Cloning and Characterization of KCC3 and KCC4, New Members of the Cation-Chloride Cotransporter Gene Family*

David B. MountDagger §, Adriana Mercadoparallel , Luyan SongDagger , Jason XuDagger , Alfred L. George Jr.Dagger **Dagger Dagger , Eric DelpireDagger Dagger §§, and Gerardo Gamba

From the Departments of Dagger  Medicine, ** Pharmacology, and §§ Anesthesiology, Vanderbilt University Medical Center, Nashville, Tennessee 37232 and the  Molecular Physiology Unit, Instituto Nacional de la Nutrición Salvador Zubirán and Instituto de Investigaciones Biomédicas UNAM, Mexico City, Mexico

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The K+-Cl- cotransporters (KCCs) belong to the gene family of electroneutral cation-chloride cotransporters, which also includes two bumetanide-sensitive Na+-K+-2Cl- cotransporters and a thiazide-sensitive Na+-Cl- cotransporter. We have cloned cDNAs encoding mouse KCC3, human KCC3, and human KCC4, three new members of this gene family. The KCC3 and KCC4 cDNAs predict proteins of 1083 and 1150 amino acids, respectively. The KCC3 and KCC4 proteins are 65-71% identical to the previously characterized transporters KCC1 and KCC2, with which they share a predicted membrane topology. The four KCC proteins differ at amino acid residues within key transmembrane domains and in the distribution of putative phosphorylation sites within the amino- and carboxyl-terminal cytoplasmic domains. The expression of mouse KCC3 in Xenopus laevis oocytes reveals the expected functional characteristics of a K+Cl- cotransporter: Cl--dependent uptake of 86Rb+ which is strongly activated by cell swelling and weakly sensitive to furosemide. A direct functional comparison of mouse KCC3 to rabbit KCC1 indicates that KCC3 has a much greater volume sensitivity. The human KCC3 and KCC4 genes are located on chromosomes 5p15 and 15q14, respectively. Although widely expressed, KCC3 transcripts are the most abundant in heart and kidney, and KCC4 is expressed in muscle, brain, lung, heart, and kidney. The unexpected molecular heterogeneity of K+-Cl- cotransport has implications for the physiology and pathophysiology of a number of tissues.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Potassium chloride cotransporters (KCCs)1 were first described as a swelling-activated K+ efflux in erythrocytes, the cell type for which functional characterization is the most complete (1, 2). The cotransport of K+ and Cl- in erythrocytes is interdependent, with a 1:1 stoichiometry and low affinity constants for both ions (2). KCCs are not influenced by membrane potential, and under most physiological conditions, they function as an efflux pathway. K+-Cl- cotransport is activated by hypotonicity and functions in regulatory volume decrease (1, 2). K+-Cl- cotransport is sensitive to the loop diuretics bumetanide and furosemide, albeit with much lower drug affinities than Na+-K+-2Cl- cotransport (3, 4). A number of other important characteristics serve to distinguish K+-Cl- cotransport from Na+-K+-2Cl- cotransport, in particular the response to protein phosphorylation and dephosphorylation (1).

A major impediment to the study of K+-Cl- cotransport has been the lack of specific high affinity inhibitors. Convincing evidence for K+-Cl- cotransport has nonetheless been reported for a number of tissues, including epithelia (5, 6), heart (7, 8), skeletal muscle (9), and brain (4, 10). In addition to cell volume regulation, non-erythroid K+-Cl- cotransport has been implicated in trans-epithelial salt absorption (5, 11), renal K+ secretion (12, 13), and the regulation of both intra- and extracellular K+-Cl- (4, 9, 10, 14).

Full-length cDNAs encoding two K+-Cl- cotransporters, KCC1 and KCC2, were recently reported (3, 4, 15-17). Both proteins are homologous to the other electroneutral cation-chloride cotransporters, the bumetanide-sensitive Na+-K+-2Cl- cotransporters BSC1 and BSC2 (also known as NKCC2 and NKCC1, respectively), and the thiazide-sensitive Na+-Cl- cotransporter TSC (also known as NCC) (18, 19). Heterologous expression of KCC1 in HEK-293 cells results in Cl--dependent, furosemide-sensitive uptake of 86Rb+ that is activated by cell swelling (3, 16). KCC1 transcript is abundant in both mouse and human erythroleukemia cell lines, indicating that KCC1 is the major erythroid K+-Cl- cotransporter (17). The widespread expression of KCC1 also suggests a significant role in non-erythroid K+-Cl- cotransport (3, 16, 20). In contrast to KCC1, the expression of KCC2 is restricted to neurons within the central nervous system (15, 21). Along with other neuronal pathways for chloride (21, 22), KCC2 plays an important role in the regulation of the transmembrane chloride gradient and thus affects the neuronal response to stimuli such as gamma -aminobutyric acid (14, 23).

