocytes. Activation or inhibition of protein kinase A (PKA) does not affect the activity of the C9 isoforms. The coinjection of mBSC1-A4 with mBSC1-F9 reduces tracer uptake, compared with mBSC1-F9 alone, an effect of C4 isoforms that is partially reversed by the addition of cAMP-IBMX to the uptake medium. The inhibitory effect of C4 isoforms is a dose-dependent function of the alternatively spliced COOH terminus. Isoforms with a C4 COOH terminus thus exert a dominant negative effect on Na+-K+-2Cl- cotransport, a property that is reversed by the activation of PKA. This interaction between coexpressed COOH-terminal isoforms of mBSC1 may account for the regulation of Na+-K+-2Cl- cotransport in the mouse TAL by hormones that generate cAMP.

sodium-potassium-chloride cotransporter; bumetanide; protein kinase A; adenosine 3',5'-cyclic monophosphate; thick ascending limb of Henle; alternative splicing


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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SALT REABSORPTION in the thick ascending limb of Henle (TAL) involves the cooperation of apical bumetanide-sensitive Na+-K+-2Cl- cotransport and apical K+ channels with basolateral Cl- permeation pathways and basolateral Na+-K+-ATPase (33). The molecular identity of the involved transporters and channels is at least partially known, following the cloning of the renal bumetanide-sensitive Na+-K+-2Cl- cotransporter (BSC1/NKCC2) (8, 25), an apical inwardly rectifying K+ channel (ROMK) (12), and basolateral Cl- channels (CLC-K1/K2 and CLCN-KA/KB; Ref. 27). Bartter's syndrome, an inherited metabolic alkalosis with profound defects in the function of the TAL, can be caused by mutations in BSC1/NKCC2 (30), ROMK (14, 31), or CLC-KNB (29), emphasizing the functional importance of each transport pathway.

We previously identified a cDNA (rBSC1) encoding the apical Na+-K+-2Cl- cotransporter from outer medulla of rat kidney (8), and we detail the identification of six alternatively spliced isoforms of mouse BSC1 in the companion article (22). Similar cDNAs, designated NKCC2, have been isolated from rabbit (25), mouse (13), and human kidney (30). The BSC1 nomenclature will be used in this study. BSC1 is a renal-specific gene, and BSC1 protein is expressed at the apical membrane of medullary and cortical TAL (MTAL and CTAL, respectively) (6, 17). BSC1 is a member of the cation-chloride cotransporter gene family (23), with which it shares a putative membrane topology. Alternative splicing of coding exon 4 of the BSC1 gene, which encodes a portion of the predicted second transmembrane segment and the contiguous intracellular loop, involves three mutually exclusive cassette exons A, B, and F (13, 25). Heterologous expression of rBSC1-F in Xenopus laevis oocytes clearly induces a bumetanide-sensitive Na+- and Cl--dependent uptake of 86Rb+, a substitute for K+ (8). A chimeric rabbit BSC2/BSC1-A cDNA, which directs the synthesis of a protein with the amino-terminal domain of shark BSC2 fused to the remainder of rabbit BSC1-A, is also active as a bumetanide-sensitive Na+-K+-2Cl- cotransporter (15). However, functional expression of all three cassette exons in a full-length BSC1 cDNA has not been reported.

Several hormones, including vasopressin, activate adenylate cyclase in mouse TAL and thus generate cAMP (3). The subsequent activation of protein kinase A (PKA) stimulates transepithelial ion transport. Vasopressin stimulates apical K+ conductance in mouse TAL (10), and direct phosphorylation by PKA is required for full activity of the ROMK K+ channel (34). Basolateral chloride conductance can also be stimulated by PKA, although this mechanism may be redundant at physiological intracellular Cl- concentrations (27). Vasopressin appears to directly activate the apical Na+-K+-2Cl- cotransporter (BSC1) in mouse TAL (21); however, the mechanism of this stimulation has not been addressed at the molecular level. Moreover, intriguing functional observations have been made in mouse TAL. First, there appears to be heterogeneity in at least the magnitude of the effect of cAMP in MTAL vs. CTAL, since vasopressin has little to no effect on Na+-Cl- absorption in the latter segment (11). Second, vasopressin can switch cotransport in mouse MTAL from a completely K+-independent, but bumetanide-sensitive Na+-Cl- mode to a K+-dependent, bumetanide-sensitive Na+-K+-2Cl- mode (32). This latter phenomenon has implications for the cellular physiology of the MTAL, where K+-recycling is largely responsible for the generation of the positive luminal potential difference that drives paracellular cation transport (9, 33). Evidence for K+-independent Na+-Cl- cotransport has also been obtained for rabbit (1) and rat outer medulla (20).

Another level of regulation of apical Na+-K+-2Cl- cotransport appears to involve alternative splicing, as characterized in the companion article (22). In addition to the three full-length mBSC1 isoforms with the previously described A, B, and F cassettes, three new mBSC1 isoforms have been identified, resulting from the combination of alternative splicing at the 5' end with alternative splicing of the 3' end. This alternative 3' end arises from the utilization of a polyadenylation site in the intron between coding exons 16 and 17 and predicts a protein with a shorter COOH-terminal domain. This splicing event appears to be independent of the splicing of exon 4, such that a total of six isoforms are produced in mouse kidney. We have designated the longer "default" COOH-terminal domain as "C9" and the truncated COOH-terminal domain as "C4." In the present study, we describe the functional characteristics of these isoforms, demonstrating Na+-K+-2Cl- cotransport for the C9 isoforms and a dominant negative effect of the C4 isoforms that is modulated by cAMP-dependent processes.


