1 Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán and Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Tlalpan 14000, Mexico City, Mexico; and 2 Division of Nephrology and Hypertension, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232
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ABSTRACT |
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The K-Cl cotransporters
(KCCs) have a broad range of physiological roles, in a number of cells
and species. We report here that Xenopus laevis oocytes
express a K-Cl cotransporter with significant functional and molecular
similarity to mammalian KCCs. Under isotonic conditions, defolliculated
oocytes exhibit a Cl-dependent
86Rb+ uptake mechanism after activation by the
cysteine-reactive compounds N-ethylmaleimide (NEM) and
mercuric chloride (HgCl2). The activation of this K-Cl
cotransporter by cell swelling is prevented by inhibition of protein
phosphatase-1 with calyculin A; NEM activation of the transporter was
not blocked by phosphatase inhibition. Kinetic characterization reveals
apparent values for the Michaelis-Menten constant of 27.7 ± 3.0 and 15.4 ± 4.7 mM for Rb+ and Cl
,
respectively, with an anion selectivity for K+ transport of
Cl
= PO
> I
> SCN
> gluconate. The
oocyte K-Cl cotransporter was sensitive to several inhibitors,
including loop diuretics, with apparent half-maximal inhibition values
of 200 and 500 µM for furosemide and bumetanide, respectively. A
partial cDNA encoding the Xenopus K-Cl cotransporter was
cloned from oocyte RNA; the corresponding transcript is widely expressed in Xenopus tissues. The predicted COOH-terminal
protein fragment exhibited particular homology to the KCC1/KCC3
subgroup of the mammalian KCCs, and the functional characteristics are the most similar to those of KCC1 (Mercado A, Song L, Vazquez N, Mount
DB, and Gamba G. J Biol Chem 275: 30326-30334, 2000).
potassium-chloride cotransport; cell volume; cell swelling
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INTRODUCTION |
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THE ELECTRONEUTRAL
COTRANSPORT of K+ and Cl is
largely accomplished by parallel K+ and
Cl
channels or via the operation of K-Cl cotransporters.
K-Cl cotransport was first defined in the red blood cell (10,
32), the tissue for which functional characterization is the
most complete. Red blood cell K-Cl cotransport shares a number of
functional properties with Na-K-2Cl cotransport, including
electroneutral characteristics, a functional dependence on the presence
of each transported ion, and sensitivity to loop diuretics. However,
these two transport mechanisms diverge significantly in several
characteristics, in particular their response to cellular volume
changes and to modulation of protein phosphorylation and
dephosphorylation. The Na-K-2Cl cotransport is thus shrinkage activated
and inhibited by protein phosphatases (45), whereas K-Cl
cotransport is activated by cell swelling and completely abolished by
inhibitors of serine/threonine protein phosphatases (10).
In addition to red blood cells, K-Cl cotransport has been detected in a variety of different tissues and cells, including neurons (48), epithelia (3, 17), myocardium (59), skeletal muscle (58), and vascular smooth muscle (2). Four mammalian K-Cl cotransporter isoforms were recently cloned and designated KCC1 (16), KCC2 (44), KCC3 (20, 40), and KCC4 (40).1 K-Cl cotransport activity has also been demonstrated in several nonmammalian cells, including teleost erythrocytes and hepatocytes (5, 8, 18, 26), amphibian red blood cells (19), lobster neurons (56), and malpighian tubules from both Drosophila melanogaster (34) and the forest ant Formica polyctena (33). The physiological roles of K-Cl cotransport remain poorly understood. However, activation by cell swelling suggests a prominent role for KCCs in the regulatory volume decrease of cells exposed to hypotonic conditions or swollen by cellular insults such as ischemia. There is also evolving evidence for the participation of K-Cl cotransport in transepithelial salt transport and intracellular ion homeostasis (3, 17, 48, 56).