An increasing amount of data suggest further heterogeneity in the proteins that mediate K+-Cl- cotransport. At the functional level, KCC1 and KCC2 differ in K+ affinities (3, 4) (Km > 25 and 5.2 mM, respectively), and a dramatically higher K+ affinity (Km 0.9 mM) has also been reported for insulin-stimulated K+-Cl- cotransport in a mouse muscle cell line (9). Erythroid K+-Cl- cotransport is heterogeneous with respect to inhibition by the K+-channel blocker quinidine, with a significant quinidine-insensitive fraction of Cl--dependent K+ flux (24). At the molecular level, cDNA probes that include human KCC1 coding sequences detect a number of transcripts, indicative of either alternative splicing or the expression of closely related gene products. Thus, a rat 3'-UTR probe detects transcripts of 3.8 and 4.4 kb in size (3), and hKCC1 probes from within the open reading frame also detect transcripts of 5.5 (20, 25) and 6-7 kb (3) in various human tissues and cell lines. Using the mouse and human EST data bases as a starting point, we have cloned cDNAs corresponding to two of these new KCC isoforms, denoted KCC3 and KCC4. Functional expression in Xenopus laevis oocytes confirms that mouse KCC3 is a furosemide-sensitive K+-Cl- cotransporter, with much greater volume sensitivity than rabbit KCC1. Although highly homologous, KCCs 1-4 differ in potentially important transmembrane (TM) segments and in the distribution of putative cytoplasmic phosphorylation sites. Northern blot analysis with specific 3'-UTR probes indicates that the KCCs have distinct but overlapping expression patterns. The identification of all four KCCs thus reveals significant differences in structure, function, and expression.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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cDNA Cloning

mKCC3-- A BLAST (basic alignment and search tool) search (26) of the EST data base revealed a number of mouse ESTs that were homologous to rat KCC1 and KCC2. The cDNAs corresponding to four of these ESTs (IMAGE clones 568084, 633794, 313521, and 698105) were obtained from the IMAGE consortium (Research Genetics, Genome Systems and/or the ATCC) and sequenced. These ESTs contained partial open reading frames homologous to various segments of KCC1 and KCC2 (Fig. 1). The 3'-end of the open reading frame and the entire 3'-UTR were identified in a fifth IMAGE cDNA (clone 1314678). The tissue distribution of mKCC3 was then assessed by RT-PCR, and widespread expression of mKCC3 was detected with the S1/AS1 primer pair (Table I), with particularly abundant transcripts in kidney and heart (data not shown). Using a primer pair (S2 and AS2 primers, Table I) spanning the gap between the IMAGE clones 313521 and 568084, a 1.2-kb fragment was amplified from C57BL/6J kidneys and subcloned into the EcoRV site of pBluescript by blunt-end ligation. PCR conditions for these and other gene-specific primers were optimized using Taq 2000 and the Opti-Prime buffer system (Stratagene). The following amplification protocol was followed, unless specified otherwise: 30 cycles of denaturation (92 °C, 2 min), annealing (54 °C, 1 min), and extension (72 °C, 1 min), followed by a final extension step (72 °C, 8 min). The extreme 5'-end of mKCC3 was cloned from BALB/c mouse kidney 5'-RACE template (CLONTECH), using two antisense primers (primers AS3 and AS4, Table I) and the AP1 adaptor primer from CLONTECH (S3 primer). This PCR utilized AmpliTaq-Gold DNA polymerase (Perkin-Elmer) and a hot-start amplification protocol, consisting of a 9-min enzyme activation step at 94 °C, followed by 35 cycles of 94 °C for 1 min and 68 °C for 2.5 min, and a final 10-min extension at 72 °C.

                              
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Table I
Oligonucleotide primers for PCR

hKCC3-- A human KCC EST clone (TIGR clone 150738) was obtained from the ATCC. Sequencing revealed that this cDNA is derived from the human ortholog of mKCC3. Another human 5'-EST (GenBankTM accession number F12342) exhibited strong amino acid homology with the extreme 3' coding sequence of the other KCCs. This EST overlaps with a large number of ESTs from the 3'-UTR of hKCC3, including the IMAGE clones 22250 and 51311 (Fig. 1). The gap between these cDNAs and the TIGR clone 150738 (Fig. 1) was bridged by RT-PCR with the primer pair S4/AS5 (Table I), using a human kidney template (CLONTECH). The PCR products for this reaction were subcloned into pBluescript. Finally, the 5'-end of hKCC3 was cloned by sequential RT-PCR of human kidney with the primer pairs S5/AS6 and S6/AS7. S5 and S6 were derived from IMAGE clones 1403108 and mKCC3, respectively.

hKCC4-- Sequence analysis of another human EST cDNA (TIGR clone 150620) indicated the existence of a fourth KCC, hKCC4. Northern blot analysis revealed significant expression in muscle, and a single 5'-RACE cDNA was cloned from a human muscle template (CLONTECH) using the S3/AS8 primer pair. This 5'-RACE PCR used Advantage polymerase mix (CLONTECH) and a hot-start protocol consisting of the following: 94 °C for 1 min, followed by 35 cycles of 94 °C for 30 s, 62 °C for 30 s, and 68 °C for 3 min. Screening a random-primed human muscle cDNA library (CLONTECH) with a probe encompassing nucleotides 741-871 of hKCC4 yielded a single cDNA that extended 5' of the start codon. Finally, two overlapping PCR fragments were amplified from human brain template (CLONTECH) with the S7/AS9 and S8/AS10 primer pairs (Table I), and subcloned into pCR2.1 by TA cloning (Invitrogen).

