©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Primary Structure, Functional Expression, and Chromosomal Localization of the Bumetanide-sensitive Na-K-Cl Cotransporter in Human Colon (*)

(Received for publication, February 23, 1995; and in revised form, May 12, 1995)

John A. Payne(§)(¶) Jian-Chao Xu(§)(**) Melanie Haas (1) Christian Y. Lytle (§§) David Ward (1) Bliss Forbush , III

From the Departments of Cellular and Molecular Physiology and Genetics, Yale University School of Medicine, New Haven, Connecticut 06510

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

By moving chloride into epithelial cells, the Na-K-Cl cotransporter aids transcellular movement of chloride across both secretory and absorptive epithelia. Using cDNA probes from the recently identified elasmobranch secretory Na-K-Cl cotransporter (sNKCC1) (Xu, J. C., Lytle, C. Zhu, T. T., Payne, J. A., Benz, E., and Forbush, B., III(1994) Proc. Natl. Acad. Sci. 91, 2201-2205), we have identified the human homologue. By screening cDNA libraries of a human colonic carcinoma line, T84 cell, we identified a sequence of 4115 bases from overlapping clones. The deduced protein is 1212 amino acids in length, and analysis of the primary structure indicates 12 transmembrane segments. The primary structure is 74% identical to sNKCC1, 91% identical to a mouse Na-K-Cl cotransporter (mNKCC1), 58% identical to rabbit and rat renal Na-K-Cl cotransporters (NKCC2), and 43% identical to the thiazide-sensitive Na-Cl cotransporters from flounder urinary bladder and rat kidney. Similar to sNKCC1 and mNKCC1, the 5`-end of the human colonic cotransporter is rich in G+C content. Interestingly, a triple repeat (GCG) occurs within the 5`-coding region and contributes to a large alanine repeat (Ala). Two sites for N-linked glycosylation are predicted on an extracellular loop between putative transmembrane segments 7 and 8. A single potential site for phosphorylation by protein kinase A is present in the predicted cytoplasmic C-terminal domain. Northern blot analysis revealed a 7.4-7.5-kilobase transcript in T84 cells and shark rectal gland and a 7.2-kilobase transcript in mammalian colon, kidney, lung, and stomach. Metaphase spreads from lymphocytes were probed with biotin-labeled cDNA and avidin fluorescein (the cotransporter gene was localized to human chromosome 5 at position 5q23.3). Human embryonic kidney cells stably transfected with the full-length cDNA expressed a 170-kDa protein recognized by anti-cotransporter antibodies. Following treatment with N-glycosidase F, the molecular mass of the expressed protein was similar to that predicted for the core protein from the cDNA sequence (132-kDa) and identical to that of deglycosylated T84 cotransporter (135-kDa). The stably transfected cells exhibited a 15-fold greater bumetanide-sensitive Rb influx than control cells, and this flux required external sodium and chloride. Flux kinetics were consistent with an electroneutral cotransport of 1Na:1K:2Cl. Preincubation in chloride-free media was necessary to activate fully the expressed cotransporter, suggesting a [Cl]-dependent regulatory mechanism.


INTRODUCTION

The vectorial transport of chloride across epithelia is a prominent mechanism in the maintenance of water and electrolyte homeostasis. Chloride transport is involved in reabsorption of NaCl in the thick ascending limb of the loop of Henle in mammalian kidney (1) and in secretion of NaCl in a diverse array of secretory epithelia, including the intestine, trachea, and parotid and the avian and elasmobranch salt glands(2, 3, 4, 5, 6) . In all of these tissues, the chloride entry into the epithelia cell is mediated by a Na-K-Cl cotransport protein, which couples the electroneutral movement of sodium, potassium, and chloride ions. In order to carry out net salt transport, the Na-K-Cl cotransporter functions in concert with three other membrane proteins: chloride and potassium channels and the sodium pump. The importance of the proper functioning of these ion transport mechanisms in chloride secretory epithelia is exemplified by the disease states of cystic fibrosis and secretory diarrhea, where there are defects in the regulation of ion transport(7) .

The Na-K-Cl cotransporter is characterized by a sensitivity to the sulfamoylbenzoic acid ``loop'' diuretics, furosemide, and bumetanide and a strict ion dependence of transport (for review, see (8) ). Among the Na-K-Cl cotransporters that have been described in various cells and tissues, there is substantial variation in both molecular weight and ``loop'' diuretic sensitivity(9) , suggesting the presence of different isoforms. For example, studies using the photosensitive bumetanide analog, 4-[H]benzoyl-5-sulfamoyl-3-(3-thenyloxy) benzoic acid, have demonstrated the specific labeling of a 150-kDa protein in membranes from dog kidney(10) , mouse kidney(11) , and duck red blood cells(12) , whereas in shark rectal gland membranes, a 195-kDa protein is observed(13) . Secretory epithelia, such as the rectal gland and parotid gland typically show a 10-fold lower affinity for bumetanide than absorptive epithelia, such as the thick ascending limb of the loop of Henle of mammalian kidney and flounder intestine(8) . Additionally, in absorptive and secretory epithelia, the Na-K-Cl cotransporter displays distinct differences in its polarized membrane distribution. In secretory epithelia, the Na-K-Cl cotransporter is an exclusively basolateral membrane protein, whereas in absorptive epithelia it is localized to the apical membrane(14) .

Recently, we reported the cloning, sequencing, and expression of a cDNA encoding the basolateral Na-K-Cl cotransporter from the shark rectal gland (sNKCC1)()(15) . In addition, we (16) and others (17) have identified the apical Na-K-Cl cotransporter from mammalian kidney (NKCC2). We proposed that NKCC1 and NKCC2 represented distinct isoforms, since they display only 61% amino acid identity and are encoded by mRNAs of different sizes and tissue specificities(15, 16) . In the present report, we have utilized homology to sNKCC1 to obtain the cDNA encoding the basolateral Na-K-Cl cotransporter (hNKCC1) from a human intestinal cell line, the T84 cell. The human Na-K-Cl cotransporter displays remarkable identity to sNKCC1 within the predicted transmembrane segments (88%), yet when examined in the same expression system, hNKCC1 exhibits considerably different ion affinities compared with sNKCC1.


