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
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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METHODS |
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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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RESULTS |
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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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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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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 -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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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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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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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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DISCUSSION |
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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
(2I) of the
2-subunit of soluble guanylyl
cyclase with a 31 amino acid alternatively spliced insert has been
identified (4). The
2I isoform
competes with the
2-subunit for
dimerization with the
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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