Sequence Analysis of KCC3 and KCC4 cDNAs

All cDNA clones were sequenced on both strands using fluorescent dye terminator chemistry (Applied Biosystems). For cDNA sequence derived exclusively from PCR, at least two cDNAs from two separate PCR reactions were sequenced. Analyses of the nucleotide and amino acid sequences were performed using the GeneWorks 2.5 and MacVector 6.5 software packages (Oxford Molecular). Alignments and other analyses also made use of the computer programs BLAST (26), DNAStar, and SMART (simple modular architecture research tool) (27).

Chromosomal Localization and Genomic Structure of hKCC3 and hKCC4

Chromosomal assignments for the human KCC3 and KCC4 genes were made using a PCR-based screening approach with the National Institute of General Medical Sciences human/rodent somatic cell hybrid mapping panel 1 (28). The primers used for hKCC3 mapping (S9 and AS11) amplify a 377-bp segment of the 3' noncoding region from genomic DNA, and the hKCC4 mapping primers (S10 and AS12) amplify a 561-bp segment of the 3' noncoding sequence. PCR reactions using DNA from the NIGMS panel were scored for the presence or absence of the appropriately sized product using agarose gel electrophoresis.

The chromosomal localization of hKCC3 was verified by sequencing the chromosome 5 genomic clone pMS621, a gift of Dr. John Armour (see "Results"). Fine mapping of hKCC4 was performed by PCR using radiation hybrid analysis with the Stanford G3 panel (Research Genetics) and the S10/AS12 primer pair. Reaction products generated by PCR were alkali-denatured, applied to a nylon membrane using a dot-blot apparatus, and subjected to Southern blotting with a 32P-labeled internal oligonucleotide probe (primer S11, Table I). Results were analyzed by querying the Stanford radiation hybrid map (http://www-shgc.stanford.edu/RH/).

Northern Blots and RT-PCR

RNA was extracted from mouse tissues (C57BL/6J strain) using guanidine isothiocyanate and cesium chloride. Total RNA (10 µg/lane) was size-fractionated by electrophoresis (5% formaldehyde, 1% agarose), transferred to a nylon membrane (Stratagene), and probed sequentially with 32P-labeled randomly primed probes corresponding to full-length glyceraldehyde-3-phosphate dehydrogenase and nucleotides 4417-5062 of mKCC3 (3'-UTR). Human multiple-tissue Northern blots containing 2 µg/lane poly(A)+ RNA (CLONTECH) were hybridized to probes generated by PCR from the 3'-UTRs of hKCC3 (nucleotides 4598-4957) and hKCC4 (nucleotides 3624-4185) and to a human beta -actin probe. Hybridization for all blots was performed overnight at 42 °C in 4× SSCP, 40% formamide, 4× Denhardt's solution, 0.5% SDS, and 200 µg of salmon sperm DNA, and membranes were washed twice for 10 min at room temperature in 2× SSCP, 0.1% SDS and twice for 1 h at 65 °C in 0.1× SSCP, 0.1% SDS. Exposure times varied as noted in the legends of Figs. 4 and 5.

In Vitro Translation of mKCC3 Protein

1.0 µg of the full-length mKCC3 cDNA (see below) was translated in vitro using [35S]methionine and T7 RNA polymerase-coupled rabbit reticulocyte lysate (TNTTM T7, Promega), both with and without pancreatic microsomes, for 90 min at 30 °C. Protein was resolved by 7% SDS-polyacrylamide gel electrophoresis followed by autoradiography.

Expression of mKCC3 in X. laevis Oocytes

For functional expression and in vitro translation, a full-length mKCC3 cDNA was assembled in the Xenopus expression vector pGEMHE (29). The resulting construct contains nucleotides 55-3812 of mKCC3. For functional comparison with mKCC3, a full-length rabKCC1 cDNA, the gift of Dr. Bliss Forbush III, was subcloned into pGEMHE. To prepare a template for cRNA, the rabbit KCC1 and mKCC3 cDNAs were linearized at the 3'-end using NheI, and cRNA was transcribed in vitro using the T7 RNA polymerase and the mMESSAGE mMACHINE kit (Ambion).

Defolliculated 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). Oocytes were incubated at 17 °C in ND96 (96 mM Na+-Cl-, 2 mM K+-Cl-, 1.8 mM Ca2+-Cl-, 1.0 mM Mg2+-Cl-, and 5 mM Hepes/Tris, pH 7.4), supplemented with 2.5 mM sodium pyruvate and 5 mg/100 ml gentamicin, for 3 days.