During the course of the cloning and characterization of electroneutral
cation-chloride cotransporters (13, 14, 38-40, 46),
we initially observed that oocytes from the frog Xenopus laevis do not contain thiazide-sensitive Na-Cl cotransport
(14, 39) but do express an endogenous bumetanide-sensitive
Na-K-2Cl cotransporter that can be activated by hypertonic conditions
(13) and inhibited by activation of protein kinase C
(47). Suvitayavat et al. (54) and Shetlar et
al. (51) have reported similar findings. More recently we
and others have obtained preliminary evidence that Xenopus
oocytes also contain an endogenous K-Cl cotransporter (38, 40,
53). During reproduction frogs place their oocytes in hypotonic
pond water, resulting in profound cellular swelling in the absence of a
compensatory response. Although swelling-activated K-Cl cotransport has
not been reported in oocytes, these cells are known to possess
hypotonically activated Cl channels (1).
Once the oocytes become fertilized eggs, they are particularly
resistant to cell swelling, potentially because of conformational
changes of the cytoskeleton that reduce the capacity of the cell to
swell (28). A physiological response to hypotonicity has
also been demonstrated in X. laevis spermatozoa, which
remain immotile until the osmolarity of the semen is diluted in pond
water (21).
To begin to study the role of K-Cl cotransport in X. laevis, we initiated a molecular and functional study of the oocyte transporter. In addition, since Xenopus oocytes are used for the characterization of other KCCs, functional characterization was necessary to understand the regulation of the endogenous transporter and to minimize the misinterpretation of heterologous expression. We report here that Xenopus oocytes exhibit an endogenous K-Cl cotransporter that can be activated by hypotonicity. We also describe the major kinetic parameters of the cotransporter, as well as its inhibitory and regulatory profile. Xenopus oocytes also express mRNA encoding a K-Cl cotransporter protein with significant homology to the mammalian KCCs. This constitutes the first detailed functional characterization of K-Cl cotransport in a nonmammalian, nonerythroid cell.
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METHODS |
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Preparation of Xenopus laevis oocytes.
Adult female normal and albino X. laevis frogs were
purchased from Carolina Biological Supply (Burlington, NC) and
maintained at the Institution 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 Carolina Biological Supply, and water was changed twice a week. Oocytes were surgically collected from anesthetized animals under 0.17% tricaine and incubated for 1 h with vigorous shaking in ND96 (in mM: 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES/Tris, pH 7.4) and 2 mg/ml of collagenase B, after which oocytes were washed four times in
ND96 and manually defolliculated. Stage V-VI oocytes (11)
were incubated for 2-4 days in ND96 at 18°C supplemented with
2.5 mM sodium pyruvate and 5 mg/100 ml of gentamicin. The incubation
medium was changed every 24 h. The day of the influx experiment,
oocytes were switched to a Cl-free ND96 (in mM: 96 Na+ isethionate, 2 K+-gluconate, 1.8 Ca2+-gluconate, 1 Mg2+-gluconate, 5 HEPES, 2.5 sodium pyruvate, and 5 mg% gentamicin, pH 7.4) for 2 h before the assay.
Assessment of K-Cl cotransporter function.
Functional analysis of the K-Cl cotransporter consisted of measuring
tracer 86Rb+ uptake (New England Nuclear) in
groups of at least 15 oocytes. 86Rb+ uptake was
measured under both isotonic and hypotonic conditions with the
following general protocol: a 30-min incubation period in a hypotonic
K+ and Cl-free medium [in mM: 50 N-methyl-D-glucamine (NMDG)-gluconate, 4.6 Ca2+-gluconate, 1.0 Mg2+-gluconate, 5 HEPES/Tris, pH 7.4] with 1 mM ouabain, followed by a 60-min uptake
period in a hypotonic Na+-free and KCl-containing medium
with variable K+ and Cl
concentrations (in
mM: 0-50 NMDG-Cl, 0-50 KCl, 0-50 NMDG-gluconate, 1.8 CaCl2, 1 MgCl2, 5 HEPES, pH 7.4), supplemented
with 1 mM ouabain, and 2.5 µCi of 86Rb+.
Experiments in isotonic conditions were performed using the same
solutions but supplemented with sucrose at 3.5 g/100 ml to increase the
osmolality of the solutions to the isosmolal conditions for oocytes
(~210 mosmol/kgH2O). Ouabain was added to prevent 86Rb+ uptake via the
Na+-K+-ATPase. The absence of extracellular
Na+ and the hypotonicity of the uptake medium prevented
86Rb+ uptake or
86Rb+-K+ exchange by the endogenous
Na-K-2Cl cotransporter that is present in oocytes (13).
