Colonic H-K-ATPase beta -subunit: identification in apical membranes and regulation by dietary K depletion

Pitchai Sangan, Sarah S. Kolla, Vazhaikkurichi M. Rajendran, Michael Kashgarian, and Henry J. Binder

Departments of Internal Medicine and Pathology, Yale University, New Haven, Connecticut 06520-8019


    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

P-type ATPases require both alpha - and beta -subunits for functional activity. Although an alpha -subunit for colonic apical membrane H-K-ATPase (HKcalpha ) has been identified and studied, its beta -subunit has not been identified. We cloned putative beta -subunit rat colonic H-K-ATPase (HKcbeta ) cDNA that encodes a 279-amino-acid protein with a single transmembrane domain and sequence homology to other rat beta -subunits. Northern blot analysis demonstrates that this HKcbeta is expressed in several rat tissues, including distal and proximal colon, and is highly expressed in testis and lung. HKcbeta mRNA abundance is upregulated threefold compared with normal in distal colon but not proximal colon, testis, or lung of K-depleted rats. In contrast, Na-K-ATPase beta 1 mRNA abundance is unaltered in distal colon of K-depleted rats. Na depletion, which also stimulates active K absorption in distal colon, does not increase HKcbeta mRNA abundance. Western blot analyses using a polyclonal antibody raised to a glutathione S-transferase-HKcbeta fusion protein established expression of a 45-kDa HKcbeta protein in both apical and basolateral membranes of rat distal colon, but K depletion increased HKcbeta protein expression only in apical membranes. Physical association between HKcbeta and HKcalpha proteins was demonstrated by Western blot analysis performed with HKcbeta antibody on immunoprecipitate of apical membranes of rat distal colon and HKcalpha antibody. Tissue-specific upregulation of this beta -subunit mRNA in response to K depletion, localization of its protein, its upregulation by K depletion in apical membranes of distal colon, and its physical association with HKcalpha protein provide compelling evidence that HKcbeta is the putative beta -subunit of colonic H-K-ATPase.

active potassium absorption; rat distal colon; hydrogen-potassium-adenosinetriphosphatase alpha -subunit


    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

ACTIVE K ABSORPTION AND secretion are important transport processes of mammalian large intestine (2). Active K absorption, a unique function of the distal colon, is energized and regulated by H-K-ATPase, an apical membrane P-type ATPase (8, 11). This colonic H-K-ATPase is a member of a gene family of related P-type ATPases that include Na-K-ATPase and gastric H-K-ATPase (7, 14, 35, 36). ATPases in this gene family usually are heterodimers that consist of alpha - and beta -subunits. The alpha -subunit contains the catalytic function of these ATPases, whereas the specific function of the heavily glycosylated beta -subunit is not completely known. Recent studies indicate that important functional properties of beta -subunits include an essential role in the stabilization, maturation, and enzymatic activity of both Na-K-ATPase and H-K-ATPase (4).

Colonic K transport is modified by several factors, including changes in dietary K and aldosterone (11, 12). Increases in dietary K induce active K secretion, whereas dietary K depletion enhances active K absorption (12). Aldosterone, either as a result of dietary Na depletion or following its subcutaneous administration, markedly stimulates active K absorption in the rat distal colon (11, 37). The recent cloning of a cDNA that encodes the alpha -subunit of the rat distal colon H-K-ATPase (HKcalpha ) (7, 14) led to studies that have assessed the regulation by which dietary Na and dietary K depletion modify active K absorption and colonic H-K-ATPase (15, 33). Dietary Na depletion increases HKcalpha message and protein abundance and H-K-ATPase activity in the rat distal colon. In contrast, dietary K depletion did not increase the abundance of HKcalpha message or protein in the distal colon (15, 33).

One possible explanation to account for the absence of an upregulation of HKcalpha message by dietary K depletion is that the effect of dietary K depletion on active K absorption is mediated by the beta -subunit of the colonic H-K-ATPase, as beta -subunits are required for the catalytic activity of P-type ATPases. Although a specific beta -subunit for the colonic H-K-ATPase has not been isolated, conflicting observations exist as to whether a colon-specific beta -subunit is required for maximal colonic H-K-ATPase activity (5, 6, 20). Thus, although Lee et al. (20) recently expressed HKcalpha in Sf9 cells as ouabain-insensitive H-K-ATPase activity in the apparent absence of any beta -subunit, other studies in Xenopus oocytes have demonstrated that the expression of HKcalpha required noncolonic beta -subunits (5, 6). This requirement for such beta -subunits may either represent a promiscuous expression by a related beta -subunit, indicating that the colonic beta -subunit is closely related, or indicate that colonic H-K-ATPase activity requires a beta -subunit that is not colon specific.