Functional expression of rabbit KCC1 and mKCC3 was assessed by measuring tracer 86Rb+ uptake in groups of 20-25 oocytes 3 days after water or cRNA injection. Because K+-Cl- cotransport is activated by cell swelling, both hypotonic and isotonic conditions were used. 86Rb+ uptake was measured after a 30-min incubation period in either hypotonic or isotonic Na+-free and Cl--free medium. Hypotonic medium (~125 mosmol/kg) contained 48 mM N-methyl-D-glucamine gluconate, 2 mM K+ gluconate, 4.0 mM Ca2+ gluconate, 1.0 mM Mg2+ gluconate, and 5 mM Hepes/Tris, pH 7.4. To generate the isotonic conditions (~220 mosmol/kg) sucrose was added to a concentration of 100 mM. Ouabain was added to both solutions at a concentration of 1 mM. This incubation period was followed by a 60-min uptake period using Na+-free uptake medium, both hypotonic and isotonic. Hypotonic uptake medium (~125 mosmol/kg) contained 48 mM N-methyl-D-glucamine-Cl-, 2 mM K+-Cl-, 1.8 mM Ca2+-Cl-2, 1 mM Mg2+-Cl-2, and 5 mM Hepes/Tris, pH 7.4. The isotonic conditions (~220 mosmol/kg) included 100 mM sucrose. Both uptake solutions contained 5.0 µCi/ml of 86Rb+ (New England Nuclear) and 1 mM ouabain. To determine the Cl--dependent fraction of 86Rb+ uptake, paired groups of oocytes were incubated in uptake medium without Cl- (substituted with gluconate). When indicated, 2 mM furosemide was added to both the incubation and uptake media. All uptakes were performed at 30 °C, after which oocytes were washed five times in ice-cold uptake solution without isotope to remove tracer activity in the extracellular fluid. After the oocytes were dissolved in 10% SDS, tracer activity was determined by beta -scintillation counting.

    RESULTS
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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
REFERENCES

Sequence of KCC3 and KCC4-- Full-length KCC3 and KCC4 sequences (deposited in the GenBankTM data base under accession numbers AF087436 (mKCC3), AF105365 (hKCC3), and AF105366 (hKCC4)) were determined from a number of overlapping cDNA clones (Fig. 1). The complete mouse and human KCC3 cDNAs are 5132 and 5230 nucleotides long, respectively, which is close in size to the KCC3 transcripts seen on Northern blots (Figs. 5A and 6A). Both cDNAs contain open reading frames of 3248 nucleotides, with 85% identity, and the predicted proteins consist of 1083 amino acids and exhibit 91% identity. The KCC3 3'-UTRs are only 34% identical, close to the lower limit of conservation between mouse and human orthologs (30).


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Fig. 1.   Schematic representation of KCC3 and KCC4 cDNAs. A full-length cDNA is shown for each isoform. Coding sequences are boxed, and solid lines represent the 5'- and 3'-UTR. The relative positions of partial cDNAs, derived from EST cDNA clones, RT-PCR, 5'-RACE RT-PCR, and library screening, are shown below each full-length sequence. The IMAGE or TIGR clone numbers are displayed below EST cDNAs. The figure is drawn to the scale indicated.

The hKCC4 cDNA sequence is 4237 nucleotides in length, with a 5'-UTR of 165 bp and a 3'-UTR of 622 bp. The discrepancy between the cDNA size and the size of the KCC4 transcripts (Fig. 6C), as well as the lack of a consensus polyadenylation site within the 3'-UTR, suggests that the 3'-UTR sequence is incomplete. There are three in-frame start codons between nucleotides 165 and 195 of the hKCC4 cDNA, at which translational initiation would result in proteins of 1150, 1141, or 1135 amino acids in length. However, homology to KCC1 and KCC3 begins before the third methionine (Fig. 2), which is thus an unlikely translational start site. Comparison with the mouse KCC4 sequence2 indicates significant conservation of the first nine codons, which also contain a PKC site (Fig. 2), and hence translation likely occurs at the first start codon.


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Fig. 2.   Protein alignments of the four mammalian K+-Cl- cotransporters. The deduced amino acid sequences of mKCC3, hKCC3, and hKCC4 are aligned together along with hKCC1 and rat KCC2. Identical segments are boxed in gray, and putative TM segments are underlined in black. Consensus sites for N-linked glycosylation are boxed in green within the predicted extracellular loop between TM-5 and TM-6. Consensus phosphorylation sites for PKC are boxed in red, protein kinase A sites are in yellow, and tyrosine kinases are boxed in blue.

At least two KCC4 transcripts of 6-7 kb are detected by Northern blot analysis (Fig. 6C), consistent with alternative splicing. In comparison with other KCC4 5' cDNAs (Fig. 1), the single 5'-RACE cDNA contained a deletion of nucleotides 708-854, encoding TM-1 and TM-2 in the predicted KCC4 protein. The deleted region corresponds precisely to exon 4 of hKCC1 (16), and hence at least part of the heterogeneity in KCC4 transcripts is the result of the alternative splicing of coding exons.

The four KCC proteins are 65-71% identical (Fig. 2). Sequence alignments indicate that hKCC3 shares 69% identity with rat KCC2, 65% identity with hKCC1, and 66% identity with hKCC4. The hKCC4 protein shares 71% identity with hKCC1, 66% identity with rat KCC2, and 66% identity with hKCC3. The identity between the KCCs and other cation-chloride cotransporters is in the range of 27 to 33%. A phylogenetic tree indicates that the mammalian cation-chloride cotransporters fall into two groups, one composed of the Na+-K+-2Cl- cotransporters and the Na+-Cl- cotransporter, and the other encompassing the four K+-Cl- cotransporters. As indicated by direct sequence alignments, the four KCCs form two subgroups, KCC1 paired with KCC4 and KCC3 paired with KCC2 (Fig. 3).