RT-PCR amplification of KCCs. A BLAST search of the National Center for Biotechnology expressed sequence tag (EST) database revealed a 549-bp coding sequence EST (GenBank accession no. AW646505) from X. laevis oocytes with significant homology to the mammalian KCCs, and we amplified this 549-bp fragment from Xenopus oocyte total RNA by RT-PCR. Xenopus KCC oligonucleotide primers were designed using the EST sequence. The sense primer 5'-ACAGTACTTCTGGGAGACTACC-3' and antisense primer 5'-GATACGTAAATGATAAAGAAAGG-3' amplified a fragment of 528 bp. The amplified band spans a region of the KCC proteins that is encoded by three exons in a Drosophila homologue (see GenBank accession no. AE003462) and all four mammalian KCCs (D. B. Mount, unpublished data). Given the level of genomic conservation between this region of the KCC genes in mammals and Drosophila, a similar genomic structure is likely for this KCC gene in X. laevis, such that the PCR primers used for the initial RT-PCR reaction will amplify a larger DNA fragment from contaminating genomic DNA, if at all. However, reverse transcriptase was omitted in control samples to verify that contaminating genomic DNA was not amplified in the RT-PCR samples. To increase the specificity of the PCR amplification, nested PCR of the amplified band was performed with two internal primers that amplify a band of 359 bp; the sense primer for nested PCR was 5'-AGCAGAGCAGGCACTGAAACAC-3' and the antisense primer was 5'-GGAAGGGCAGAAGCATAAGC-3'. A second overlapping EST (GenBank accession no. BE576764) was subsequently identified, obtained from Research Genetics, and sequenced in entirety using fluorescent dye termination chemistry (BigDye, Applied Biosystems).
Total RNA was extracted from defolliculated oocytes and other tissues using the guanidine isothiocyanate-cesium chloride method (49). PCR was performed with 1 µg of reverse-transcribed RNA in 20-µl reactions containing 1× PCR buffer (in mM: 10 Tris · HCl, 1.5 MgCl2, 50 KCl, pH 8.3), 0.1 mM of each dNTP, 10 µM of each primer, and one unit of Taq DNA polymerase (Life Technologies). Thirty-five PCR cycles were performed with the following profile: 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C. The last cycle was followed by a final extension step of 5 min at 72°C. The PCR product from oocytes was gel purified from a 1.5% agarose gel and sequenced by the dideoxy chain termination method (50) using the Sequenase DNA sequencing kit (USB). Once the nature of the single PCR band from oocytes was confirmed by DNA sequencing as a Xenopus homologue of the mammalian KCC cotransporters (see RESULTS), Southern blot of all tissue PCR products was performed under high-stringency conditions using the 528-bp fragment to generate a nonradioactive probe by using the PCR DIG probe synthesis kit (Boehringer Mannheim, Germany). Hybridization bands were detected by an immunoperoxidase reaction.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 was 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's method for multiple comparison procedures, as needed.
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RESULTS |
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Expression of an endogenous K-Cl cotransporter in Xenopus oocytes.
Figure 1 shows a summary from
seven experiments in which 86Rb+ uptake was
assessed using an uptake solution containing 2 mM and 50 mM of
extracellular K+ and Cl, respectively.
Uptakes were performed under both isotonic conditions (220 mosmol/kgH2O for Xenopus oocytes) and hypotonic
conditions (110 mosmol/kgH2O). Under isotonic conditions,
oocytes exhibited a Rb+ uptake of 10.1 ± 1.4 pmol · oocyte
1 · h
1 that
was reduced to 6.3 ± 1.3 pmol · oocyte
1 · h
1 when
oocytes were incubated in Cl
-free medium. However, the
difference did not reach significance. Under hypotonic conditions,
Rb+ uptake in the presence of extracellular
Cl
increased to 52.5 ± 11.0 pmol · oocyte
1 · h
1
(P < 0.00001); the increased Rb+ uptake
was Cl
dependent, since uptake under hypotonic
Cl
-free conditions was 5.22 ± 1.0 pmol · oocyte
1 · h
1
(P < 0.001). Therefore, oocytes exhibit a
Cl
-dependent 86Rb+ uptake
mechanism that is activated by cell swelling (hypotonic conditions). A
similar observation was made using oocytes harvested from the albino
type of X. laevis (data not shown). In addition, Fig. 1,
inset, shows the result of a single experiment in which 36Cl
uptake was assessed in the presence of
10 mM and 50 mM of extracellular K+ and Cl
,
respectively. In isotonicity, 36Cl
uptake was
similar in the presence or absence of extracellular K+
[908 ± 124 vs. 912 ± 346 pmol · oocyte
1 · h
1,
P = nonsignificant (NS)]. In contrast, incubation in
hypotonicity induced an increase in 36Cl
uptake to 2,960 ± 455 pmol · oocyte
1 · h
1
(P < 0.01) that was partially but significantly
K+ dependent (1,615 ± 296 pmol · oocyte
1 · h
1,
P < 0.01). Thus oocytes also exhibit a
K+-dependent 36Cl
uptake
mechanism that is only apparent during cell swelling.