To identify a beta -subunit for colonic H-K-ATPase, a series of low-stringency Northern blot analyses with gastric H-K-ATPase beta -subunit (HKgbeta ) and Na-K-ATPase beta 1- and beta 2-subunit (NaKbeta 1 and NaKbeta 2) cDNAs were performed. These analyses demonstrated either no hybridization (HKgbeta , NaKbeta 2) or only a single band (NaKbeta 1) with mRNA from rat distal colon. A novel beta -subunit cDNA that had been recently cloned from a rat astrocytoma cell line (38) and that had hybridized with guinea pig colon mRNA (Y. Suzuki, personal communication) was also used as a probe in additional Northern blot analyses using mRNA from rat distal colon. Because this cDNA hybridized with mRNA from rat distal colon at the size of 1.9 kb, we initiated experiments to clone a rat colon-derived beta -subunit using the rat astrocytoma beta -subunit cDNA as a probe.

The present study demonstrates the following. 1) A full-length beta -subunit cDNA (HKcbeta ) isolated from a rat colon cDNA library is identical to a cDNA isolated from rat astrocytoma cells (38). In studies using this cDNA, dietary K depletion selectively increases the abundance of HKcbeta message in the rat distal colon. 2) A polyclonal antibody to HKcbeta protein identifies a protein in apical and basolateral membranes of rat distal colon. 3) This HKcbeta protein expression is selectively increased in K depletion in apical but not in basolateral membranes. 4) Coimmunoprecipitation experiments reveal a physical interaction between HKcalpha and HKcbeta proteins. As a result, these observations suggest that this cDNA encodes the colonic H-K-ATPase beta -subunit.


    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Male Sprague-Dawley rats (200-250 g body wt; Charles River Laboratories, Wilmington, MA) were divided into three experimental groups: the control group was fed normal rat food that contained 4.4 g Na/kg and 9.5 g K/kg. The Na-depleted group was given a Na-free diet for 1 wk. The K-depleted group was given a K-free diet (0.6 mg K/kg) for 3 wk. All animals were allowed free access to water.

At the end of the experimental diet periods, the animals were killed and proximal colon, distal colon, ileum, jejunum, stomach, kidney, brain, lung, testis, liver, spleen, and heart from normal rats; proximal and distal colon, testis, and lung from K-depleted rats; and proximal and distal colon from Na-depleted rats were immediately removed and washed with diethyl pyrocarbonate-treated sterilized saline. Colonocytes from proximal and distal colon were obtained, as previously described (33), and all tissues were then homogenized using a Polytron homogenizer in 4 M guanidine isothiocyanate buffer for 60 s and centrifuged at 3,000 rpm for 10 min to remove any unbroken cells. Total RNA was prepared from the supernatant by ultracentrifugation through CsCl (32). Poly(A)+ mRNA was isolated by passing total RNA through oligo(dT) cellulose columns according to the method described by Sambrook et al. (32). Total RNA and mRNA were quantitated by absorbance at 260 nm in an ultraviolet-visible double-beam spectrophotometer (Shimadzu).

Northern blot analysis. Northern blot analyses were performed using poly(A)+ mRNA, as previously described (33). A 32P-labeled full-length HKcbeta probe [1 × 106 counts · min-1 · ml-1 (cpm/ml)] was added to the membrane for hybridization at 42°C in a Hybaid oven for 18 h. Blots were washed for 15 min in 0.1× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) and 0.1% SDS at 65°C and were exposed to Hyperfilm (Amersham, La Jolla, CA) using a Bioplus intensifying screen at 70°C.