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Fig. 3.   Phylogenetic relationships between mammalian cation-chloride cotransporters. The six human cation-chloride cotransporters are compared with rat KCC2 in a phylogenetic tree generated with the DNAStar program.

The seven mammalian cation-chloride cotransporters share a predicted membrane topology. A central core of 12 TM domains is flanked by hydrophilic amino- and carboxyl-terminal domains that are known to have a cytoplasmic orientation (18). The major structural difference between the KCCs and the Na+-linked cotransporters is the position of a large glycosylated extracellular loop, which is predicted to occur between TM-5 and TM-6 in the KCCs and between TM-7 and TM-8 in the Na+-K+-2Cl- cotransporters and the Na+-Cl- cotransporter (18). Homology is most marked in the TM domains, the intracellular loops, and the cytoplasmic carboxyl terminus (Fig. 2).

A comparison of the four KCCs reveals a number of intriguing differences. KCC4 is the longest of the four because of an extension of ~60 amino acids at the extreme amino-terminal end (Fig. 2). Although highly conserved, none of the TM domains in the four KCCs are completely identical. Within the cytoplasmic domains the four KCCs differ significantly in the distribution of consensus phosphorylation sites for tyrosine kinases, protein kinase A, and protein kinase C (PKC). A carboxyl-terminal tyrosine phosphorylation site in KCC2 (Tyr1087) is conserved in mouse and human KCC3 (Tyr1054) (Fig. 2). The KCC4 sequence predicts a total of 11 PKC sites, 7 contained within the first 90 amino acids (Fig. 2). KCC4 has two potential protein kinase A sites, one of which (Ser939) is a predicted substrate for both protein kinase A and C. The KCC3 sequences predict fewer PKC sites, of which only two are conserved in both mouse and human (Thr814 and Ser1006).

KCC1 (3), KCC2 (4), and other members of the cation-chloride cotransporter gene family (18) are known to be glycoproteins, and the four KCC sequences contain three identical N-linked glycosylation sites in the otherwise poorly conserved extracellular loops (Fig. 2). The in vitro translation of mKCC3 results in a protein with an apparent molecular mass of ~115 kDa, slightly lower than the predicted core weight of 119 kDa. The addition of canine pancreatic microsomes results in the appearance of an additional band of higher molecular mass (Fig. 5C), which is consistent with in vitro glycosylation.

Chromosomal Localization of hKCC3 and hKCC4-- Chromosomal assignments for human KCC3 and KCC4 were defined using a PCR-based somatic cell hybrid mapping strategy. The genes for KCC3 and KCC4 were assigned to chromosomes 5 and 15, respectively. Fine mapping by radiation hybrid analysis places the hKCC4 gene on chromosome 15q14 between the markers D15S1040 and D15S118. Further localization of KCC3 was facilitated by the finding that D5S110 (31), a chromosome 5 VNTR (variable number of tandem repeats) minisatellite marker, is contained within the gene. The corresponding genomic subclone, pMS621 (the gift of Dr. John Armour), was sequenced to verify that it contained hKCC3 exons just 5' of the VNTR (Fig. 4). The exon/intron boundaries in the hKCC1 gene (16) are conserved in this portion of the hKCC3 gene, and the exons in pMS621 correspond to exons 14-17 of hKCC1 (Table II). The chromosome 5 summary map generated by the Wessex Human Genetics Institute (http://cedar.genetics.soton, ac.uk/pub/chrom5/map.html) indicates that D5S110 is on 5p15.3, between D5S678 and the telomere.


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Fig. 4.   Structure of the pMS621 genomic subclone. The pMS621 clone contains exons 14-17 of the hKCC3 gene, which flank the D5S110 VNTR. The polymorphic tandem repeat, 1.8 kb in length in the pMS621 clone, can vary between 0.5 and 10 kb (31). Arrows and GenBankTM/EBI accession numbers indicate the previously reported DNA sequence flanking the repeat.

                              
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Table II
Exon/intron boundaries of hKCC3 exons 14-17

Tissue Distribution of KCC3 and KCC4-- Northern blot analysis was performed with probes derived from the 3'-UTRs of KCC3 and KCC4. KCC3 probes detect 5.3-kb transcripts in a number of tissues, most prominently in the heart and kidney (Figs. 5A and 6A). Very little KCC3 transcript is detectable in adult brain. KCC4 has a more restricted expression pattern, with significant amounts of transcript found only in muscle, brain, lung, heart, and kidney (Fig. 6C). At least two different transcripts of 6-7 kb hybridize to KCC4 probes, consistent with alternative splicing (see above).


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Fig. 5.   A, Northern blot analysis of mKCC3 (48-h exposure). The transcript is abundant in kidney and heart and undetectable in brain. B, the same blot was stripped and reprobed with glyceraldehyde-3-phosphate dehydrogenase (12-h exposure). C, In vitro translation of mKCC3 protein. 35S-labeled mKCC3 polypeptide was translated from cDNA by rabbit reticulocyte lysate, both with (+) and without (-) pancreatic microsomes, resolved by 7% SDS-polyacrylamide gel electrophoresis, and visualized by autoradiography (12-h exposure).