MATERIALS AND METHODS

Cloning and Sequence Analysis

A human colonic (T84 epithelial cell line) cDNA library in Uni-Zap XR was obtained from Stratagene (La Jolla, CA). This library was screened with two P-labeled cDNA probes derived from the shark rectal gland Na-K-Cl cotransporter (nucleotides -298 to 1235 and 1236 to 3411; see (15) ). Using low stringency hybridization (34 °C in 50% formamide, 5 SSPE, 5 Denhardt's solution, 0.1% SDS, and 100 µg/ml fish sperm DNA) and high stringency wash conditions (55 °C in 0.5 SSC and 0.1% SDS), a single positive clone (TEF 1-1) was identified, carried through two rounds of plaque purification, and successfully excised into Bluescript SK with the helper phage R408. A second T84 cDNA library in obtained from Dr. John R. Riordan (Hospital for Sick Children; Toronto, Ontario, Canada) was screened with a 5`-end 0.7-kb XbaI-XbaI fragment of TEF 1-1. Using similar screening procedures as described above, 23 positive cDNAs were obtained in Bluescript SK (see Fig. 1). All cDNAs were characterized by Southern blot analysis and partial sequencing.


Figure 1: Schematic diagram of selected clones encoding the Na-K-Cl cotransporter isolated from T84 cDNA libraries. The 3636-nucleotide open reading frame is displayed at top. The shadedregion of the clones indicates the open reading frame. The solidregion indicates untranslated sequence, and the hatchedregion indicates intronic sequence. The sequence corresponding to consensus splice acceptor and donor sites (28) are displayed underneath each intronic region, where intronic sequence is in lowercase, and coding sequence is in uppercase. Two exons are displayed as open regions A and B in clones TEF 11a and TEF 2a.



The full-length cDNA was sequenced bidirectionally by the dideoxy chain termination method (18) using a combination of manual sequencing with Sequenase II (U. S. Biochemical Corp.) and automated sequencing (Applied Biosystems Inc.) with synthetic oligonucleotide primers and fluorescent dideoxy terminators. Analysis of the nucleotide sequence and deduced amino acid sequence were performed with programs from the Genetics Computer Group software. The program TBLASTN (19) was used to search GenBank. Identities with other sequences are given as the fraction of residues in hNKCC1, which are found to be identical in the matched sequence.

Northern Blot Analysis

T84 cells were obtained from Dr. James L. Madara (Brigham and Women's Hospital, Boston, MA) and cultured as described previously(20) . Total RNA was isolated from fresh rabbit tissues, spiny dogfish (Squalus acanthias) rectal gland, and T84 cells by the guanidine thiocyanate method(21) . Poly(A) RNA was purified from total RNA using magnetic beads (PolyATtract, Promega Corp.).

Samples of rabbit tissue mRNA were denatured by heating to 65 °C in formamide and formaldehyde and size-fractionated on a 1% agarose gel. Fractionated mRNA was transferred to a nylon membrane by semidry blotting. The rabbit tissue Northern blot used in this study was the one used in (16) . The membrane was completely stripped of previously hybridized probe by incubating in 0.1 SSC and 0.5% SDS at 75 °C, and confirmation of probe removal was determined by film exposure (48 h at -70 °C). A blot of human tissue mRNA was obtained commercially (Clonetech, human MTN blot, 7760-1, lot 52805). The blots were prehybridized 4 h at 45 °C in 50% formamide, 2 SSPE, 2 Denhardt's, 1% SDS, 200 µg/ml yeast RNA, 100 µg/ml fish sperm DNA and then hybridized for 24 h in fresh hybridization solution containing 10 cpm/ml P-labeled cRNA probe. Antisense cRNA probes were produced as run-off transcripts (MAXIscript, Ambion Inc) (nucleotides 650-1102 for Fig. 5a and nucleotides 796-1926 for Fig. 5b) from cDNA clones. The blots were subjected to a final wash of 20 min at 65 °C in 0.1 SSC and 0.5% SDS and analyzed by autoradiography at -70 °C with an intensifying screen.


Figure 5: Northern blot analysis of expression of the human colonic Na-K-Cl cotransporter and related mRNAs. A, rabbit tissues and shark rectal gland. B, human tissues. Poly(A)-selected RNA (5-10 µg in A, 2 µg in B) was hybridized with antisense cRNA probes transcribed in vitro from hNKCC1 (upperpanels). The blots were reprobed with a -actin cDNA probe as a control for RNA integrity (lower panels); note that an additional band in some lanes of the -actin control panel reflects a muscle-specific actin transcript, 1.8-kb.



Chromosomal Localization

Fluorescence in situ hybridization was performed as described previously(22) . Metaphase spreads were prepared from human peripheral blood lymphocytes after methotrexate synchronization and bromodeoxyuridine incorporation. Probes were assigned to R bands generated by bromodeoxyuridine incorporation and subsequent photolysis(23) . Briefly, TEF 1-1 cDNA was labeled with 11-biotin dUTP by nick translation. The probe DNA (60 ng) was coprecipitated with 3 µg of human Cot 1 DNA plus 6 µg of salmon sperm DNA. After resuspension in 12 µl of hybridization solution (50% formamide, 2 SSC, 10% dextran sulfate) the DNA was denatured at 75 °C for 10 min. Denatured DNA was applied directly to denatured metaphase spreads (50% formamide and 2 SSC for 2 min at 70 °C). The slides were incubated for 12 h at 37 °C, washed 3 times in 42% formamide and 2 SSC at 42 °C, washed 3 additional times at low stringency (1 SSC, 40 °C), blocked with bovine serum albumin (3% in 4 SSC for 30 min at 37 °C), and stained with Hoechst 33258 (2.5 mg/ml) for 15 min. After washing briefly with distilled water, they were placed under a UV lamp at a distance of 10 cm for 1 h; they were subsequently incubated for an additional hour in 2 SSC at 42 °C. Avidin-DCS-fluorescein isothiocynate (Vector Laboratories) in 1% bovine serum albumin and 4 SSC was used to detect probe sequences in which 11-biotin dUTP had been incorporated, and, after incubation for 30 min at 37 °C, the slides were washed 3 times in 4 SSC and 1% Tween 20 for 5 min at 42 °C. Propidium iodide (200 ng/ml) in antifade (Dabco) was used to counterstain chromosomes. A CCD camera (PM512, Photometrics, Tucson, AZ) was used to visualize fluorescent signals; gray scale images were obtained sequentially for fluorescein and propidium iodide with precision filter sets (C. Zeiss), and the images were pseudocolored and merged.

