Functional and molecular characterization of the K-Cl cotransporter of Xenopus laevis oocytes

Adriana Mercado1, Paola de los Heros1, Norma Vázquez1, Patricia Meade1, David B. Mount2, and Gerardo Gamba1

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


    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<UP><SUB>4</SUB><SUP>3−</SUP></UP> = Br- > 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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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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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).

All uptakes were performed at room temperature. At the end of the uptake period, oocytes were washed five times in ice-cold uptake solution without isotope to remove extracellular tracer. Oocytes were dissolved in 10% sodium dodecyl sulfate, and tracer activity was determined for each oocyte by beta scintillation counting.

To determine the ion transport kinetics of the K-Cl cotransporter we performed experiments using varying concentrations of K+ and Cl-. To maintain osmolality and ionic strength, gluconate was used as a Cl- substitute and NMDG as a K+ substitute. The sensitivity and kinetics for several inhibitors were assessed by exposing groups of oocytes to inhibitor at concentrations varying from 20 µM to 2 mM. For these experiments, the desired concentration of the inhibitor was present during both the incubation and uptake periods. We also assessed the effect of several drugs on the activation of the K-Cl cotransporter by adding the drug during 15 min before the uptake period.

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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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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Fig. 1.   K-Cl cotransport activity in Xenopus laevis oocytes. 86Rb+ uptakes were measured under hypotonic or isotonic conditions, as indicated, in the presence of 2 mM K+, with (open bars) or without (filled bars) extracellular Cl-. Each bar represents mean ± SE of 7 experiments from different frogs. * Significantly different from uptake in control group at 110 mosmol/kgH2O (P < 0.001). Inset: a single experiment in which 36Cl- uptake was assessed in hypotonic or isotonic conditions, in the presence (open bars) or absence (hatched bars) of 10 mM extracellular K+. For 36Cl- uptake experiment, oocytes were exposed to a 30-min incubation period in a hypotonic or isotonic K+ and Cl--free medium, followed by 30-min uptake period in an isotonic or hypotonic Na+-free medium in the presence of 1 µCi/ml of H36Cl. * P < 0.01 vs. uptake in 220 mosmol/kgH2O. ** P < 0.01 vs. uptake in control group in 110 mosmol/kgH2O.

K-Cl cotransport in several cells is uniquely activated by the N-alkylating agent N-ethylmaleimide (NEM) (32), whereas Na-K-2Cl cotransport is inhibited by this agent (55). We therefore analyzed the effect of NEM upon 86Rb+ uptake in oocytes incubated in isotonic conditions. In the presence of 10 mM K+ and 50 mM Cl-, exposing oocytes to 1 mM NEM before the influx period increased the 86Rb+ uptake by ~2-fold from 117 ± 6.3 pmol · oocyte-1 · h-1 in the control group to 227 ± 29 pmol · oocyte-1 · h-1 in the NEM-treated group (P < 0.001). The increased uptake induced by NEM was Cl- dependent. In the absence of extracellular Cl-, influx was similar between the control and NEM-treated oocytes (88 ± 7 vs. 60 ± 11 pmol · oocyte-1 · h-1, P = NS). Therefore, under isotonic conditions, addition of NEM resulted in increased activity of the K-Cl cotransporter.

Figure 2 illustrates the time course of 86Rb+ uptake when oocytes were exposed to a hypotonic uptake medium containing 10 mM of extracellular K+, in the presence or absence of 50 mM Cl-. When Cl- was present in the extracellular medium we observed increased 86Rb+ uptake that was linear for 90 min. This uptake was due to the K-Cl cotransporter since no uptake was observed in the Cl--free uptake medium.


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Fig. 2.   Time course (in min) of 86Rb+ influx in X. laevis oocytes. Oocytes were incubated for 30 min in K+ and Cl--free hypotonic medium and then in a 10 mM K+-containing medium in the presence (open circle ) or absence () of 50 mM Cl-. Uptake periods are indicated. Each point represents mean ± SE of 15 oocytes. * P < 0.001 vs. Cl--free group.