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Fig. 6.   A, Northern blot analysis of hKCC3. Human multiple-tissue Northern blots (CLONTECH) were probed with a cDNA probe from the 3'-UTR of hKCC3 (4-day exposure). A 5.3-kb transcript is seen in multiple tissues. B, the same Northern blots reprobed with a human beta -actin probe (12-h exposure). C, Northern blot analysis of hKCC4, using a 3'-UTR probe (2-day exposure). Only one multiple-tissue blot was positive. Variable expression of two 6-7 kb transcripts is detected in muscle, heart, kidney, lung, and brain.

Functional Expression of mKCC3 and Rabbit KCC1-- Microinjection of Xenopus oocytes with cRNA in vitro transcribed from mKCC3 and rabbit KCC1 resulted in significant increases in 86Rb+ uptake compared with water-injected oocytes. The increased 86Rb+ uptake was evident only when oocytes were incubated in hypotonic medium. Fig. 7A shows 86Rb+ uptake in H2O, rabbit KCC1, and mKCC3-injected oocytes, using isotonic and hypotonic conditions. In H2O-injected oocytes 86Rb+ uptake increased from 26.3 ± 5.33 pmol/oocyte/h under isotonic conditions to 46.8 ± 2.79 pmol/oocyte/h in hypotonic conditions (p < 0.01). In hypotonic medium, however, tracer Rb+ uptake decreased to 4.2 ± 0.43 pmol/oocyte/h in the absence of extracellular Cl- (p < 0.001) and to 17.9 ± 1.6 pmol/oocyte/h in the presence of 2 mM furosemide, suggesting the presence of an endogenous K+-Cl- cotransporter in Xenopus oocytes. In rabbit KCC1-injected oocytes, 86Rb+ uptake increased from 8.8 ± 0. 5 pmol/oocyte/h in isotonic medium to 200 ± 13.8 in hypotonic medium (p < 0.001). The observed uptake was Cl--dependent and furosemide-sensitive. 86Rb+ uptake in mKCC3-injected oocytes increased from 15.3 ± 1.4 pmol/oocyte/h when oocytes were incubated in isotonic conditions (~220 mosmol/liter) to 2,552 ± 126 pmol/oocyte/h when incubated in hypotonic medium (~125 mosmol/liter). Most of the 86Rb+ uptake observed in mKCC3-injected oocytes was Cl--dependent, because uptake in mKCC3 oocytes in Cl--free medium was 65 ± 5.8 pmol/oocyte/h. The activation of mKCC3 by cell swelling is significantly higher than that observed for H2O-injected oocytes or for rabbit KCC1-injected oocytes. Fig. 7B shows that the Cl--dependent fraction of 86Rb+ uptake was 1.9 ± 0.1-fold higher than the isotonic base line in control oocytes, 38.6 ± 2.8-fold higher in rabbit KCC1-injected oocytes, and 226 ± 11.5-fold higher than base line in oocytes injected with mKCC3.


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Fig. 7.   Functional expression of mKCC3 and rabKCC1 in X. laevis oocytes. A, 86Rb+ uptake in oocytes injected with H20, rabKCC1 or mKCC3. Uptakes were performed under both isotonic (200 mosmol/kg) and hypotonic (100 mosmol/kg) conditions in the presence (open bars) or absence (gray bars) of extracellular chloride and in the presence of 2 mM furosemide (black bars). There is a significant difference in the vertical scale between the three graphs. B, relative activation by hypotonicity of K+-Cl- cotransport expressed as a multiple of the respective uptakes in isotonic conditions. The endogenous K+-Cl- cotransporter (H20) exhibits only a 2-fold activation, compared with the 39-fold activation of rabbit KCC1 (rabKCC1) and a 226-fold activation of mKCC3.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We report here the cloning of KCC3 and KCC4, expanding the family of mammalian cation-chloride cotransporters to a total of seven members (Fig. 3). All of the known cation-chloride transporters belong functionally and/or structurally in one of three groups, encompassing two Na+-K+-2Cl- cotransporters, one Na+-Cl- cotransporter, and four K+-Cl- cotransporters. A phylogenetic comparison of protein sequences indicates that the four K+-Cl- cotransporters evolved separately from the Na+-dependent transporters, forming a distinct subfamily (Fig. 3). This separation is also evident at the genomic level, since the exon/intron structure of the BSC/NKCC genes (32, 33) is similar to that of the TSC/NCC gene (34), but differs significantly from that of the KCC1 gene (16). Partial characterizations of the mouse KCC2 gene (GenBankTM accession number AJ011033) and the human KCC3 gene (Table II) indicate that the KCCs have a conserved genomic structure.