Stable Expression in a Mammalian Cell Line

A full-length construct of the sequence for the human colonic Na-K-Cl cotransporter was prepared from the two cDNAs, TEF 11a and TEF 1-1 (see Fig. 1). Much of the 5`-untranslated region and all of the intronic DNA were removed from TEF 11a by subcloning a PstI-PstI fragment into Bluescript KS (nucleotides -24 to 831). The small subcloned fragment of TEF 11a and the cDNA of TEF 1-1 were ligated at a common NcoI site and subcloned into the mammalian expression vector pJB20 at EcoRI and KpnI restriction sites of the polylinker(24) . Transcription of the insert is under the control of the cytomegalovirus promoter, and the vector contains a neomycin (G418) resistance gene.

The human embryonic kidney cell line (HEK-293) was maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, penicillin (50 units/ml), and streptomycin (50 µg/ml) in a humidified incubator (5% CO at 37 °C). The full-length construct was transfected into HEK-293 cells by calcium phosphate precipitation(25) . Nearly confluent cells were split 1:6 the day before transfection and were 50-70% confluent in a 10-cm dish immediately prior to transfection with 20 µg of DNA in 140 mM NaCl, 25 mM HEPES, 0.75 mM NaHPO, 125 mM CaCl, pH 7.05. 48 h after transfection, cells were selected for neomycin resistance by growth in G418 (900 µg/ml), and after 3 weeks, single resistant colonies were amplified and screened by Rb influx measurements and Western blotting.

Enzymatic Modification of Protein

CHAPS-solubilized membrane protein (50 µg) was deglycosylated by incubation (4 h at 37 °C) with 20 units/ml of N-glycosidase F (Boehringer Mannheim) in a 0.05-ml solution containing 4% CHAPS, 0.15 M sodium phosphate buffer, pH 7.8, 2.5 mM EDTA, and protease inhibitors. Enzymatic treatment was terminated by the addition of electrophoresis sample buffer.

Rb Influx Assay

HEK-293 cells were subcultured into 96-well plates (1:5 split) and grown to confluency (2-3 days). Prior to the flux assay, the cells were preincubated for 1 h in a low chloride hypotonic medium (1:2 dilution of isotonic low chloride medium but lacking ouabain) and then briefly washed in a low chloride isotonic medium containing 135 mM sodium gluconate, 5 mM potassium gluconate, 1 mM CaCl, 1 mM MgCl, 1 mM NaHPO, 2 mM NaSO, 15 mM HEPES, pH 7.4, and 0.1 mM ouabain. The initial rate of Rb influx was determined in quadruplicate wells in the presence or absence of 200 µM bumetanide. Uptake was determined in isotonic medium in which Rb (1 µCi/ml) replaced potassium. Influx was terminated after 0.5 min by the addition of an ice-cold, high potassium saline followed by five rinses in phosphate-buffered saline. Cellular extracts (80 µl of 2% SDS) were assayed for Rb by Cherenkov radiation, for protein with the Micro BCA protein kit (Pierce) and for Na-K-Cl cotransporter production by Western blot analysis(13) . Reported K and K values were obtained from nonlinear least squares fits to the data using one- or two-site (for chloride) models. Western blot analysis made use of a monoclonal antibody (T10) produced against a glutathione S-transferase fusion protein containing the hydrophilic C terminus of hNKCC1 (amino acids 902-1212).()


RESULTS

Cloning and Sequencing of hNKCC1

Clones encoding the bumetanide-sensitive Na-K-Cl cotransporter in human colon (the T84 cell) were initially obtained by screening a cDNA library under low stringency conditions with nonoverlapping fragments of the shark rectal gland Na-K-Cl cotransporter (see ``Materials and Methods'' and (15) ). The first library screening produced a cDNA (TEF 1-1; Fig. 1) with homology to the rectal gland Na-K-Cl cotransporter with a large open reading frame and poly(A) tail but that lacked the 5`-end of the coding region. A second library was screened with a 5`-end fragment of TEF 1-1 (0.7-kb; see ``Materials and Methods''). 23 cDNAs were plaque-purified and excised into Bluescript SK. Two independent cDNAs (TEF 11a and 20a) extended the 5`-end of TEF 1-1, providing the complete coding region of the human colonic Na-K-Cl cotransporter (Fig. 1). An overlapping nucleotide sequence of 4115-bp was identified, hNKCC1. The 5`-end of hNKCC1 is exceedingly rich in guanine and cytosine nucleotides with the first 900 bases containing 74% (G+C) content. We have reported previously the presence of a (G+C)-rich region at the 5`-end of sNKCC1(15) . Interestingly, within this (G+C)-rich region of hNKCC1 is a triple repeat (GCG), which occurs within the coding region and contributes to a large alanine repeat (Ala- Ala; Fig. 2). This same triple repeat is observed in the putative basolateral Na-K-Cl cotransporter from mouse inner medullary collecting duct cells, mNKCC1(26) .()Since alanine is assigned a positive hydropathic index (i.e. hydrophobic residue) by Kyte and Doolittle(42) , the large alanine repeat encoded by the GCG trinucleotide repeat is responsible for the very hydrophobic region (residues color-coded red), which appears within the otherwise hydrophilic N-terminal domain of Fig. 3A.