The K-Cl cotransporter is one of the efflux pathways that are activated by cell swelling, as part of the regulatory volume decrease (RVD) response. With this in mind, we also examined 86Rb+ efflux when oocytes were exposed to hypotonicity (Fig. 3). Over a 150-min period we observed a progressive reduction in the amount of 86Rb+ remaining in the cells, together with a gradual increase in the tracer Rb+ in the extracellular medium. As shown in Fig. 3, A and B, the addition of the loop diuretic furosemide significantly reduced the efflux of 86Rb+ from the cells, indicating the proportion of efflux that was through the K-Cl cotransporter.


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Fig. 3.   86Rb+ efflux in X. laevis oocytes. A: single experiment showing time course of tracer Rb+ remaining in oocytes (pmol/oocytes). B: mean of 3 experiments showing time course for appearance of 86Rb+ in the extracellular medium [counts per min (cpm)]. For these experiments, oocytes were incubated for 1 h in regular isotonic ND96 containing 5.0 µCi/ml of 86Rb+. At the end of this loading period, oocytes were washed 5 times and transferred to a hypotonic solution containing (in mM) 48 N-methyl-D-glucamine gluconate, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5.0 HEPES, and 1 ouabain, without extracellular 86Rb+, in the absence (open circle ) or presence () of 2 mM extracellular furosemide. * P < 0.01 vs. same time point in presence of furosemide. In A each point represents mean ± SE of 15 oocytes. In B each point represents mean ± SE of 3 experiments.

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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Fig. 4.   Kinetic analysis of Rb+ uptake in Xenopus oocytes. A: Rb+ dependency of Rb+ uptake. Uptakes were performed over a 60-min period in 110 mosmol/kgH2O with a fixed concentration of Cl- at 50 mM, changing extracellular concentration of Rb+ ([Rb+]e) from 0 to 50 mM. B: Cl- dependency of 86Rb+ uptake. Uptakes were performed over a 60-min period in 110 mosmol/kgH2O with a fixed concentration of K+ at 20 mM and changing extracellular concentration of Cl- ([Cl-]e) from 0 to 50 mM. For data on A, uptakes were also measured in groups of oocytes in the absence of Cl- (data not shown), and the mean values for these groups were subtracted in corresponding RbCl-containing groups to analyze only the Cl--dependent 86Rb+ uptake. For data on B, the uptake observed in a solution containing 20 mM K+ and 0 mM Cl- was subtracted from uptakes in all other groups. Lines were fit using the Michaelis-Menten equation. The Hill coefficients for K+ and Cl- were close to unity: 1.04 ± 0.16 and 1.07 ± 0.14 for K+ and Cl-, respectively. Each point represents the mean ± SE of 15 oocytes.

It has been shown that some extracellular anions other than Cl- can support ion translocation through the K-Cl cotransporter of sheep and human erythrocytes (43). It was thus of interest to study the anion series for the oocyte K-Cl cotransporter, assessed as the relative amount of 86Rb+ influx in the presence of anions other than Cl-. We observed no significant difference in tracer 86Rb+ uptake in the presence of Cl- (129 ± 18 pmol · oocyte-1 · h-1), PO<UP><SUB>4</SUB><SUP>3−</SUP></UP> (130 ± 21 pmol · oocyte-1 · h-1), or Br- (101 ± 16 pmol · oocyte-1 · h-1), whereas a significant reduction (P < 0.001) was observed in the presence of I- (67 ± 15 pmol · oocyte-1 · h-1), SCN- (44 ± 12 pmol · oocyte-1 · h-1), and gluconate (25 ± 5 pmol · oocyte-1 · h-1). The function profile in the presence of different anions of the oocyte transporter was thus Cl- = PO<UP><SUB>4</SUB><SUP>3−</SUP></UP> = Br- > I- > SCN- > gluconate.

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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Fig. 5.   Effect of transport inhibitors on Cl--dependent 86Rb+ uptake. Uptakes were assessed in 10 mM K+ and 50 mM Cl--hypotonic medium. In the Cl--free group gluconate was used as substitute. The concentration of all inhibitors was 100 µM. * P < 0.05 vs. uptake in control conditions (1st bar). Each bar represents mean ± SE of 15 oocytes.