    METHODS
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INTRODUCTION
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Animals and materials. Adult female X. laevis frogs were purchased from Carolina Biological Supply (Burlington, NC) and 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. Frogs were fed with frog brittle dry food from Nasco, and water was changed twice a week. Tracer sodium (22Na+) and rubidium (86Rb+) were purchased from DuPont-NEN (Boston, MA). Ouabain, amiloride, bumetanide, IBMX, and general chemicals were from Sigma (St. Louis, MO). Dibutyryl-cAMP, collagenase B, and all restriction enzymes and other enzymes were from Boehringer (Mannheim, Germany) and New England Biolabs (Beverly, MA). N-(2{[3-(4-bromophenyl)-2-propenyl]-amino}-ethyl)-5-isoquinolinesulfonamide (H-89) was from Calbiochem (La Jolla, CA).

X. laevis oocyte preparation and injection. Clusters of oocytes were surgically harvested from anesthetized frogs under 0.17% tricaine and incubated for 1 h with vigorous shaking at room temperature in Ca2+-free ND-96 (in mM: 96 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES-Tris, pH 7.4) and 2 mg/ml of collagenase B, after which oocytes were manually defolliculated. Oocytes were incubated overnight at 17°C in ND-96 supplemented with 2.5 mM sodium pyruvate and 5 mg/100 ml of gentamicin. On the next day, stage V-VI oocytes were injected with 50 nl of water or a solution containing cRNA at a concentration of 0.5 µg/µl (25 ng/oocyte). In coinjection experiments, oocytes were injected with 50 nl of a solution containing 0.5 µg/µl of each coinjected cRNA (50 ng/oocyte). After injection, oocytes were incubated at 17°C in ND-96 supplemented with 2.5 mM sodium pyruvate and 5 mg/100 ml of gentamicin for 3 days. The oocyte incubation medium was changed every day. The day before the isotopic uptake experiment, oocytes were incubated overnight in Cl--free frog Ringer (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), to reduce cell Cl- activity, increasing the driving force for tracer uptake and/or activating Na+-K+-2Cl- cotransport (8).

Generation of mBSC1 cDNAs. The mBSC1 isoforms identified in the companion study (22) were assessed for functional activity. These isoforms are denoted by the relevant cassette exon (A, B, F) and alternative COOH terminus (C4 or C9) (22). Since mBSC1-A4 and mBSC1-F9 were isolated as full-length cDNA clones, they were not modified for this purpose. Full-length mBSC1-A9 and mBSC1-F4 were generated by interchanging A and F cassettes between mBSC1-A4 and mBSC1-F9, using unique Nsi I (nucleotide 752 of mBSC1-A4, 730 of mBSC1-F9; GenBank accession nos. U61381 and U94518, respectively) and Bsm I (nucleotide 1837 of mBSC1-A4; nucleotide 1859 of mBSC1-F9) sites on either side of the cassettes. The short B cassette cDNA (22) was lengthened by PCR to 318 nucleotides, using long primers which overlapped with its 5' and 3' ends. This PCR product was digested with Nsi I and Afl II (nucleotide 1049 of mBSC1-A4) and ligated into the Nsi I and Afl II sites of mBSC1-A4 to obtain mBSC1-B4; sequencing of the DNA between these two restriction sites verified the fidelity of the PCR. Full-length mBSC1-B9 was then generated by inserting the B cassette and flanking DNA into mBSC1-F9, using the unique Bsm I and Nsi I sites. The mBSC1 isoforms used in the present study are all in the plasmid pSPORT1 (GIBCO-BRL). To address the functional effect of the CCC-CAC (proline-histidine) polymorphism identified at codon 11 (22), this segment of the mBSC1-A9, mBSC1-B9, and mBSC1F9 cDNAs was replaced with that of mBSC1-A4, using the Nsi I site and the Kpn I site in the 5' multicloning site of pSPORT1.

For preparation of cRNA template, each isoform cDNA was first linearized at the 3' end using Not I or Xba I restriction enzymes, and then cRNA was transcribed in vitro, using the T7 RNA polymerase in the presence of Cap analog (7-methyl GpppG; Boehringer Mannheim). Transcription product integrity was confirmed on agarose gels, and concentration was determined by absorbance reading at 260 nm (model DU 640; Beckman, Fullerton, CA). cRNA was stored frozen in aliquots at -80°C.

Functional characterization of mBSC1 isoforms. Functional expression of mBSC1 isoforms was assessed by measuring tracer 22Na+ or 86Rb+ uptake in groups of 20-25 oocytes 4 days after water or cRNA injection. 22Na+ uptake was measured with the following protocol: a 30-min incubation period in a hypotonic K+- and Cl--free medium (in mM: 75 sodium gluconate, 6.0 calcium gluconate, 1.0 magnesium gluconate, and 5 HEPES-Tris, pH 7.4) with 1 mM ouabain, 100 µM amiloride, ±100 µM bumetanide, ±1 mM dibutyryl-cAMP + 1 mM IBMX; followed by a 60-min uptake period in a hypotonic uptake medium (in mM: 62 NaCl, 10 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES-Tris, pH 7.4) containing 2.5 µCi/ml of 22Na+ (DuPont-NEN) and the same drugs used during incubation period. The hypotonic conditions inhibit the endogenous oocyte Na+-K+-2Cl- cotransporter (8). Ouabain was added to prevent Na+ exit via Na+-K+-ATPase, and amiloride prevents Na+ uptake via Na+ channels or Na+/H+ antiporters. To determine the K+- and Cl--dependent fraction of Na+ uptake, paired groups of oocytes were incubated in uptake media without Cl- (substituted with gluconate) or without K+ [substituted with N-methyl-D-glucamine (NMDG)].