Figure 2: Sequence alignment of the deduced primary structure of Na-K-Cl cotransporters in human colon (hNKCC1), mouse inner medullary collecting duct cells (mNKCC1; Ref. 26), shark rectal gland (sNKCC1; (15) ), and rabbit kidney (rNKCC2; splice variant A, (16) ). Lower case letters identify residues not identical to hNKCC1. The predicted transmembrane segments are underlined. Two putative N-linked glycosylation sites between predicted transmembrane segments 7 and 8 are identified by a verticalbar. The single potential cAMP-dependent protein kinase phosphorylation site is identified by an asterisk. Two threonines that correspond to known phosphoacceptors in sNKCC1 are identified by solidtriangles. The regions encoded by the two exons identified from the cDNA libraries are distinguished by the hatchedlinesabove the sequence (from Fig. 1; exon A and B).




Figure 3: Hypothetical model of the human colonic Na-K-Cl cotransporter. The amino acid residues are color-coded by hydropathic index (A) as determined by the Kyte-Doolittle algorithm (42) or by the fractional identity of hNKCC1 to sNKCC1 (B). Both the hydropathic index and identity are averaged over a running window of 15 amino acids. Potential glycosylation sites between putative transmembrane segments 7 and 8 are indicated by branchedlines. Secondary structural elements predicted by the PHD program (45, 46) are shown (helices, ; wavy lines, ). No attempt has been made to pair structural elements.



The hNKCC1 cDNA includes a full-length open reading frame encoding 1212 amino acids, beginning with the first ATG (GCTATGG) downstream of a stop codon. The predicted molecular mass is 132 kDa, which is very similar to the core polypeptide identified in N-glycosidase-F-treated membranes from T84 cells with monoclonal antibodies developed against the shark rectal gland Na-K-Cl cotransporter (135-kDa)(27) . The full-length amino acid sequence of hNKCC1 is 93% identical to mNKCC1 and 73% identical to sNKCC1 (Fig. 2). The most divergent region is the N terminus, which includes a number of insertions and deletions when compared with mNKCC1 and sNKCC1. Outside of this area, hNKCC1 aligns perfectly with mNKCC1.

We noted that a number of the clones isolated from one of the libraries contained intronic sequences with consensus splice sites for intron-exon boundaries ((28) , Fig. 1). Many of these clones were undoubtedly produced from incompletely processed mRNA during library construction (see (29) ). This finding allowed us to identify some of the exons in the hNKCC1 gene (Fig. 1). Interestingly, one cDNA contained consensus splice sites and intronic DNA on either side of a 96-bp exon (Fig. 1, exon B), which corresponds exactly to the position of an alternatively spliced cassette exon in the rabbit kidney Na-K-Cl cotransporter(16) .

Structure of hNKCC1

Similar to previously identified cation-chloride cotransport proteins(15, 16, 17, 26, 30) , hydropathy analysis of hNKCC1 predicts a large central hydrophobic region bounded by N- and C-terminal hydrophilic domains (Fig. 3A). As displayed in Fig. 3, 12 membrane-spanning helices are predicted in our model of hNKCC1. Based on homology with sNKCC1, we predict that both terminal domains reside within the cytoplasm. The polypeptide sequence of hNKCC1 has five potential N-linked glycosylation sites. Two of these sites are located in a predicted extracellular hydrophilic region of the protein between putative transmembrane segments 7 and 8 ( Fig. 2and Fig. 3). In comparing the primary structure of hNKCC1 to related proteins, we note that among the loops connecting predicted transmembrane domains, those that are predicted to be intracellular are considerably better conserved than are those that are predicted to be extracellular (e.g. sNKCC1, Fig. 3B).

The Na-K-Cl cotransporter in the T84 cell is known to be stimulated by agents such as vasoactive intestinal peptide that activate adenylate cyclase(2) . Unlike sNKCC1, which has no consensus sites for phosphorylation by cAMP-dependent protein kinase, there is a single potential cAMP-dependent protein kinase site in hNKCC1 located within the predicted cytoplasmic C-terminal domain (Ser; Fig. 2). This residue corresponds to one of three potential cAMP-dependent protein kinase phosphorylation sites in the rabbit kidney Na-K-Cl cotransporter (rNKCC2, Ser; (16) ). In addition, hNKCC1 contains two threonines (Thr and Thr; Fig. 2), which correspond to known phosphoacceptors in sNKCC1 (Thr and Thr; (15) ). The fact that the region around both of these threonine residues in hNKCC and sNKCC1 is very well conserved suggests that these residues are also phosphacceptors in hNKCC1 (Fig. 3B).

Chromosomal Localization of the hNKCC1 Gene

Using the TEF 1-1 cDNA, we localized the hNKCC1 gene to human chromosome 5 at position 5q23.3 by fluorescence in situ hybridization on R-banded chromosomes (Fig. 4). Five separate metaphase spreads were investigated. Two had distinct signals on both homologues of chromosome 5, with signals on all four chromatids. Three others had distinct signals on only one homologue with discrete signals on each chromatid. Evidence of hybridization at other chromosomal regions with TEF 1-1 was not observed. Delpire et al.(1994) have recently mapped mNKCC1 to mouse chromosome 18 in a region that they reported to be syntenic with human chromosome 5q31-33. Interestingly, the murine renal absorptive isoform, mNKCC2, has recently been cloned, and the gene localized to mouse chromosome 2(43) . These studies clearly distinguish NKCC1 and NKCC2 as separate gene products.


Figure 4: Chromosomal localization of hNKCC1 gene. The hNKCC1 gene is localized to chromosome 5 at position 5q23.3 on metaphase spreads of human peripheral lymphocytes. Note red signal against the green R-banded chromosomes.



Tissue Northern Blot Analysis

The level of expression of hNKCC1 and related transcripts in T84 cells, shark rectal gland, and several rabbit and human tissues was examined by Northern blot analysis (Fig. 5). Using antisense cRNA probes, a 7.2-7.5-kb mRNA was expressed at high levels in T84 cells, rectal gland, rabbit stomach, rabbit kidney, and rabbit large intestine (Fig. 5A) and in all of the human tissues (Fig. 5B). A prominent message is also found at 6.3 kb in human heart and skeletal muscle, and a signal is detected in human kidney at 5.1 kb (Fig. 5B). After longer development of the blot shown in Fig. 5A, transcripts were detected at 7.2 kb in rabbit brain, lung, and small intestine and at 5.1 kb in rabbit kidney (data not shown).