cDNA screening and sequencing. A rat colon cDNA library with randomly primed cDNA inserts >1.0 kb in length was cloned in pcDNAI (Invitrogen, San Diego, CA) and was used in the cloning of the beta -subunit; 300,000 colonies were plated onto 20 Luria-Bertani [LB; containing 50 µg/ml ampicillin and 7 µg/ml tetracycline (amp/tet)] plates 150 mm in diameter. A microwave colony-screening protocol from Invitrogen was strictly followed. Briefly, colonies were transferred to nylon membrane (UV Duralon membrane, Stratagene, La Jolla, CA) after nylon membrane had been placed on top of the colonies in the plate. Membrane filters were lifted carefully and were laid on a fresh LB (amp/tet) agar plate by facing the colony side up; the plates were incubated with filters at 37°C for 1 h to allow colonies to grow. To regrow the colonies on the agar plates that were lifted, plates were incubated at 37°C for about 6 h and then stored at 4°C. The filters were carefully removed from the agar plate and placed colony side up on a Whatman 3 filter paper prewetted in lysis buffer (2× SSC-5% SDS, pH 7.0). The filter paper and the membrane with colonies were placed in a turntable microwave oven and heated at high power for 6 min. The filters were then placed in prehybridization solution containing 2× SSC (pH 7.0), 1% SDS, and 0.5% nonfat dry milk at 65°C for 1 h. The filters were removed from the prehybridization solution; any cellular debris was removed. The filters were then hybridized in buffer containing 6× SSC, 1% SDS, 0.5% nonfat dry milk, 100 µg denatured salmon sperm DNA, and 1 × 106 cpm/ml 32P-labeled full-length beta -subunit cDNA probe from a rat astrocytoma cell line (38) labeled by random primer method (32) at 65°C for at least 16 h. The filters were then washed at room temperature for 10 min by gentle shaking in a buffer containing 2× SSC (pH 7.0) and 1% SDS, followed by 1× SSC (pH 7.0) and 1% SDS; the filters were transferred to prewarmed buffer (45°C) containing 0.1× SSC (pH 7.0) and 1% SDS and washed by gentle shaking at 45°C for 15 min. The filters were air dried, covered with Saran wrap, and exposed to Hyperfilm (Amersham) using a Bioplus intensifying screen for 18 h. After development of the film, positive colonies were identified and screened two more times to obtain positive clones. Plasmids were prepared according to the method of Morelle (27) using 2 ml of "terrific broth" (32), and the plasmid DNA containing inserts was sequenced in both strands using an automated fluorescence sequencing machine at the Yale Sequencing Facility.

Computer analysis of the nucleotide sequences was performed using Blast search [Genetics Computer Group (GCG) program, University of Wisconsin]. Hydropathy analysis was performed by the procedure of Kyte and Doolittle (19). Multiple sequence alignment of beta -subunits was performed by the Pileup program (GCG software) (9).

Antibody production. The cDNA sequence (165 bp) corresponding to the amino acid sequence between Pro-87 and Ser-142 of HKcbeta was amplified using the full-length colon-derived HKcbeta as a template with a sense primer (5'-GGGGGGATCCCACCGACTGCCTTGGATTATACATA-3') to which a BamH I site was appended at the 5' end and an antisense primer (5'-GGGGGGAATTCACTATAGTCTGGACCCTCCTG-3') to which an EcoR I site was appended at the 5' end. The expected size 165-bp fragment was gel purified, digested with BamH I and EcoR I, and ligated using T4 DNA ligase into PGEX-KG vector that had previously been digested with BamH I and EcoR I and dephosphorylated with calf intestine phosphatase. The ligated DNA was transformed into XL-1 blue Escherichia coli cells and spread onto LB-ampicillin plates. Plasmid DNAs were prepared from recombinant colonies, digested with BamH I and EcoR I, and electrophoresed onto 1.5% agarose gels. Positive clones containing the 165-nucleotide HKcbeta fragment were selected and sequenced. Clones with correct reading frame and with correct cDNA sequence were selected to prepare glutathione S-transferase (GST)-HKcbeta fusion protein. The recombinant colonies were grown in LB-ampicillin medium to an absorbance of 0.8 at 600 nm; the GST-HKcbeta fusion proteins were then overexpressed after induction with 1.0 mM isopropyl beta -D-thiogalactopyranoside at 37°C for 3 h. After induction, the cells were harvested, washed with 50 mM Tris · HCl (pH 7.4) containing 10 mM EDTA, and resuspended in the lysis buffer containing 50 mM Tris · HCl (pH 7.4), 100 mM NaCl, 5 mM dithiothreitol, 2 mM EDTA, 2 mM EGTA, and 1 mg/ml lysozyme, mixed 45 min at 4°C, frozen at 80°C, and thawed. Then 1% Triton X-100, 10 mM MgCl2, and 0.1 mg/ml DNase I were added and incubated at 4°C for an additional 30 min. After centrifugation, the GST-HKcbeta fusion protein was purified from the supernatant by passage through a glutathione-agarose column; after the column was washed separately with 1× PBS and 1× PBS containing 1% Triton X-100 and with 50 mM Tris · HCl (pH 8.0) containing 1 mM EDTA, the GST-HKcbeta fusion protein was eluted from the column using 10 mM reduced glutathione (pH 8.0) by incubating at 4°C with gentle nutation. The eluted GST-HKcbeta fusion protein was dialyzed against 1× PBS at 4°C and concentrated using Centriprep 10. At this stage, the purified GST-HKcbeta fusion proteins were run on SDS-PAGE gels and analyzed for homogeneity. The homogeneous preparation of GST-HKcbeta fusion proteins was used to inject rabbits to raise polyclonal antibodies.