86Rb+ uptake was assessed using the following protocol. A 30-min incubation period in a hypotonic Na+/K+-free solution (in mM: 75 NMDG-Cl-, 1.8 CaCl2, 1 MgCl2, and 5 HEPES-Tris, pH 7.4) with 1 mM ouabain ± 100 µM bumetanide, followed by a 60-min uptake period using a hypotonic uptake medium (in mM: 58 NaCl, 2 KCl, 10 BaCl2, 1.8 CaCl2, 1.0 MgCl2, and 5.0 HEPES, pH 7.4) with 1 mM ouabain, ±100 µM bumetanide, and 10 µCi/ml of 86Rb+, specific activity 0.57 µCi/nmol (DuPont-NEN). For ion-dependency experiments, Na+ was replaced in the uptake solution by NMDG and Cl- was replaced by gluconate.

All uptakes were performed at 30°C and were linear over the first 60 min. At the end of the uptake period, oocytes were washed five times in ice-cold uptake solution without isotope to remove extracellular fluid tracer. After the oocytes were dissolved in 10% SDS, tracer activity was determined by beta scintillation counting. The degree of variability between experiments is due to seasonal variations in the quality of oocytes, as well as the relative quality of cRNA. Thus experiments were compared with internal controls done under identical conditions, using oocytes prepared from the same donor.

Statistical analysis. Statistical significance is defined as two-tailed P < 0.05, and the results are presented as means ± SE. The significance of the differences between groups were tested by one-way ANOVA with multiple comparison using Bonferroni correction or by the Kruskal-Wallis one-way analysis of variance on ranks with the Dunn method for multiple comparison procedure, as needed.


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ABSTRACT
INTRODUCTION
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Expression of mBSC1 C9 isoforms in Xenopus oocytes. Microinjection of Xenopus oocytes with cRNA in vitro transcribed from the C9 isoforms mBSC1-F9, mBSC1-A9, and mBSC1-B9 resulted in significant increases in 22Na+ uptake when compared with water-injected oocytes. As Fig. 1A shows, the increased Na+ uptake observed in oocytes with the three C9 isoforms was K+ dependent, Cl- dependent, and bumetanide sensitive. In addition, Fig. 1B shows that the three C9 isoforms also exhibit increased tracer Rb+ uptake that was Na+-dependent, Cl- dependent, and bumetanide sensitive. Furthermore, the mBSC1-F9, mBSC1-A9, and mBSC1-B9 cDNAs containing the polymorphism CCC (proline) or CAC (histidine) at the codon 11 in the NH2-terminal domain (22) also induced Na+-K+-2Cl- cotransport in oocytes (data not shown). Therefore, the three C9 isoform cDNAs containing the A, B, or F cassette exons within the putative second transmembrane domain encode bumetanide-sensitive Na+-K+-2Cl- cotransporters, under the conditions utilized.


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Fig. 1.   Functional expression of mBSC1-9 isoforms in X. laevis oocytes injected with water (solid bars) or with 25 ng of cRNA from either mBSC1-F9 (open bars), mBSC1-A9 (hatched bars), or mBSC1-B9 (shaded bars). A: 22Na+ uptake in groups of oocytes that were exposed to tracer Na+ in presence or absence of extracellular K+ (i.e., [K+]e), Cl- ([Cl-]), or bumetanide, as indicated. B: 86Rb+ uptake in absence or presence of extracellular Na+, Cl- or bumetanide. * Significant difference (P < 0.001) from uptakes in presence of Na+, K+, and Cl-.

Since Na+-K+-2Cl- transport in TAL is affected by cAMP and PKA (21), initial experiments addressed the regulation of Na+-K+-2Cl- transport by PKA, using oocytes expressing the full-length, functional C9 isoforms. Figure 2A depicts the pooled results from seven experiments in which oocytes were obtained from different frogs. mBSC1-F9-injected oocytes expressed increased tracer Na+ uptake that was completely abolished in the presence of 100 µM bumetanide but which was not affected by the addition of a cell membrane-permeable analog of cAMP plus the phosphodiesterase inhibitor IBMX to the uptake medium (2,090 ± 93 vs. 2,102 ± 87 pmol · oocyte-1 · h-1, respectively; P = not significant). In addition to this finding, Fig. 2B shows the pooled results from three experiments in which increased Na+ uptake induced by mBSC1-F9 cRNA injection into oocytes was not affected by the addition of the PKA inhibitor H-89 to the uptake medium. Equivalent results were obtained with mBSC1-A9 (data not shown). Thus the Na+-K+-2Cl- cotransport induced by heterologous expression of mBSC1 C9 isoforms was not affected by PKA activation with cAMP-IBMX or by direct inhibition of the endogenous PKA activity.


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Fig. 2.   Effects of cAMP on 22Na+ uptake of Xenopus oocytes injected with water (hatched bar) or with mBSC1-F9 cRNA (open bars). A: effect of protein kinase A (PKA) activation by the addition of 1 mM dibutyryl-cAMP + 1 mM IBMX to uptake medium. B: effect of PKA inhibition by addition of 20 µM H-89. Each bar represents the mean ± SE from 7 different experiments in A and 3 experiments in B.

Expression of mBSC1 C4 isoforms in oocytes. Figure 3 depicts the results of 11 pooled experiments showing that oocytes injected with up to 25 ng of mBSC-A4 isoform cRNA do not express any increase in 22Na+ uptake. A similar observation was made assessing 86Rb+ uptake in oocytes injected with cRNA from the mBSC-F4 isoform (data not shown). Thus oocytes injected with the truncated C4 isoforms mBSC1-F4 and mBSC1-A4 did not express significantly increased 22Na+ or 86Rb+ uptake, compared with water-injected oocytes. Insignificant functional activity was also observed in oocytes injected with mBSC1-F4 cRNA transcribed from a vector containing Xenopus beta -globin 5'- and 3'-untranslated regions (19) (data not shown). Since the C4 and C9 isoforms differ in consensus PKA and PKC sites (22), the effect of phorbol ester and dibutyryl-cAMP-IBMX was examined; these agents did not affect transport in mBSC1-A4-injected oocytes (data not shown).