Functional Expression of hNKCC1 in HEK-293 Cells

The function of the protein encoded by hNKCC1 was examined by isolating a stably expressing cell line (HEK-293) following transfection with a full-length construct in the mammalian expression vector, pJB20 (see ``Materials and Methods''). Of the 40 cells lines that were screened, 37 exhibited various levels of increased bumetanide-sensitive Rb influx above untransfected control cells (data not shown). The cell line that displayed the largest bumetanide-sensitive Rb influx was used in subsequent studies to characterize the functional expression of hNKCC1.

Western blot analysis using monoclonal antibody T10 (see ``Materials and Methods'') revealed that HEK-293 cells stably expressing hNKCC1 produced a glycoprotein with very similar mass to that observed in native T84 membranes, 170-kDa (Fig. 6). We also immunodetected a similar sized band in untransfected HEK-293 cells but at a much reduced level (note different protein loads). We presume that this 170-kDa band in untransfected HEK-293 cells is the endogenous Na-K-Cl cotransport protein we have previously identified in these cells by bumetanide-sensitive Rb influx analysis(15) . Two bands were immunodetected in the lane containing membranes from the stable cell line (Fig. 6). The lower molecular weight band is indicative of incomplete glycosylation of the expressed protein, a probable result of the large amount of protein being produced. We presume that the higher molecular weight band is the functional, glycosylated cotransporter delivered to the plasma membrane, since removal of N-linked oligosaccharides with N-glycosidase F resulted in a single smaller molecular weight band indistinguishable from that of the native deglycosylated T84 cotransporter (135-kDa; Fig. 6).


Figure 6: Western blot analysis of membranes from native T84 cells, HEK-293 cells stably expressing hNKCC1, and untransfected HEK-293 cells. Membrane protein was separated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane, and probed with monoclonal antibody T10 (see ``Materials and Methods''). Membrane protein from each cell line was treated with (+) or without(-) N-glycosidase F prior to gel electrophoresis. Molecular mass standards are indicated in kDa.



Fig. 7presents the time course of Rb uptake in HEK-293 untransfected cells and HEK-293 cells stably expressing hNKCC1. The stably expressing cells exhibited a linear Rb uptake for about 30 s, and the initial rate of bumetanide-sensitive Rb influx was 15-fold greater in cells expressing hNKCC1 than control untransfected cells. As we have noted previously in expression studies of the shark rectal gland cotransporter(15) , full activity of the expressed Na-K-Cl cotransporter from human colon required a preincubation in low chloride media prior to the Rb influx assay (Fig. 7). This supports the hypothesis that intracellular chloride is an important regulator of cotransporter activity in secretory epithelia(15, 31) . The expressed T84 cotransport protein displayed definitive characteristics of a Na-K-Cl cotransport mechanism. As displayed in Fig. 8(bottomrow), these include Rb influx required the simultaneous presence of sodium and chloride in the flux medium, and Rb influx was almost completely inhibited by bumetanide (200 µM). Additionally, the Rb influx kinetics are consistent with a single transport site for sodium and rubidium (hyperbolic curves) and two sites for chloride (sigmoidal curve, see legend of Fig. 8).


Figure 7: Time course of Rb uptake into untransfected HEK-293 cells and HEK-293 cells stably expressing hNKCC1 following a 1-h preincubation in chloride-free (closed symbol) or control media (open symbol). Crosssymbols indicate Rb uptake in the presence of 200 µM bumetanide. Lines are drawn by eye. Samples were taken in triplicate. Data are representative of three separate experiments.




Figure 8: The dependence of the initial rate of Rb influx on external [Na], [Cl], and [Rb] for untransfected HEK-293 cells and HEK-293 cells stably expressing either sNKCC1 (HEK 15; Ref. 15) or hNKCC1. Cations were replaced with N-methyl glucamine and chloride with gluconate. Curves represent the best fits of the data to a model of activation at single sodium and rubidium sites and at two identical chloride sites; K values for this representative experiment are displayed in each panel (summary data are presented in the text).



The results of an experiment examining the ion dependences of cotransport in cells expressing sNKCC1 or hNKCC1, as well as in untransfected HEK-293 cells are presented in Fig. 8. The affinities for sodium, rubidium, and chloride are 4-9-fold greater in cells expressing hNKCC1 compared with those expressing sNKCC1 (in three experiments, the values (millimolar) for hNKCC1-transfected cells were as follows: K = 19.6 ± 4.9, K = 2.68 ± 0.72, K = 26.5 ± 1.3); and for sNKCC1-transfected cells K = 165 ± 34, K = 14.3 ± 8.0, K = 101 ± 24; see also (15) ). These findings are consistent with much lower ion affinities observed for cotransport in the shark rectal gland (40) compared with mammalian systems(32, 44) . Ion affinities for sodium and chloride in the untransfected cells (K = 22.7 ± 9.5, K = 12.8 ± 7.0, K = 41.5 ± 5.8; see also (15) ) were similar to those for hNKCC1-transfected cells.

Fig. 9displays the bumetanide inhibition curves for HEK-293 untransfected cells and cells expressing hNKCC1. The stably expressing cells displayed a K for bumetanide of 0.16 ± 0.05 µM that was not significantly different than that observed for the endogenous cotransport protein of untransfected HEK-293 cells, 0.09 ± 0.03 µM (t test; p > 0.05). The bumetanide affinities of untransfected HEK-293 cells and of cells expressing hNKCC1 are greater than that for sNKCC1 expressed in HEK-293 cells (0.57 µM, (15) ). The bumetanide affinity of expressed hNKCC1 is 10-fold higher than that reported by Dharmsathaphorn et al.(2) for T84 cells; this discrepancy can be attributed to a difference in experimental protocol.()


Figure 9: Bumetanide inhibition of the initial rate of Rb influx into untransfected HEK cells and HEK-293 cells stably expressing hNKCC1. Cells were incubated in chloride-free medium for 1 h and then prior to the flux assay; cells were allowed to bind bumetanide at the indicated concentration for 20 min in the presence of 72 mM chloride. Results are fit by a model of bumetanide inhibition at a single site. Summary data of separate experiments are as follows (mean ± S.E.): untransfected HEK-293 cells, K = 0.09 µM ± 0.02, V = 8.34 nmol/mg of protein/min ± 0.98; and HEK-293 cells stably expressing hNKCC1, K = 0.16 µM ± 0.03, V = 154 nmol/mg of protein/min ± 16.