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Kinetics properties and anion dependence of the oocyte K-Cl
cotransporter.
Figure 4 shows the Rb+ and
Cl dependency of the K-Cl cotransporter in
Xenopus oocytes. To assess the Rb+ kinetics,
uptakes were performed in extracellular media with fixed concentration
of Cl
at 50 mM, changing concentrations of
Rb+ from 0 to 50 mM. In contrast, to assess the
Cl
kinetics, uptakes were done with a fixed concentration
of K+ at 20 mM, changing the concentration of
Cl
from 0 to 50. As illustrated in Fig. 4,
Rb+ uptake showed Michaelis-Menten behavior. The calculated
Michaelis-Menten constant (Km) and maximal
velocity (Vmax) for extracellular
Rb+ concentration were 27.7 ± 3.0 mM and 1,531 ± 78 pmol · oocyte
1 · h
1,
respectively, and the apparent Km and
Vmax values for extracellular Cl
concentration were 15.4 ± 4.7 mM and 318 ± 39 pmol · oocyte
1 · h
1,
respectively. Consistent with electroneutrality of the transport process, the Hill coefficient for both ions remained close to unity:
1.04 ± 0.17 and 1.07 ± 0.14 for K+ and
Cl
, respectively.
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Sensitivity to inhibitors.
Figure 5 illustrates the effect of a 100 µM concentration of several inhibitors of cation-chloride
cotransporters on the oocyte K-Cl cotransporter. At a 100 µM
concentration, DIDS had no effect on 86Rb+
uptake, whereas the mammalian KCCs are sensitive to DIDS at this concentration (32, 38, 42). The addition of a 100 µM
concentration of the loop diuretic furosemide or bumetanide to the
uptake medium resulted in a 22 and 20% reduction in the
Cl-dependent tracer Rb+ uptake, respectively,
compared with uptake observed in control group. The reduction was
statistically significant (P < 0.001). In contrast, a
100 µM concentration of [(dihydroindenyl)oxy]alkanoic acid (DIOA)
reduced the uptake by 76% (P < 0.000001). Thus the oocyte K-Cl cotransporter is more sensitive to DIOA than to loop diuretics, as is the case for the mammalian isoforms (38).
Figure 6 shows the
concentration-dependent inhibition of the oocyte K-Cl cotransporter by
loop diuretics. The IC50 values for furosemide and
bumetanide were calculated at 200 and 500 µM, respectively. Although
the four mammalian KCCs differ in relative sensitivity to the two loop
diuretics, bumetanide is always less effective than furosemide, as is
the case for the oocyte K-Cl cotransporter.
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Regulation of the oocyte K-Cl cotransporter.
In red blood cells of several species, swelling-induced activation of
K-Cl cotransport appears to involve a protein dephosphorylation step,
presumably of the transporter protein itself (10). With this in mind, we assessed the functional effect of inhibiting the
protein phosphatases. To discriminate between phosphatases, we used 100 nM of calyculin A, which inhibits the function of both protein
phosphatases 1 and 2A (PP1 and PP2A), 1 nM of okadaic acid, a
concentration that inhibits only PP2A (4), and 100 pM
cypermethrin, which inhibits the function of protein phosphatase 2B
(PP2B) (12). As Fig. 7
shows, the increased 86Rb+ uptake induced by
hypotonicity was completely abrogated by calyculin A. In contrast, no
effect was observed with okadaic acid and cypermethrin.