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Fig. 3.   Functional expression of mBSC-A4 isoform. 22Na+ uptake in oocytes injected either with water or with cRNA from mBSC1-A4 and mBSC1-F9, as indicated. Tracer uptake was performed in absence (open bars) or presence (solid bar) of 100 µM bumetanide. Each bar represents mean ± SE from 11 experiments.

Coexpression of mBSC1 isoforms and regulation by cAMP. As detailed in the companion study, mBSC1 isoforms with both the C4 and C9 COOH termini are coexpressed within TAL cells. Given the lack of transporter function in oocytes injected with cRNA from the C4-type isoforms, as well as the hypothesis that the COOH terminus of the Na+-K+-2Cl- cotransporter could be involved in functional regulation of the protein, we measured 22Na+ uptake in Xenopus oocytes coinjected with cRNA from one of the long COOH-terminal C9-type isoforms together with cRNA from one of the C4-type truncated isoforms. When the full-length mBSC1-F9 isoform was coinjected with the shorter mBSC1-A4 truncated cRNA, a significant reduction in Na+ uptake was observed, compared with the mBSC1-F9-injected oocytes. Figure 4 shows the effect of increased amount of mBSC1-A4 cRNA in oocytes that were injected with mBSC1-F9 cRNA. Oocytes were coinjected with a fixed amount of mBSC1-F9 cRNA (25 ng/oocyte) plus increasing concentrations of mBSC1-A4 cRNA, from 6.25 ng to 50 ng per oocyte. Increasing the concentration of mBSC1-A4 cRNA progressively inhibited the increased uptake that was observed in oocytes injected with mBSC1-F9 cRNA. Thus the reduction in Na+-K+-2Cl- cotransport induced by coinjection with mBSC1 C4 isoforms is dose dependent. Furthermore, Fig. 5 shows that the negative effect of mBSC1 C4 cRNAs in C4/C9 coinjected oocytes is not due to simple competition for translation, since unrelated cRNA, specifically 25 ng of renin cRNA or Shaker K+ channel, did not decrease Na+ uptake when coinjected with 25 ng of mBSC1-F9 cRNA. In addition, the interaction between C9 and C4 isoforms is not affected by the presence of the 5' A/B/F mutually exclusive cassettes. As Fig. 6A shows, both mBSC1-F4 and -A4 isoforms equally reduced increased Na+ uptake induced by mBSC1-F9 cRNA expression. Similarly, Fig. 6B shows that the function of the Na+-K+-2Cl- cotransporter induced by mBSC1-A9 cRNA was reduced by mBSC1-A4 and -F4.


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Fig. 4.   Effect of increasing concentrations of mBSC1-A4 cRNA in Xenopus oocytes on functional expression of the Na+-K+-2Cl- cotransporter. Tracer Na+ uptake was assessed on groups of 25 oocytes that were injected with water (hatched bar), with 25 ng of mBSC1-F9 cRNA alone or together with increasing amounts of mBSC1-A4 cRNA (open bars). Uptake in the same groups of oocytes was also assessed in presence of 100 µM bumetanide (solid bars). * P < 0.01 vs. respective control group.


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Fig. 5.   22Na+ uptake in Xenopus oocytes injected with mBSC1-F9 cRNA and unrelated cRNAs. A: oocytes injected with water (open bar), 25 ng of mBSC1-F9 cRNA alone (hatched bar), or 25 ng of mBSC1-F9 with 25 ng of renin cRNA (shaded). B: oocytes injected with water (open bar), 25 ng of mBSC1-F9 cRNA alone (hatched bar), or 25 ng of mBSC1-F9 with 25 ng of Shaker K+ channel cRNA (solid bar).


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Fig. 6.   22Na+ uptake in Xenopus oocytes coinjected with mBSC1 C4 and C9 isoforms alone or in combination. A: 22Na+ uptake in oocytes injected with water (hatched bars), mBSC1-F9, mBSC1-F9 + mBSC1-F4, or mBSC1-F9 + mBSC1-A4, as indicated. B: oocytes injected with water (hatched bar), mBSC1-A9, mBSC1-A9 + mBSC1-A4, and mBSC1-A9 + mBSC1-F4, as indicated. In both A and B, the solid bar depicts uptake in presence of 100 µM bumetanide. * P < 0.001 vs. control uptake in mBSC1-F9 or mBSC1-A9 groups.

Since K+-independent bumetanide-sensitive Na+-Cl- cotransport has been described in mouse TAL (32), the ion dependency of the 22Na+ uptake in coinjected oocytes was examined. Figure 7 shows that Na+ uptake observed in C9/C4 coinjected oocytes remains K+ dependent, Cl- dependent, and bumetanide sensitive, indicating that the two isoforms together form a K+-dependent, bumetanide-sensitive Na+-K+-2Cl- cotransporter.


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Fig. 7.   Ion dependency and bumetanide sensitivity of Na+ uptake in Xenopus oocytes injected with mBSC1-F9 cRNA or coinjected with mBSC1-F9 + mBSC1-A4 cRNA. Tracer uptake was performed in presence of the 3 cotransported ions (open bars), in absence of extracellular K+ (hatched bars), in absence of extracellular Cl- (shaded bars), or in presence of 100 µM bumetanide (solid bars). * P < 0.0001 vs. C9 control. ** P < 0.001 vs. mBSC1-F9 + mBSC1-A4 control.