The four KCC proteins share a 65-71% identity in primary structure. Within the central hydrophobic core, TM-2 and TM-4 show the most variation (Fig. 2); data from an extensive study of ion affinity in the Na+-K+-2Cl- cotransporters implicates TM-2 in cation transport (35) and TM-4 in anion transport (36). The cytoplasmic carboxyl termini contain segments of substantial homology interspersed with a number of more variable regions. The extreme carboxyl terminus is identical in the four KCCs. Indeed, this homology was crucial in identifying ESTs that contained the 3'-end of hKCC3 (see "Materials and Methods"). Presumably some of the conserved cytoplasmic segments are involved in protein-protein interactions; however, none of the KCCs contain recognizable protein signaling domains (27). The predicted amino and carboxyl termini differ significantly in the distribution of putative phosphorylation sites (see Fig. 2 and "Results"). KCC4, in particular, is rich in potential PKC sites. The KCC2 and KCC3 sequences predict a tyrosine phosphorylation site near the extreme carboxyl terminus. This consensus phosphorylation site is altered in KCC1 and KCC4 by variation in amino acids flanking the conserved tyrosine (Fig. 2).

The structural similarity between the four KCCs strongly suggests that both KCC3 and KCC4 encode K+-Cl- cotransporters. The functional expression of both KCC1 (3, 16) and KCC2 (4) has already been reported, using heterologous expression in HEK-293 cells. Using modification of a previously published protocol (37), we have utilized X. laevis oocytes for the functional expression of rabbit KCC1 and mKCC3. The significant identity between hKCC4 and the other KCCs suggests that it too encodes a K+-Cl- cotransporter; however, functional expression of this cDNA has not yet been examined. Xenopus oocytes have an endogenous bumetanide-sensitive Na+-K+-2Cl- cotransporter that is activated by hypertonicity and inhibited by hypotonicity (37). The activity of this cotransporter was abolished in the present study by the use of Na+-free conditions. The lack of extracellular Na+ also exposes an endogenous K+-Cl- cotransporter that is only weakly activated by cell swelling (Fig. 7A), permitting a clear distinction between oocytes injected with KCC cRNA and controls injected with water. Oocytes injected with cRNA generated from the rabbit KCC1 and mKCC3 cDNAs express Cl--dependent uptake of 86Rb+, which is partially sensitive to furosemide. A furosemide concentration of 2 mM inhibited the K+-Cl- cotransport activity of rabbit KCC1 and mKCC3 by 81 and 69%, respectively. Rabbit KCC1 and mKCC3 are both activated by cell swelling but differ markedly in the degree of activation over base-line isotonic conditions. Thus, rabbit KCC1 is activated 35-fold by hypotonic conditions, compared with the 226-fold activation of mKCC3 (Fig. 7B). Functional expression in HEK-293 cells suggests that rat KCC2 is insensitive to cell swelling (4). However, this KCC has not yet been compared with KCC1 and KCC3 using the Xenopus expression system. Although direct phosphorylation of KCC proteins has not been demonstrated, it is known that protein phosphorylation and dephosphorylation play important roles in the regulation of red cell K+-Cl- cotransport (1, 38). KCC1 and KCC3 differ in the distribution of putative phosphorylation sites for a number of protein kinases (Fig. 2), which may account in part for their differential sensitivity to cell swelling.

The 5.3-kb KCC3 transcript is widely expressed in both mouse and human, and ESTs have been reported from an array of cDNA libraries. However, in both species KCC3 transcript is most abundant in the heart, followed by the kidney. Cultured chick cardiac cells possess a K+-Cl- cotransporter capable of both inward and outward K+-Cl- transport (7). The role of this transport pathway in cardiac physiology is not yet clear. However, alpha 1-adrenoreceptor stimulation of isolated rat hearts stimulates a K+ efflux pathway that is partially sensitive to loop diuretics (39). The cell swelling induced by cardiac ischemia also stimulates a significant efflux of K+ that is mediated predominantly by K+-Cl- cotransport. This efflux of K+ may play an important role in the genesis of arrhythmias following myocardial ischemia (8).

KCC4 has a more restricted expression pattern than KCC1 and KCC3, confined primarily to muscle, brain, heart, and kidney. An insulin-stimulated K+-Cl- cotransport system with a high affinity for extracellular K+ has been described in mouse skeletal muscle cells (9); however, the physiological roles of K+-Cl- cotransport in this tissue have not been extensively studied. KCC4 cDNAs were also cloned by RT-PCR from human brain (Fig. 1), in which a single transcript of ~6 kb has been detected by Northern analysis. Like KCC2, KCC4 may function in neuronal Cl- homeostasis, with secondary effects on the response to gamma -aminobutyric acid and other stimuli (10, 14, 21, 23).

The combined results from this and other reports indicate that the mammalian kidney expresses KCC1 (3, 16), KCC3, and KCC4 (Figs. 5 and 6). There is strong physiological evidence for K+-Cl- cotransport in several nephron segments, including proximal tubule (5, 40), thick ascending limb (6, 11), distal convoluted tubule (12), and cortical collecting duct (13). The expression of three KCCs in the kidney suggests the possibility that there is an overlap in their intrarenal distribution. Moreover, one or more KCCs may target to the apical membrane of renal tubular cells, particularly within the cortical nephron (12, 13). The intrarenal distribution of hKCC1 was recently studied by in situ hybridization with a coding sequence probe (20). Although the probe chosen cross-hybridizes with KCC3, it is evident from this study that KCCs are expressed along the entire nephron, including within glomeruli (20). The role of K+-Cl- cotransport within the glomerulus is unknown. However, glomerular mesangial cells express BSC2/NKCC1 in vivo and exhibit hormone-sensitive Na+-K+-2Cl- cotransport (18). There is also preliminary evidence that cation-chloride cotransporters help set intracellular chloride activity in mesangial cells (41).