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Xenopus oocytes express a homologue of the mammalian KCCs.
A BLAST search of EST databases revealed the existence of one X. laevis EST (accession no. AW646505) from oocytes that was
homologous to KCC sequences, exhibiting 76% identity to the rat KCC1
sequence. On the basis of this Xenopus EST, we designed a
primer pair to amplify a fragment of 528 bp (see METHODS).
A single band of the expected size was amplified whereas no band was
observed in the absence of reverse transcription (data not shown). A
second primer pair was used for nested PCR-amplifying of 349 bp using
the first band as a template. In addition, the 528-bp PCR band was gel
purified and the DNA sequence was confirmed. A second overlapping EST
was subsequently identified, obtained from Research Genetics, and
sequenced in entirety; the composite cDNA (accession no. AF325505)
encodes the COOH-terminal 358 amino acids of the Xenopus KCC
and the entire 3'-untranslated region. Figure
9 shows the alignment of the deduced
amino acid sequence of each of the mammalian KCC cDNAs with the partial
sequence of the Xenopus KCC; this COOH-terminal fragment
exhibits 68% identity with rat KCC1 (accession no. U55815), 56% with
rat KCC2 (accession no. U55816), 76% with human KCC3 (accession no.
AF105366), and 62% with mouse KCC4 (accession no. AF087436). We also
performed RT-PCR and Southern blot analysis of RT-PCR products obtained from several tissues, using the 528-bp product from oocytes as a
nonradioactive probe. Figure
10A shows the amplification
of a ~528-bp product from all tested tissues. Control PCR reactions in the absence of reverse transcriptase were negative for all tissues
(data not shown, except for oocytes mRNA in last lane of Fig.
10A), indicating that the amplified product was not due to
contamination with genomic DNA. In addition, Fig. 10B shows that the PCR product obtained from all tissues was able to hybridize with the KCC-specific probe on a Southern blot of an agarose gel.
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DISCUSSION |
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In the present study we have defined the principal functional,
pharmacological, and regulatory properties of the K-Cl cotransport pathway that is expressed in X. laevis oocytes. Under
isotonic conditions, some individual experiments (e.g., Fig. 7) showed a small but significant Cl-dependent
86Rb+ uptake. This is potentially due to weak
volume-independent activation of the K-Cl cotransporter; however,
experimental variability at this low level of transport may also play a
role. In addition, this is theoretically due to activity of the oocyte
Na-K-2Cl cotransporter, since Lytle et al. (35) have
proposed that in the absence of external Na, as was the case in our
experiments, the Na-K-2Cl cotransporter can potentially catalyze
Cl
-dependent K/Rb exchange. Regardless, when data from
multiple experiments are analyzed (Fig. 1), the difference between
isotonic Rb+ uptake in the presence and absence of
Cl
does not reach statistical significance. Under
hypotonic conditions, oocytes exhibited a very significant
Cl
-dependent 86Rb+ uptake. In
these circumstances, it is particularly unlikely that 86Rb+ transport activity in the absence of
Na+ was due to the Na-K-2Cl cotransporter, because this
cotransporter in oocytes is inhibited by hypotonicity
(13). In addition, oocytes exhibit a
K+-dependent 36Cl
uptake pathway
that is activated by hypotonicity. The Cl
-dependent
86Rb+ uptake in oocytes exhibited the following
characteristics: 1) transport can be activated by cell
swelling and to a lesser extent by NEM, which are the classic
activators of K-Cl cotransport in several cell types and species
(32); 2) the transport of K+ and
Cl
exhibits interdependency and electroneutrality, with
Hill coefficients of 1; 3) the K-Cl cotransporter is
sensitive to loop diuretics, with higher affinity for furosemide over
bumetanide, a common feature of the K-Cl cotransporters (16,
44); 4) it is also sensitive to the specific K-Cl
inhibitor DIOA (15); and 5) activation by
hypotonicity can be prevented by the protein phosphatase inhibitor calyculin A. At the molecular level, we have identified a partial cDNA
clone that encodes a protein with a high degree of identity (>75%)
with mammalian KCC sequences. Although we show no direct evidence that
this gene is responsible for the Cl
-dependent
86Rb+ uptake observed in this study, our data
clearly indicate that Xenopus oocytes express a K-Cl
cotransporter that shares functional and molecular properties with the
mammalian KCCs.