Given the lack of effect of cAMP on Na+-K+-2Cl- cotransporter function induced by C9 isoforms in Xenopus oocytes (Fig. 2), we measured the effect of cAMP on tracer uptake in C4/C9 coinjected oocytes. Figure 8 shows the pooled results from seven experiments in which oocytes were injected with mBSC1-F9 cRNA alone or together with mBSC1-A4 cRNA. The reduction in Na+ uptake in C9/C4 coinjected oocytes was partially reversed by the addition of cAMP-IBMX to the uptake medium (1,023 ± 62 vs. 1,514 ± 97 pmol · oocyte-1 · h-1, respectively; P < 0.001). A similar result was obtained when mBSC1-F9 and mBSC1-F4 were coexpressed. Thus, in Xenopus oocytes, the function of the Na+-K+-2Cl- cotransporter can be modulated by cAMP only when mBSC1-9 and mBSC1-4 isoforms are coexpressed, but not when a mBSC1-9 isoform is expressed alone (Fig. 2). Note in Fig. 8 that Na+ uptake in the coinjected oocytes, with or without cAMP-IBMX, exhibited bumetanide sensitivity, consistent with Na+-K+-2Cl- cotransport activity.


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Fig. 8.   Effects of cAMP + IBMX in mBSC1-A4-induced reduction of Na+-K+-2Cl- cotransport activity. 22Na+ uptake of Xenopus oocytes injected with water (hatched bar), mBSC1-F9 isoform cRNA alone (open bars), or with mBSC1-F9 + mBSC1-A4 cRNAs (solid bars). Either 1 mM dibutyryl-cAMP + 1 mM IBMX or 100 µM bumetanide was added to the uptake medium as indicated. Each bar represents mean ± SE from 7 different experiments with oocytes obtained from 7 frogs. * P < 0.001 vs. mBSC1-F9-injected group. ** P < 0.001 vs. mBSC1-F9 + mBSC1-A4 injected oocytes.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present work describes the functional properties of alternatively spliced mBSC1 isoforms that we have identified from mouse kidney mRNA (22). The cDNA sequence of three of these isoforms predicts proteins of 1,095 amino acid residues containing a long COOH-terminal domain of 457 amino acids in length, referred to here as the C9 COOH-terminal domain. The difference between the individual C9 isoforms arises from the alternative splicing of three variants of coding exon 4, denoted A, B, and F by Payne and Forbush (25). The data in the present article show that the three C9 mBSC1 isoforms from mouse kidney encode bumetanide-sensitive Na+-K+-2Cl- cotransporters. These alternatively spliced isoforms were potential candidates (25) for the K+-independent bumetanide-sensitive Na+-Cl- transport activity, for which there is functional evidence in mouse, rabbit, and rat kidney (1, 20, 32). However, all three isoforms catalyze K+-dependent uptake of 22Na+ and bumetanide-sensitive uptake of 86Rb+ in oocytes and thus do not encode K+-independent Na+-Cl- cotransporters under the conditions utilized. In the experiments using 22Na+ uptake (Fig. 1A), all three isoforms showed some residual K+-independent uptake, presumably due to the difficulty in preparing solutions that are completely devoid of this and equivalent ions (e.g., NH+4). The functional differences between these isoforms are not completely clear. However, reproducible differences in uptake were observed, such that oocytes injected with mBSC1-F9 consistently had the highest transport activity (Fig. 1). A difference in translational efficiency and membrane expression is possible, although these isoforms differ only in the 96-bp cassette exons. The individual isoforms may differ in the affinity for a transported ion or ions; however, thus far we have not been able to use the oocyte expression system for kinetic analysis of BSC1 (unpublished data). Kinetic analysis of a chimeric rabbit BSC1-A9 construct, expressed in HEK-293 cells, has recently been reported (15). Unfortunately, chimeric rabbit BSC1-F9 and BSC1-B9 constructs cannot be expressed in mammalian cells, nor can full-length nonchimeric BSC1 cDNAs (15). Therefore, kinetic comparison of the cassette exons in BSC1 constructs is not possible at this time.

The sequence of another class of cDNA clones isolated from mouse outer medulla predicts proteins of 770 amino acid residues, containing a shorter COOH-terminal domain of 129 amino acids in length (22). We have called this alternative COOH-terminal domain C4. RT-PCR clones detailed in the companion study demonstrate that all three of the 5' A/B/F cassettes can pair with the C4 3' end in renal mRNA. An isoform-specific antibody demonstrates that mBSC1 proteins with the C4 COOH terminus are expressed in mouse kidney along the length of the TAL (22). Isoforms of mBSC1 with a C4 COOH terminus did not express Na+-K+-2Cl- cotransport in Xenopus oocytes (Fig. 3). Of potential significance, the 55 amino acid residues at the end of the C4 COOH-terminal domain are distinct, and predict different consensus sites for PKC- and PKA-dependent phosphorylation (see figure 1 in Ref. 26). Since it is possible that these isoforms are differentially affected by phosphorylation processes, we also assessed the effects of modulating PKA and PKC on the function of mBSC1-A4; no increase in 22Na+ uptake was observed (data not shown).

The stimulation of cAMP formation by hormones such as vasopressin activates transepithelial salt transport in the murine TAL (3). Initial experiments thus examined the effect of cAMP on mBSC1 isoforms expressed in Xenopus oocytes. In oocytes injected with any of the three C9 isoforms, however, activation of PKA with cAMP did not affect 22Na+ uptake. In addition, since previous work with the ROMK (34) and ENaC (28) cation channels had suggested that PKA is active in the unstimulated Xenopus oocyte, the effect of inhibiting basal PKA activity was examined. However, the PKA inhibitor H-89 did not affect transport in oocytes expressing mBSC1 isoforms (Fig. 2B). These combined observations suggest that other factors are required to reconstitute the observed cAMP activation of apical Na+-K+-2Cl- cotransport in murine TAL.