DISCUSSION

This paper presents the cloning, functional expression, and chromosomal localization of the basolateral isoform of the Na-K-Cl cotransporter in the human T84 colonic carcinoma cell line. We have previously cloned the basolateral Na-K-Cl cotransporter from the shark rectal gland (NKCC1; (15) ) as well as the apical isoform of this protein from the rabbit kidney (NKCC2; (16) ).

Two T84 cell cDNA libraries were screened under low stringency conditions to obtain the full-length coding region of human NKCC1 (hNKCC1) from overlapping clones. Over the entire length, the amino acid sequence is 91% identical to a putative basolateral Na-K-Cl cotransporter from mouse(26) , 74% identical to the shark rectal gland Na-K-Cl cotransporter(15) , 58% identical to the rabbit and rat kidney Na-K-Cl cotransporters(16, 17) , and 43% identical to the flounder and rat thiazide-sensitive Na-Cl cotransporters(17, 30) . Analysis of the primary structure of all NKCC and thiazide-sensitive Na-Cl cotransporter proteins cloned to date suggests that they have very similar overall structures with large N- and C-terminal hydrophilic regions bounding a central core hydrophobic domain containing 12 putative transmembrane segments (Fig. 3). These cotransporter proteins display significant homology to predicted protein sequences encoded by open reading frames identified in sequencing projects of a cyanobacterium (Synococcus sp.; (33) ), yeast (Saccharomyces cerevisiae, unpublished, Z36104), a nematode (Caenorhabditis elegans, Refs. 34 and 35), and a moth (Manduca sexta, unpublished, U17344). An expressed sequence tag from a human colorectal tumor (unpublished, T25163) was identified from the data base, and the nucleotide sequence is 100% identical to hNKCC1. Another expressed sequence tag from human brain (unpublished, T16107) displays 38% amino acid identity to hNKCC1.

The N terminus is the most divergent region among the different NKCC proteins ( Fig. 2and Fig. 3B) with numerous insertions and deletions present. Interestingly, NKCC1 is a significantly larger protein than NKCC2, due primarily to an additional 80 amino acids at the N terminus. The wide interspecies sequence divergence at the N terminus suggests that much of this region is not directly involved in carrying out ion translocation. Since the Na-K-Cl cotransporter is known to be regulated by various factors in a cell-specific fashion (9) , the divergence at the N terminus might reflect differences in modulation by phosphorylation and other regulatory processes.

Despite great variation in the N-terminal hydrophilic region of the NKCC proteins, all of these proteins contain a short 11-amino acid segment (for sNKCC1, TFGHNTIDAVP) that is very well conserved (note the high identity red region of the N terminus in Fig. 3B). Within this short peptide, Ile in sNKCC1 is the only variant residue, being replaced by methionine in hNKCC1, mNKCC1, and NKCC2. Interestingly, the strictly conserved Thr is known to be phosphorylated in sNKCC1 by stimuli that promote secretion through activation of cAMP-dependent protein kinase(36) . Since Thr and the region around it are so highly conserved, it is likely that this phosphorylation site is important for activation of both secretory and absorptive NKCC isoforms. Our evidence support the hypothesis that the response to cAMP in the rectal gland is an indirect result of changes in cell [Cl] and not a direct effect of cAMP-dependent protein kinase on the cotransporter(31, 37) . This hypothesis is consistent with the finding that no region of sNKCC1 conforms to the consensus motif of cAMP-dependent protein kinase(15) . Human NKCC1 contains a single strong cAMP-dependent protein kinase consensus site at Ser (KKES), and it is possible that this cAMP-dependent protein kinase site plays a role in regulation; recent work with dog tracheal epithelial cells has provided evidence for the direct activation of the Na-K-Cl cotransporter via a cAMP-dependent protein kinase-dependent pathway in addition to activation via a reduction in cell [Cl](38) .

The 5`-end of hNKCC1 is very rich in cytosine and guanine nucleotides. This appears to be a characteristic of the secretory isoform of NKCC since it is also observed in sNKCC1 and mNKCC1 but not in the rabbit or rat renal absorptive isoforms, NKCC2. We have noted that within this (G+C)-rich region of hNKCC1 is a triple repeat (GCG) that occurs within the coding region of the protein. A growing number of human genes are known to contain five or more consecutive triple repeats and several human genetic diseases, including fragile X syndrome and myotonic dystrophy, are associated with mutations that result in a tremendous expansion of the trinucleotide repeat(39) .

The central core hydrophobic domain of hNKCC1, containing the 12 putative transmembrane (TM) segments, displays high identity to NKCC2 (79%) consistent with the importance of the transmembrane segments in ion translocation. Six of the 12 putative TM segments of hNKCC1 are greater than 90% identical to sNKCC1, and putative TM segments 1, 3, 6, and 10 show complete conservation of all residues between these two secretory cotransporters. One of the putative TM segments, which displays lower identity among the different NKCC and thiazide-sensitive Na-Cl cotransporter proteins is TM2 (50-80%). We have previously reported that putative TM2 in rNKCC2 is encoded largely by an alternatively spliced 96-bp exon, resulting in three separate renal NKCC2 splice variants that are structurally different only over the 31 amino acids encoded by this exon(16) . Although we have no evidence of alternative splicing in the human colonic Na-K-Cl cotransporter, we have identified a similar 96-bp exon encoding the identical region of hNKCC1 (exon B in Fig. 1and Fig. 2). We have hypothesized that the portion of TM2 encoded by this exon may form part of the ion translocation pocket and thus may confer differences in ion affinities among the NKCC proteins(16) . It may be significant, therefore, that sNKCC1 and hNKCC1 stably expressed in HEK-293 cells display very different affinities for the three cotransported ions ( (15) and see Fig. 8).