To be fertilized, the Xenopus female lays the oocytes into pond water of very low osmolarity. Because of still not very well understand mechanisms that include conformational changes of the cytoskeleton, when oocytes become fertilized eggs they develop a complete resistance to cell swelling. Kelly et al. (28) showed that frog fertilized eggs transferred to dilute buffer with osmolality of 10 mosmol/kgH2O for several hours developed no changes in cell volume, whereas oocytes exhibit a slow increase in cell volume over time and eventually burst. Since it was shown in the same study that oocytes do not develop a clear RVD response, the authors suggested that in oocytes exposed to dilute buffer osmolyte efflux occurs and limits swelling. Thus oocytes clearly possess the transport mechanisms to release intracellular ions to reduce cell swelling while they become fertilized eggs, since they express a swelling-activated K-Cl cotransporter that is capable of K-Cl efflux (Fig. 3).
Kinetic analysis of the 86Rb+ uptake in swollen
oocytes showed that both K+ and Cl are
required. The Hill coefficients for both ions were close to unity,
indicating an electroneutral transport process with a stoichiometry of
1:1. The affinity for extracellular ions revealed an apparent
Km for extracellular K+ of ~22 mM
and for extracellular Cl
of ~15 mM. These values are
similar to those of the mammalian KCC1 isoform (38).
Whereas all of the KCC isoforms studied thus far exhibit similar
affinity for extracellular Cl
(14 to 17 mM), they differ
dramatically in the affinity for extracellular K+. The
isoform with the highest K+ affinity is rat KCC2, with a
Km for extracellular K+ of ~5 mM
(42), followed by KCC4 (Km of ~17
mM) (38), KCC1 (Km of ~25 mM)
(38), and KCC3 (Km of ~51 mM)
(37). Although the direction of transport is ultimately
dictated by gradients for the transported ions, it has been proposed
that KCCs with a higher cation affinity (KCC2 and KCC4) can transport
K+ in both directions at higher rates. This has been
verified experimentally in neurons, which predominantly express KCC2
(23). In contrast, isoforms with lower K+
affinity (KCC1 and KCC3) are more suited to function as extrusion mechanisms, when the gradient for K+ will favor efflux
(42).
Despite the lack of variation in Cl affinity, K-Cl
cotransporters differ in the anion series of rubidium transport, i.e., the relative activity in the presence of anions other than
Cl
(37). The Xenopus transporter
is similar in this respect, in that anions other than Cl
could also support K+ translocation. In fact, the anion
series of the endogenous oocyte K-Cl cotransporter is very similar to
the anion series observed in KCC1-injected oocytes (38).
As shown for K-Cl cotransport in other species, the Xenopus transporter is sensitive to loop diuretics and other inhibitors of anion transport (16, 20, 42). In addition, 86Rb+ uptake was also sensitive to DIOA, which appears to be a specific inhibitor of K-Cl cotransport (15); this compound has no effect on the oocyte Na-K-2Cl cotransporter (data not shown).
Inhibition of protein phosphatases prevents the swelling- and NEM-induced activation of the K-Cl cotransporter in several cells (6, 25, 27, 29, 52), and it is widely accepted that dephosphorylation is required for activation of the cotransporter. Consistent with the data from other cells, we observed in the present study that the protein phosphatase inhibitor calyculin A abolishes hypotonic activation of the oocyte cotransporter (Fig. 8). Calyculin A inhibits two types of phosphatase, both PP1 and PP2A (9). To determine the phosphatase involved, we tested the effect of okadaic acid, which is a specific PP2A inhibitor at the 1 nM concentration used, and the specific PP2B inhibitor cypermethrin (4, 12); neither inhibitor affected the activation of the cotransporter by hypotonicity. Therefore, it is likely that in Xenopus oocytes, PP1 is the phosphatase involved in activation of the K-Cl cotransporter during cell swelling (6, 25).