Given that the two alternative COOH-terminal mBSC1 isoforms are coexpressed in TAL (see companion article, Ref. 22), the effect of coinjecting both types of mBSC1 isoforms was examined. In multiple experiments, C4 isoforms reduced the transport activity of the high-expressing mBSC1-F9 isoform. This effect occurred irrespective of which of the 5' cassettes were included in the coexpressed cRNAs (Fig. 6). Thus mBSC1-F4 cRNA inhibits the uptake of oocytes expressing either mBSC1-F9 or mBSC1-A9 isoforms, and mBSC1-A4 reduces uptake expressed by mBSC1-F9 or by mBSC1-A9. The 22Na+ uptake in C9/C4 coinjected oocytes is K+ and Cl- dependent (Fig. 7), indicating that together the two isoforms do not form a bumetanide-sensitive Na+-Cl- cotransporter under the experimental conditions used here. The possibility of competition for translation in C4/C9 coinjected oocytes is unlikely to account for the reduced 22Na+ uptake, since coinjecting mBSC1-F9 with renin or Shaker K+ channel, unrelated cRNAs, did not significantly reduce uptake (Fig. 6). Moreover, in contrast to the lack of a cAMP effect in oocytes injected with C9 isoforms alone, cAMP abrogated the inhibitory effect of C4 isoforms, suggesting a specific functional effect. Therefore, COOH-terminal truncated isoforms exert a dominant negative function on the ion transport expressed by full-length C9 isoforms, a property that is reversed by cAMP. Heterogeneity of immunostaining for the C4 antibody is described in the companion study (22), such that not all cells are positive for the C4 antigen. Moreover, the intensity of staining and distribution of positive cells is axially distributed, such that CTAL appears to express less C4 than outer medullary segments (22). This heterogeneity may underlie the observed difference in vasopressin-sensitive Na+-Cl- cotransport in CTAL vs. MTAL (11).

There is precedence for the dominant negative effect of the truncated C4 isoforms of mBSC1. For example, two groups have reported a dominant negative property for an alternatively spliced isoform of the long-QT syndrome K+ channel (5, 16). This isoform, which predicts an NH2-terminal truncation of the channel protein, is inactive on its own, but has a dominant negative effect on the channel activity of the full-length isoform, both in Xenopus oocytes (16) and in mammalian cells (5). The generation of dominant negative isoforms by alternative splicing has also been demonstrated in nonmembrane proteins, particularly for transcription factors (2). Finally, an isoform (alpha 2I) of the alpha 2-subunit of soluble guanylyl cyclase with a 31 amino acid alternatively spliced insert has been identified (4). The alpha 2I isoform competes with the alpha 2-subunit for dimerization with the beta 1-subunit, producing a nonfunctional guanylyl cyclase that may play a role in the regulation of nitric oxide-sensitive guanylyl cyclase activity.

The functional effects observed here suggest a number of testable hypotheses. First, BSC1 proteins likely form a multimeric complex; indeed, preliminary immunoprecipitation experiments with an amino-terminal antibody suggest that they may form oligomers (18). Like thiazide-sensitive cotransporter, TSC (26), BSC1 proteins are detected within subapical vesicles (24), and trafficking of these vesicles to the apical membrane may play a role in regulation. The staining of mouse kidney with anti-C4 antibody suggests that a significant proportion of these isoforms have a subapical distribution (22), and it is possible that the modification of the mBSC1 COOH terminus by alternative splicing affects the interaction of the transporter proteins with the cytoskeleton (7) and/or the vesicular trafficking machinery. If so, then coassociation of C4 isoforms with C9 isoforms may play a role at this level of regulation. Specifically, C4 isoforms may either "retrieve" the BSC1 complex from the apical membrane or "trap" it within vesicles. Alternatively, heteromeric complexes of C4 and C9 isoforms may be inactive in transport at the apical membrane. The results obtained in Xenopus oocytes suggest that such interactions would be sensitive to cAMP, perhaps due to direct transporter phosphorylation. Regulation of Na+-K+-2Cl- transport could also occur at the level of alternative splicing, and changing the ratio of alternatively spliced isoforms may play a role in the response to physiological stimuli such as water deprivation and water loading. Finally, if equivalent splicing events exist in humans, then genetic modulation of the relative efficiency of alternative splicing may affect the pathophysiology of the TAL in disorders such as Bartter's syndrome and hypertension. Such a phenomenon is likely in genetic forms of the cardiac long-QT syndrome (5).

In summary, in the present study we have addressed the functional characteristics of alternatively spliced isoforms of mBSC1 gene. The three C9 isoforms containing a full-length COOH-terminal domain and the A, B, or F cassette exons clearly encode bumetanide-sensitive Na+-K+-2Cl- cotransporters, when expressed in Xenopus oocytes. In contrast, the C4 isoforms containing the truncated C4 COOH-terminal domain do not induce cotransporter activity by themselves, but give rise to a dominant negative effect on Na+-K+-2Cl- cotransporter function that can be modulated by cAMP. These processes may play a role in the regulation of Na+-K+-2Cl- cotransport in TAL by vasopressin and other hormones.


    ACKNOWLEDGEMENTS

We are grateful to Dr. Octavio Villanueva for help with animal care and to members of the Molecular Physiology Unit for suggestions and stimulating discussion.


    FOOTNOTES

This work was supported by Research Grant 3840 from the Mexican Council of Science and Technology (CONACYT) and Grant 75197-553601 from Howard Hughes Medical Institute to G. Gamba, and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-36803 and DK-02103 to S. C. Hebert and D. B. Mount, respectively. C. Plata is supported by scholarship grants from CONACYT and Dirección, General del Personal Académico of the National University of Mexico.