In contrast to the variability of the N-terminal hydrophilic region, the C terminus displays remarkable homology among the NKCC proteins ( Fig. 2and Fig. 3B). Outside of a small region where there is considerable divergence and a 6-bp deletion in comparison with sNKCC1 (residues 946-975), hNKCC1 aligns perfectly with sNKCC1 and is greater than 82% identical to it. A very well conserved region within the C-terminal domain (hNKCC1; Gln-Thr) displays between 90 and 92% identity among the different NKCC isoforms ( Fig. 2and Fig. 3B). It seems likely that this may encode a membrane-associated region involved in ion translocation.

Using Northern blot analysis to examine the tissue expression of hNKCC1, we have found that a 7.2-7.5-kb transcript is found in most tissues examined (Fig. 5), similar to the result previously obtained in shark rectal gland(15) . This result suggests that in addition to its special role in salt-secreting epithelia, NKCC1 may represent the ``housekeeping'' form of the Na-K-Cl cotransporter. A prominent 6.3-kb message was detected in human skeletal and heart muscle; based on its strength of hybridization, this transcript is most likely a muscle-specific splice variant of NKCC1. An additional transcript was weakly detected by the hNKCC1 probe in human kidney (Fig. 5B) and in rabbit kidney at 5.1-kb after longer development (data not shown). We and others have reported that this 5.1-kb transcript encodes the absorptive or apical isoform of the Na-K-Cl cotransporter which is found only in renal tissue and is found there at high levels(16, 17) .

The ion affinities obtained from similar Rb influx studies on HEK-293 cells stably expressing sNKCC1 or hNKCC1 are considerably different from one another. For example, an almost 10-fold difference in sodium affinity is observed between the two NKCC proteins (Fig. 8). Previous work in this laboratory has provided evidence that both anions and cations are occluded within a transport ``pocket'' on the Na-K-Cl cotransporter(41) . It is reasonable to propose that this transport ``pocket'' is present within the TM segments and that this region of the protein largely determines the ion affinities of the cotransporter. TM segments that are most divergent between sNKCC1 and hNKCC1 may be responsible for the ion affinity differences; these include TM2 (80% identity), TM4 (75%), TM5 (75%), and TM11 (80%). In this regard, we have proposed that the alternatively spliced exons encoding most of TM2 of rNKCC2 result in ion affinity differences for the three kidney splice variants(16) .


FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK47661 and DK17433. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 GenBank®/EMBL Data Bank with accession number(s) U30246[GenBank Link].

§
Contributed equally to this work.

Supported by a fellowship from the American Heart Association, Connecticut affiliate. To whom correspondence should be addressed: Dept. of Human Physiology, University of California, School of Medicine, Davis, CA 95616. Tel.: 916-752-1359; Fax: 916-752-5423.

**
Present address: Div. of Immunobiology, Yale University School of Medicine, New Haven, CT 06510.

§§
Present address: University of California, Div. of Biomedical Sciences, 2226 Webber Hall, Riverside, CA 92521.

The abbreviations used are: sNKCC1, basolateral Na-K-Cl cotransporter from the shark rectal gland; NKCC, Na-K-Cl cotransporter; kb, kilobase(s); bp, base pair(s); TM, transmembrane segment; HEK, human embryonic kidney; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

C. Y. Lytle, J.-C. Xu, D. Biemesderfer, and B. Forbush, manuscript in preparation.

The secretory and absorptive isoforms of the Na-K-Cl cotransporter are designated NKCC1 (15) and NKCC2 (16), respectively. The alternate designation BSC2 and BSC1 have also been employed (17, 26).

We preincubated the cells in the various bumetanide solutions for 20 min prior to the flux assay, whereas Dharmsathaphorn et al. (2) performed a 3-min flux assay directly in the presence of bumetanide. Since the rate constant of association of bumetanide is low (e.g. 2.5 10 mol/s for dog kidney, (47) ), the latter method substantially underestimates the affinity for bumetanide.


ACKNOWLEDGEMENTS

We thank Laura Roman for technical advice and many helpful discussions, Mike Caplan, Peter Igarashi, Chris Gillen, and Rachel Behnke for reading the manuscript, and Grace Jones for excellent technical assistance. We also thank John Riordan for providing a cDNA library and James Madara for providing the T84 cells. Automated sequencing was performed by the Yale University/Keck Foundation Nucleic Acid Facility.