Antibodies were produced in a New Zealand White rabbit following primary subcutaneous injection of purified GST-HKcbeta fusion protein in complete Freund's adjuvant; subsequent boosts of fusion protein were injected in incomplete Freund's adjuvant. The animal was killed and exsanguinated on day 11 after the last boost. Serum IgG was affinity purified using a protein A column. Antibodies to GST proteins were also removed by glutathione-Sepharose affinity chromatography. The purified antibody to HKcbeta protein was used for Western blot analyses.

Specificity of HKcbeta antibody. The specificity of the HKcbeta antibody was established by the following experiments. 1) In an immunodepletion experiment, GST-HKcbeta fusion protein and HKcbeta antibody were mixed and incubated at room temperature. The antigen-antibody complex mixture was then used as an antibody source for the Western blot analysis, which contained apical membranes, basolateral membranes, or the immunoprecipitate of apical membranes by HKcalpha antibody. 2) HKcbeta cDNA and NaKbeta 1 cDNA were subcloned into pcDNA 3.1 (+), which was transfected into COS-7 cells (13). The expressed HKcbeta and NaKbeta 1 proteins were used for Western blot analysis with HKcbeta antibody. 3) Highly purified Na-K-ATPase from rabbit kidney (18) was run on SDS-PAGE gels and transferred to nitrocellulose membranes, and Western blot analysis was performed using HKcbeta or NaKbeta 1 antibodies.

Isolation of apical and basolateral membranes. Apical and basolateral membranes were isolated by methods previously described in detail (29, 30).

Western blot analysis. Western blot analyses were performed with HKcalpha , HKcbeta , and NaKbeta 1 antibodies using previously described methods (20, 33). Apical or basolateral membrane proteins (50 µg) were electrophoresed on SDS-PAGE gels and transferred to nitrocellulose membranes. Western blot analysis was then performed with dilution of 1:1,000 HKcbeta antibody in Tris-buffered saline-Tween containing 5% nonfat dry milk; anti-rabbit IgG horseradish peroxidase conjugate (1:5,000 dilution) was used as the secondary antibody. HKcbeta antibody-specific protein bands were visualized by the enhanced chemiluminescence procedure.

Coimmunoprecipitation. One hundred micrograms of apical membrane proteins of rat distal colon were resuspended in 1 ml of immunoprecipitation buffer containing 10 mM Tris · HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.2 mM phenylmethylsulfonyl fluoride, and 1% Triton X-100. One microliter of HKcalpha antibody was added and incubated at room temperature for 2 h in a nutator. To the above mixture, 50 µl of protein A-Sepharose (50% suspension) were added to bind antigen-antibody complex and incubated at room temperature for 1 h in a nutator. After a 1-min centrifugation, the pellet was washed three times for 5 min each in the above immunoprecipitation buffer containing 5% nonfat dry milk. The pellet was then washed three times with immunoprecipitation buffer. Finally, the pellet was resuspended in 2× SDS sample buffer containing 2-mercaptoethanol, boiled for 5 min, and centrifuged briefly, and the supernatant was loaded onto SDS-PAGE gels. After electrophoresis, proteins were transferred onto a nitrocellulose membrane and Western blot analysis was performed with HKcbeta antibody diluted to 1:1,000.


    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Isolation and characterization of HKcbeta cDNA clones. We screened a rat colon cDNA library using a full-length novel beta -subunit cDNA isolated from a rat astrocytoma cell line (38) as a probe and obtained positive clones that were sequenced in both directions. The full-length cDNA of HKcbeta consists of 1,728 nucleotides with an open reading frame encoding 279 amino acids, an initiation codon ATG, an in-frame termination codon (TAG), a 78-nucleotide 5' untranslated region, and an 810-nucleotide 3' untranslated sequence. The sequence of this cDNA is identical to that previously cloned by Watanabe et al. (38) from a rat astrocytoma cell line.