Portions of this work were presented at the 27th, 28th, and 29th Annual Meetings of the American Society of Nephrology and have been published in abstract form (J. Am. Soc. Nephrol. 5: 282, 1994; J. Am. Soc. Nephrol. 6: 347, 1995; and J. Am. Soc. Nephrol. 7: 1288, 1996).

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. §1734 solely to indicate this fact.

Address for reprint requests: G. Gamba, Molecular Physiology Unit, Instituto Nacional de la Nutrición Salvador Zubirán, Instituto de Investigaciones Biomédicas, UNAM, Vasco de Quiroga No. 15, Tlalpan 14000, Mexico City, Mexico (E-mail: gamba{at}mailer.main.conacyt.mx).

Received 15 June 1998; accepted in final form 30 October 1998.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alvo, M., J. Calamia, and J. Eveloff. Lack of potassium effect on Na+-Cl- cotransport in the medullary thick ascending limb. Am. J. Physiol. 249 (Renal Fluid Electrolyte Physiol. 18): F34-F39, 1985[Medline].

2.   Bach, I., and M. Yaniv. More potent transcriptional activators or a transdominant inhibitor of the HNF1 homeoprotein family are generated by alternative RNA processing. Embo J 12: 4229-4242, 1993[Abstract].

3.   Bailly, C. Transducing pathways involved in the control of Na+-Cl- reabsorption in the thick ascending limb of Henle's loop. Kidney Int. Suppl. 65: S29-S35, 1998[Medline].

4.   Behrends, S., C. Harteneck, G. Schultz, and D. Koesling. A variant of the alpha 2 subunit of soluble guanylyl cyclase contains an insert homologous to a region within adenylyl cyclases and functions as a dominant negative protein. J. Biol. Chem. 270: 21109-21113, 1995[Abstract/Free Full Text].

5.   Demolombe, S., I. Baro, Y. Pereon, J. Bliek, R. Mohammad-Panah, H. Pollard, S. Morid, M. Mannens, A. Wilde, J. Barhanin, F. Charpentier, and D. Escande. A dominant negative isoform of the long QT syndrome 1 gene product. J. Biol. Chem. 273: 6837-6843, 1998[Abstract/Free Full Text].

6.   Ecelbarger, C. A., J. Terris, J. R. Hoyer, S. Nielsen, J. B. Wade, and M. A. Knepper. Localization and regulation of the rat renal Na+-K+-2Cl- cotransporter, BSC-1. Am. J. Physiol. 271 (Renal Fluid Electrolyte Physiol. 40): F619-F628, 1996[Abstract/Free Full Text].

7.   Ehlers, M. D., E. T. Fung, R. J. O'Brien, and R. L. Huganir. Splice variant-specific interaction of the NMDA receptor subunit NR1 with neuronal intermediate filaments. J. Neurosci. 18: 720-730, 1998[Abstract/Free Full Text].

8.   Gamba, G., A. Miyanoshita, M. Lombardi, J. Lytton, W. S. Lee, M. A. Hediger, and S. C. Hebert. Molecular cloning, primary structure, and characterization of two members of the mammalian electroneutral sodium-(potassium)-chloride cotransporter family expressed in kidney. J. Biol. Chem. 269: 17713-17722, 1994[Abstract/Free Full Text].

9.   Greger, R. Ion transport mechanisms in thick ascending limb of Henle's loop of mammalian nephron. Physiol. Rev. 65: 760-797, 1985[Free Full Text].

10.   Hebert, S. C., and T. E. Andreoli. Effects of antidiuretic hormone on cellular conductive pathways in mouse medullary thick ascending limbs of Henle. II. Determinants of the ADH-mediated increases in transepithelial voltage and in net Cl- absorption. J. Membr. Biol. 80: 221-233, 1984[Medline].

11.   Hebert, S. C., R. M. Culpepper, and T. E. Andreoli. NaCl transport in mouse medullary thick ascending limbs. I. Functional nephron heterogeneity and ADH-stimulated Na+-Cl- cotransport. Am. J. Physiol. 241 (Renal Fluid Electrolyte Physiol. 10): F412-F431, 1981[Abstract/Free Full Text].

12.   Ho, K., C. G. Nichols, W. J. Lederer, J. Lytton, P. M. Vassilev, M. V. Kanazirska, and S. C. Hebert. Cloning and expression of an inwardly rectifying ATP-regulated potassium channel. Nature 362: 31-38, 1993[Medline].

13.   Igarashi, P., G. B. Vanden Heuvel, J. A. Payne, and B. Forbush III. Cloning, embryonic expression, and alternative splicing of a murine kidney-specific Na+-K+-Cl- cotransporter. Am. J. Physiol. 269 (Renal Fluid Electrolyte Physiol. 38): F405-F418, 1995[Abstract/Free Full Text].

14.   International Collaborative Study Group. Mutations in the gene encoding the inwardly-rectifying renal potassium channel, ROMK, cause the antenatal variant of Bartter syndrome: evidence for genetic heterogeneity. Hum. Mol. Genet. 6: 17-26, 1997[Abstract/Free Full Text].

15.   Isenring, P., and B. Forbush III. Ion and bumetanide binding by the Na+-K+-Cl- cotransporter. Importance of transmembrane domains. J. Biol. Chem. 272: 24556-24562, 1997[Abstract/Free Full Text].

16.   Jiang, M., J. Tseng-Crank, and G. N. Tseng. Suppression of slow delayed rectifier current by a truncated isoform of KvLQT1 cloned from normal human heart. J. Biol. Chem. 272: 24109-24112, 1997[Abstract/Free Full Text].