REFERENCES

  1. Greger, R., and Schlatter, E.(1981)Pflgers Arch. 392, 92-94
  2. Dharmsathaphorn, K., Mandel, K. G., Masui, H., and McRoberts, J. A.(1985) J. Clin. Invest. 75, 462-471 [Medline] [Order article via Infotrieve]
  3. Fong, P., Chao, A. C., and Widdicombe, J. H.(1991)Am. J. Physiol. 261,L290-L295
  4. Turner, R. J., George, J. N., and Baum, B. J.(1986)J. Membr. Biol. 94, 143-152 [Medline] [Order article via Infotrieve]
  5. Torchia, J., Lytle, C., Pon, D. J., Forbush, B., III, and Sen, A. K.(1992)J. Biol. Chem. 267, 25444-25450 [Abstract/Free Full Text]
  6. Hannafin, J., Kinne-Saffran, E., Friedman, D., and Kinne, R.(1983) J. Membr. Biol. 75, 73-83 [Medline] [Order article via Infotrieve]
  7. Halm, D. R., and Frizzell, R. A. (1990) in Textbook of Secretory Diarrhea (Lebenthal, E., and Duffy, M., eds) pp. 47-58, Raven Press Ltd., New York
  8. Haas, M.(1994) Am. J. Physiol.267,C869-C885
  9. Haas, M.(1989) Annu. Rev. Physiol. 51, 443-457 [CrossRef][Medline] [Order article via Infotrieve]
  10. Haas, M., and Forbush, B., III(1987)Am. J. Physiol.253,C243-C250
  11. Haas, M., Dunham, P. B., and Forbush, B., III(1991)Am. J. Physiol. 260,C791-C804
  12. Haas, M., and Forbush, B., III(1988)Biochim. Biophys. Acta 939, 131-144 [Medline] [Order article via Infotrieve]
  13. Lytle, C., Xu, J.-C., Biemesderfer, D., Haas, M., and Forbush, B., III(1992) J. Biol. Chem. 267, 25428-25437 [Abstract/Free Full Text]
  14. O'Grady, S. M., Palfrey, C., and Field, M.(1987)Am. J. Physiol. 253,C177-C192
  15. Xu, J. C., Lytle, C., Zhu, T., Payne, J. A., Benz, E., and Forbush, B., III(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2201-2205 [Abstract]
  16. Payne, J. A., and Forbush, B., III(1994)Proc. Natl. Acad. Sci. U. S. A. 91, 4544-4548 [Abstract]
  17. Gamba, G., Miyanoshita, A., Lombardi, M., Lytton, J., Lee, W.-S., Hediger, M., and Hebert, S. C.(1994)J. Biol. Chem. 269, 17713-17722 [Abstract/Free Full Text]
  18. Sanger, F., Nicklen, S., and Coulson, A. R.(1977)Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  19. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J.(1990) J. Mol. Biol. 215, 403-441 [CrossRef][Medline] [Order article via Infotrieve]
  20. Dharmsathaphorn, K., McRoberts, J. A., Mandel, K. G., Tisdale, L. D., and Masui, H. (1984)Am. J. Physiol.246,G204-G208
  21. Chomczynski, P., and Sachi, N.(1987)Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  22. Haas, M., Ward, D., Lee, J., Roses, A. D., Clarke, V., E'Eustachio, P., Lau, D., Vega Saen de Miera, E., and Rudy, B.(1993)Mammalian Genome 4,711-715 [Medline] [Order article via Infotrieve]
  23. Arnold, N., Bhatt, M., Ried, T., Ward, D. C., and Wienberg, J. (1993) in Techniques and Methods in Molecular Biology: Nonradioactive Labelling and Detection of Biomolecules (Kessler, C. ed) pp. 324-334, SpringerVerlag, New York
  24. Beck, P. J., Orlean, P., Albright, C., Robbins, P. W., Gething, M.-J., and Sambrook, J. F. (1990)Mol. Cell. Biol. 10, 4612-4622 [Medline] [Order article via Infotrieve]
  25. Puddington, L., Woodgett, C., and Rose, J. K.(1987)Proc. Natl. Acad. Sci. U. S. A. 84, 2756-2760 [Abstract]
  26. Delpire, E., Rauchman, M. I., Beier, D. R., Hebert, S. C., and Gullans, S. R.(1994) J. Biol. Chem. 269, 25677-25683 [Abstract/Free Full Text]
  27. Lytle, C., Torchia, J., and Forbush, B., III(1992)J. Gen. Physiol. 100,39 (abstr.)
  28. Mount, S. M. (1982)Nucleic Acids Res. 10, 459-472 [Abstract]
  29. Riordan, J. R., Rommens, J. M., Kerem, B. S., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J. L., Drumm, M. L., Iannuzzi, M. C., Collins, F. S., and Trsui, L. C.(1989)Science 245, 1066-1073 [Medline] [Order article via Infotrieve]
  30. Gamba, G., Saltzberg, S. N., Lambardi, M., Miyanoshita, A., Lytton, J., Hediger, M. A., Brenner, B. M., and Hebert, S. C.(1993)Proc. Natl. Acad. Sci. U. S. A. 90, 2749-2753 [Abstract]
  31. Forbush, B., III, Haas, M., and Lytle, C.(1992)Am. J. Physiol. 262,C1000-C1008
  32. Kim, H. D., Tsai, Y.-S., Franklin, C. C., and Turner, J. T.(1988)Biochim. Biophys. Acta 946, 397-404 [Medline] [Order article via Infotrieve]
  33. Cantrell, A., and Bryant, D. A.(1987)Plant Mol. Biol. 9, 453-468
  34. Waterston, R., Martin, C., Craxton, M., Huynh, C., Coulson, A., and Hillier, L.(1992) Nature Genetics 1, 114-123 [Medline] [Order article via Infotrieve]
  35. Sulston, J., Du, Z., Thomas, K., Wilson, R., Hillier, L., Staden, R., Halloran, N., Green, P., Thierry-Mieg, J., Qiu, L., Dear, S., Coulson, A., Craxton, M., Durbin, R., Berks, M., Metzstein, M., Hawkins, T., Ainscough, R., and Waterston, R. (1992) Nature356, 37-41 [CrossRef][Medline] [Order article via Infotrieve]
  36. Lytle, C., and Forbush, B., III(1992)J. Biol. Chem. 267, 25438-25443 [Abstract/Free Full Text]
  37. Lytle, C., and Forbush, B., III(1992)Am. J. Physiol.262,C1009-C1017
  38. Haas, M., and McBrayer, D. G.(1994)Am. J. Physiol. 266,C1440-C1452
  39. Morell, V.(1993) Science 260, 1422-1423 [Medline] [Order article via Infotrieve]
  40. Silva, P., and Myers, M. A.(1986)Am. J. Physiol.250,F516-F519
  41. Forbush, B., III, and Haas, M.(1989)Biophys. J.55,422 (abstr.)
  42. Kyte, J., and Doolittle, R. F.(1982)J. Mol. Biol.157,105-132 [Medline] [Order article via Infotrieve]
  43. Quaggin, S. E., Payne, J. A., Forbush, B., III, and Igarashi, P. (1995) Mamm. Genome, in press
  44. McRoberts, J. A., Erlinger, S., Rindler, M. J., and Saier, M. H., Jr.(1982) J. Biol. Chem. 257, 2260-2266 [Abstract/Free Full Text]
  45. Rost, B., and Sander, C. (1994)Proteins19,55-77 [Medline] [Order article via Infotrieve]
  46. Rost, B. Casadio, R., Fariselli, P., and Sander, C. (1995) Protein Science, in press
  47. Forbush, B., III, and Palfrey, H. C.(1983)J. Biol. Chem.258,11787-11792 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.