Several cotransporters are known to be affected by exposure to mercury
(Hg2+). For example, transport by the NaSi cotransporter
(36) and the NaPi-3 cotransporters in Xenopus
oocytes is significantly reduced when oocytes are exposed to
Hg2+ (57). Similarly, heterologous expression
of the basolateral isoform of the Na-K-2Cl cotransporter in HEK-293
cells is sensitive to Hg2+ (22). In contrast,
when expressed in Xenopus oocytes, aquaporin-6 is activated
by this heavy metal (60). It is known that
Hg2+ affects the function of channels and transporters by
interacting with sulfhydryl groups (-SH) on cysteine residues (7,
60), and in some cases this has been proven by mutagenesis
studies (30, 41). In the present study we observed that
exposing Xenopus oocytes to 150 µM HgCl2
resulted in increased 86Rb+ uptake by
activation of at least two pathways: one that is Cl
dependent and another that is Cl
independent. Because
uptakes were performed in the absence of extracellular Na+,
it is unlikely that the opening of this Cl
-dependent
86Rb+ influx pathway represents activity of the
Na-K-2Cl cotransporter. In addition, Jacoby et al. (22)
have shown that Na-K-2Cl cotransporter is inhibited by
Hg2+. Therefore, the Cl
-dependent fraction of
the Hg2+-induced increase in 86Rb+
uptake is due to activation of K-Cl cotransport. The mechanism by which
Hg2+ affects the function of membrane proteins is not
clear, but it is known that Hg2+ interacts with cysteinyl
sulfhydryls (-SH). The observation that the effect of Hg2+
on 86Rb+ influx in oocytes was completely
prevented by the reducing agent DTT suggests that, indeed, interaction
of Hg2+ with SH groups is necessary for the stimulatory
effect. Of interest, although it has been suggested that NEM also
affects the K-Cl cotransporter function by interaction with SH groups,
the stimulatory effect of NEM upon the K-Cl cotransporter observed in
this study (Fig. 2) was significantly smaller than the effect of
Hg2+. It is still unclear, however, if the activating
effect of NEM on the cotransporter is related to NEM-induced
dephosphorylation or direct modification of SH groups. There are
reports supporting both possibilities (24, 31).
Finally, we have confirmed the expression of a KCC isoform in Xenopus oocytes and multiple other tissues. Sequence data from a Xenopus EST was used to clone a partial cDNA by RT-PCR, which overlapped with another fully sequenced EST cDNA. The predicted protein sequence of this partial cDNA was more homologous to the KCC1-KCC3 subfamily than to the KCC2-KCC4 subfamily of the K-Cl cotransporters. Cloning of the full-length cDNA will be pursued, since this will provide the means for structure-function studies of this KCC and for characterization of the physiological role(s) of this transporter in Xenopus tissues.
In summary, we have shown that X. laevis oocytes express a K-Cl cotransporter in the plasma membrane that is activated by cell swelling, NEM, and HgCl2, and inhibited by loop diuretics and DIOA. The functional properties resemble those of mammalian KCC1, and the sequence of the COOH terminus is closest to KCC3, indicating that this Xenopus KCC is the amphibian orthologue of one or both of these low-affinity K-Cl cotransporters.
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ACKNOWLEDGEMENTS |
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We are grateful to J. López for help with frog 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 Mexican Council of Science and Technology (CONACYT) Research Grant 97629m and Howard Hughes Medical Institute Research Grant 75197-553601 to G. Gamba and by National Institutes of Health Grant RO1-DK-57708 to D. B. Mount. A. Mercado and P. Meade were supported by scholarship grants from the Dirección General del Personal Académico of the National University of Mexico and CONACYT, respectively. D. B. Mount is supported by an Advanced Research Career Development Award from the Department of Veterans Affairs. G. Gamba is an International Scholar of the Howard Hughes Medical Institute.
Address for reprint requests and other correspondence: G. Gamba, Molecular Physiology Unit, Vasco de Quiroga No. 15, Tlalpan 14000, Mexico City, Mexico (E-mail: gamba{at}conacyt.mx).
1 We initially referred to the KCC on human chromosome 15q14 as KCC4 and the KCC on chromosome 5p15 as KCC3 (40). However, in deference to the earlier publication of Hiki et al (20), we reversed the numbering of our GenBank/EBI submissions to refer to the KCC on chromosome 15q14 as KCC3 and the KCC on chromosome 5p15 as KCC4 (see NOTE ADDED IN PROOF in Ref. 40).
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.
Received 10 October 2000; accepted in final form 21 March 2001.
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