The human KCC3 gene has been localized to chromosome 5. Fine mapping to 5p15.3, between the anchor marker D5S678 and the telomere, was facilitated by the identity between a segment of hKCC3 and sequence flanking the VNTR D5S110 (31). A genomic clone containing this VNTR also contains four exons of the hKCC3 gene (Fig. 4) homologous to exons 14-17 of the hKCC1 gene (Table II) (16). Polymorphism involving the D5S110 VNTR is generated by variable repetition of a core 11-bp sequence motif (31). The hKCC3 gene is not a particularly likely candidate for genetic disorders mapped to 5p15. However, KCC3 is expressed within the kidney, and it may play a role in basolateral ion transport within the thick ascending limb (6, 11). KCC3 is therefore a functional candidate gene for the remaining cases of Bartter's syndrome not caused by mutations in BSC1/NKCC2, ROMK, or CLC-NKB (42). In this regard, repeat expansion of a 12-bp motif within the cystatin B promoter has been shown to cause progressive myoclonic epilepsy (43), and expansion of the D5S110 VNTR could also conceivably affect the expression of hKCC3.

Human KCC4 has been localized to chromosome 15q14 between the markers D15S1040 and D15S118. This section of chromosome 15 has been linked to two subtypes of idiopathic generalized epilepsy (44, 45) and to a neurophysiological phenotype associated with schizophrenia (46). The markers D15S1040 and D15S118 also flank a 5-centimorgan region of chromosome 15q14 containing the gene for peripheral neuropathy with or without agenesis of the corpus callosum (Andermann's syndrome) (47).

In summary, we have cloned KCC3 and KCC4, two new members of the cation-chloride cotransporter family. The unexpected heterogeneity of K+-Cl- cotransport raises a number of important issues for future study. First, the overlapping expression patterns of the four KCCs suggests the possibility that certain cell types will express more than one isoform, and indeed this appears to be the case in some human cell lines (25). Second, the significant homology within cytoplasmic domains has implications for the generation of isoform-specific antibodies. Third, the structural and functional comparison of the KCCs suggests that they differ in a number of important characteristics, including the response to changes in cellular volume (Fig. 7). Fourth, the full characterization of the physiological role(s) of individual KCCs will likely require sequential targeted disruption of the mouse KCC genes.

    ACKNOWLEDGEMENTS

We thank Dr. Steven C. Hebert for encouragement and generous support, Drs. Bliss Forbush III and John Armour for clones, and Karen Sloan-Brown and Craig Short for technical assistance.

    Note Added in Proof

While this manuscript was in press, Hiki et al. published the characterization of human KCC3 (Hiki, K., D'Andrea, R. J., Furze, J., Crawford, J., Woollatt, E., Sutherland, G. R., Vadas, M. A., and Gamble, J. R. (1999) J. Biol. Chem. 274, 10661-10667). This KCC3 cDNA is an alternative splice form of the KCC4 cDNA reported here. In deference to the earlier publication, we have reversed the numbering of our GenBankTM/EBI submissions and will henceforth refer to the KCC on chromosome 15q14 as KCC3 and the KCC on chromosome 5p15 as KCC4.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants K-11 DK02103 (to D. B. M.) and R-29 HL-49251 (to E. D.) and by grants from the Mexican Council of Science and Technology (CONACYT, M3840) and the Howard Hughes Medical Institute (75197-553601) (to G. G).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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF087436 (mKCC3), AF105365 (hKCC3), and AF105366 (hKCC4).

§ To whom correspondence should be addressed: Div. of Nephrology, Vanderbilt University Medical Ctr., MCN S-3223, Nashville, TN 37232. Tel.: 615-343-2853; Fax: 615-343-7156; E-mail: david.mount{at}mcmail.vanderbilt.edu.

parallel Supported by a scholarship from Dirección Genaral del Personal Académico of the National University of Mexico.

Dagger Dagger Established investigator of the American Heart Association.

2 D. B. Mount and E. Delpire, unpublished data.

    ABBREVIATIONS

The abbreviations used are: KCC, K+-Cl- cotransporter; BSC or NKCC, bumetanide-sensitive Na+-K+-2Cl- cotransporter; TSC or NCC, thiazide-sensitive Na+-Cl- cotransporter; h, human (e.g. hKCC); m, mouse (e.g. mKCC); 86Rb+, rubidium; EST, expressed sequence tag; bp, base pair(s); kb, kilobase (or kilobase pairs); IMAGE, integrated molecular analysis of genomes and their expression; TIGR, The Institute for Genome Research; UTR, untranslated region; RT-PCR, reverse transcriptase polymerase chain reaction; RACE, rapid amplification of conserved ends; VNTR, variable number of tandem repeats; TM, transmembrane segment; PKC, protein kinase C.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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