17.   Kaplan, M. R., M. D. Plotkin, W. S. Lee, Z. C. Xu, J. Lytton, and S. C. Hebert. Apical localization of the Na+-K+-Cl- cotransporter, rBSC1, on rat thick ascending limbs. Kidney Int. 49: 40-47, 1996[Medline].

18.   Lee, A. J., J. M. Bradfor, J. M. Terris, M. A. Knepper, and C. A. Ecelbarger. The type 1 bumetanide-sensitive Na+-K+-2Cl- cotransporter (BSC1) exists in the plasma membrane as a high molecular weight oligomeric complex (Abstract). J. Am. Soc. Nephrol. 8: 37, 1997.

19.   Liman, E. R., J. Tytgat, and P. Hess. Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs. Neuron 9: 861-871, 1992[Medline].

20.   Ludens, J. H., M. A. Clark, and J. A. Lawson. Does ADH alter cotransporter properties in conscious rats? Evidence for a shift from K+-independent to K+-dependent cotransport (Abstract). J. Am. Soc. Nephrol. 6: 344, 1995.

21.   Molony, D. A., W. B. Reeves, S. C. Hebert, and T. E. Andreoli. ADH increases apical Na+, K+, 2Cl- entry in mouse medullary thick ascending limbs of Henle. Am. J. Physiol. 252 (Renal Fluid Electrolyte Physiol. 21): F177-F187, 1987[Abstract/Free Full Text].

22.   Mount, D. B., A. Baekgaard, A. E. Hall, C. Plata, J. Z. Xu, D. R. Beier, G. Gamba, and S. C. Hebert. Isoforms of the Na-K-2Cl cotransporter in murine TAL. I. Molecular characterization and intrarenal localization. Am. J. Physiol. 276 (Renal Physiol. 45): F347-F358, 1999[Abstract/Free Full Text].

23.   Mount, D. B., R. S. Hoover, and S. C. Hebert. The molecular physiology of electroneutral cation-chloride cotransport. J. Membr. Biol. 158: 177-186, 1997[Medline].

24.   Neilsen, S., A. B. Maunsbach, D. Ecelbarger, and M. A. Knepper. Cellular and subcellular localization of the Na+-K+-2Cl- co-transporter BSC-1 in apical plasma membrane and vesicles of thick ascending limb and macula densa cells (Abstract). J. Am. Soc. Nephrol. 8: 40, 1997[Abstract].

25.   Payne, J. A., and B. Forbush III. Alternatively spliced isoforms of the putative renal Na+-K+-Cl- cotransporter are differentially distributed within the rabbit kidney. Proc. Natl. Acad. Sci. USA 91: 4544-4548, 1994[Abstract].

26.   Plotkin, M. D., M. R. Kaplan, J. W. Verlander, W.-S. Lee, D. Brown, E. Poch, S. R. Gullans, and S. C. Hebert. Localization of the thiazide-sensitive Na+-Cl- cotransporter, rTSC1, in the rat kidney. Kidney Int. 50: 174-183, 1996[Medline].

27.   Reeves, W. B., C. J. Winters, L. Zimniak, and T. E. Andreoli. Properties and regulation of medullary thick limb basolateral Cl- channels. Kidney Int. Suppl. 65: S24-S28, 1998[Medline].

28.   Saxena, S., Y. Oh, M. W. Quick, and D. G. Warnock. cAMP regulation of wild type amiloride-sensitive sodium channels expressed in Xenopus oocytes (Abstract). J. Am. Soc. Nephrol. 7: 1289, 1996.

29.   Simon, D. B., R. S. Bindra, T. A. Mansfield, C. Nelson-Williams, E. Mendonca, R. Stone, S. Schurman, A. Nayir, H. Alpay, A. Bakkaloglu, J. Rodriguez-Soriano, J. M. Morales, S. A. Sanjad, C. M. Taylor, D. Pilz, A. Brem, H. Trachtman, W. Griswold, G. A. Richard, E. John, and R. P. Lifton. Mutations in the chloride channel gene, CLCNKB, cause Bartter's syndrome type III. Nat. Genet. 17: 171-178, 1997[Medline].

30.   Simon, D. B., F. E. Karet, J. M. Hamdan, A. DiPietro, S. A. Sanjad, and R. P. Lifton. Bartter's syndrome, hypokalaemic alkalosis with hypercalciuria, is caused by mutations in the Na+-K+-2Cl- cotransporter NKCC2. Nat. Genet. 13: 183-188, 1996[Medline].

31.   Simon, D. B., F. E. Karet, J. Rodriquez-Soriano, J. H. Hamdan, A. DiPietro, H. Trachtman, S. A. Sanjad, and R. P. Lifton. Genetic heterogeneity of Bartter's syndrome revealed by mutations in the K+ channel, ROMK. Nat. Genet. 14: 152-156, 1996[Medline].

32.   Sun, A., E. B. Grossman, M. Lombardi, and S. C. Hebert. Vasopressin alters the mechanism of apical Cl- entry from Na+:Cl- to Na+:K+:2Cl- cotransport in mouse medullary thick ascending limb. J. Membr. Biol. 120: 83-94, 1991[Medline].

33.   Winters, C. J., W. B. Reeves, and T. E. Andreoli. A survey of transport properties of the thick ascending limb. Semin. Nephrol. 11: 236-247, 1991[Medline].

34.   Xu, Z. C., Y. Yang, and S. C. Hebert. Phosphorylation of the ATP-sensitive, inwardly rectifying K+ channel, ROMK, by cyclic AMP-dependent protein kinase. J. Biol. Chem. 271: 9313-9319, 1996[Abstract/Free Full Text].


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