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Fig. 6.   Kinetic analyses of K-Cl cotransporter (KCC) inhibition by loop diuretics. Uptakes were assessed in hypotonic medium. Groups of 10 Xenopus oocytes were exposed to increasing concentrations of furosemide () or bumetanide (open circle ) in the preincubation and uptake medium, from 20 to 2,000 µM. Data were normalized as the percentage of uptake, taking 100% as the value in the absence of loop diuretics. The calculated IC50 was 200 µM and 500 µM for furosemide and bumetanide, respectively. Each point represents the mean ± SE of 15 oocytes.

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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Fig. 7.   Effect of protein phosphatase inhibition upon the hypotonicity-induced activation of the K-Cl cotransporter in Xenopus oocytes. Influxes were performed under isotonic or hypotonic conditions, exposing oocytes to calyculin A, okadaic acid, or cypermethrin during preincubation and uptake periods as stated. Uptakes were assessed in 10 mM K+-containing medium in the presence (open bars) and absence (filled bars) of extracellular Cl-. * P < 0.001 vs. uptake in control conditions in hypotonicity.

Many ion transporters are dramatically affected by exposure to Hg2+ (22, 36, 57). For example, orthologues of the basolateral isoform of the Na-K-2Cl cotransporter exhibit variable inhibition by mercury (22), whereas other proteins such as aquaporin-6 are activated by Hg2+ (60). We thus analyzed the effect of Hg2+ on the 86Rb+ uptake of Xenopus oocytes. As Fig. 8 shows, oocyte incubation in the presence of 150 µM HgCl2 under isotonic conditions for the 15 min previous to the influx period resulted in a dramatic increase in 86Rb+ uptake. We used this HgCl2 concentration since this was the dose at which maximal response was observed. This increase was completely prevented by pretreatment of oocytes with 10 mM of the reducing agent dithiothreitol (DTT) (22). The incubation of oocytes with and without extracellular Cl- during uptake revealed that HgCl2-induced increase in 86Rb+ uptake is composed of at least two distinct pathways, each accounting for ~50% of the total uptake. One pathway is Cl- dependent, consistent with K-Cl cotransport, whereas the other is Cl- independent, suggesting the opening of a cation channel.


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Fig. 8.   Effect of 150 µM HgCl2 and 10 mM dithiothreitol (DTT) on 86Rb+ uptake in Xenopus oocytes. Cells were incubated in isotonic conditions during the uptake. Before the uptake period, oocytes were exposed to 30 min of DTT alone, 15 min of HgCl2 alone, or 30 min of DTT plus HgCl2 in the last 15 min; HgCl2 or DTT was not present during the uptake period. As indicated, uptakes were performed in the presence or absence of extracellular Cl-. Each point represents the mean ± SE of 30 oocytes. * P < 0.001 vs. HgCl2 control group.

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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Fig. 9.   Protein alignments of the mammalian K-Cl cotransporters KCC1 to KCC4, with the deduced amino acid sequence of the Xenopus oocyte K-Cl cotransporter (XKCC). The fragment corresponds to a part of the carboxy terminus, which is predicted to be cytoplasmic. Identical segments are boxed. HKCC3, human KCC3; MKCC4, mouse KCC4.



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Fig. 10.   A: an acrylamide gel of RT-PCR products from several Xenopus tissues, using a KCC-specific primer pair to amplify a 528-bp fragment. The last lane shows the control PCR from oocyte RNA in the absence of reverse transcriptase [RT(-)]. B: Southern blot analysis of RT-PCR products obtained from total RNA extracted from several X. laevis tissues. Membranes were probed under high-stringency conditions with a probe constructed from the 528-bp fragment of the Xenopus K-Cl cotransporter.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

We are grateful to J. López for help with frog care and to members of the Molecular Physiology Unit for suggestions and stimulating discussion.


    FOOTNOTES

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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