A comparison of the predicted amino acid sequence of the rat HKcbeta with those of the rat NaKbeta 1, NaKbeta 2, and HKgbeta subunits is shown in Fig. 1. The predicted amino acid sequence of HKcbeta exhibits 100% identity to the astrocytoma beta -subunit probe we used for screening (38), 36.4% identity to rat HKgbeta (34), 36.5% identity to rat NaKbeta 1 (26), and 49% identity to rat NaKbeta 2 (23), all previously characterized rat beta -subunits. In addition, the HKcbeta coding region has 80% identity to human NaKbeta 3 and 100% identity to the recently cloned partial beta 3 cDNA from rat placenta (22). The Asn residues at positions Asn-124 and Asn-158 are potential sites of N-linked glycosylation that are not conserved with the other rat beta -subunits. The six Cys residues (at positions Cys-128, Cys-144, Cys-154, Cys-170, Cys-191, and Cys-250) that might interact with an alpha -subunit are present in the extracellular domain of the HKcbeta subunit and are highly conserved with rat HKgbeta , NaKbeta 1, and NaKbeta 2 subunits. Hydropathy analysis (not shown) by the Kyte and Doolittle (19) algorithm predicts that HKcbeta contains a charged cytoplasmic amino terminus, a single transmembrane domain, and a large extracellular carboxy-terminal domain. The predicted secondary structure of HKcbeta is similar to those of rat HKgbeta , NaKbeta 1, and NaKbeta 2 subunits. The hydrophobic residue-rich region of HKcbeta is between amino acids Trp-36 and Leu-62. These observations establish substantial structural similarities between HKcbeta and other P-type ATPase beta -subunits.


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Fig. 1.   Amino acid comparison of colonic H-K-ATPase beta -subunit (HKcbeta ), gastric H-K-ATPase beta -subunit (HKgbeta ), and Na-K-ATPase beta 1- and beta 2-subunits (NaKbeta 1 and NaKbeta 2). Amino acid sequences conserved between beta -subunits are marked as black boxes. Amino acids are numbered at right. Transmembrane domain region, Trp-36 to Leu-62, and amino acid sequence that was used to raise antibodies, Pro-87 to Ser-142, are underlined. Alignments were done using Pileup and Prettybox programs of University of Wisconsin Genetics Computer Group software. NaKbeta 2, HKgbeta , and NaKbeta 1 sequences were obtained from GenBank.

Expression of HKcbeta subunit mRNA in rat tissues. Expression of HKcbeta mRNA was analyzed in several tissues by Northern blot analysis (Fig. 2). HKcbeta cDNA probe hybridized with a transcript of ~1.9 kb in distal colon, proximal colon, ileum, jejunum, stomach, liver, lung, kidney, brain, testes, spleen, and heart. It should be noted that HKcbeta cDNA that was cloned from colon is 1.73 kb. HKcbeta message was highly expressed in testis and lung; the message was slightly smaller in size in testis than those identified in other tissues.


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Fig. 2.   Expression of HKcbeta mRNA in rat tissues. Poly(A)+ RNA from rat tissues was analyzed by Northern blot hybridization, as described in METHODS. Lane 1, distal colon; lane 2, proximal colon; lane 3, stomach; lane 4, jejunum; lane 5, ileum; lane 6, liver; lane 7, lung; lane 8, kidney; lane 9, brain; lane 10, testis; lane 11, spleen; and lane 12, heart. HKcbeta message is marked by arrow. Blot was stripped and reprobed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to indicate approximately equal loading of samples.

Regulation of HKcbeta expression. The identification of a colon-derived putative beta -subunit isoform permitted studies to evaluate the role of HKcbeta in the transport function of the rat distal colon. Because HKcbeta is expressed in rat distal colon, it could be associated with the regulation of K transport and/or Na transport, which are closely linked to colonic H-K-ATPase and Na-K-ATPase, respectively (2). Expression of HKcbeta was analyzed by Northern blot with mRNA isolated from distal colon of normal, dietary Na-depleted, and dietary K-depleted rats (Fig. 3). Na depletion stimulates Na and K absorption and K secretion (2, 37), whereas K depletion enhances only K absorption in distal colon (12). Figure 3A demonstrates that HKcbeta mRNA expression is increased in distal colon of K-depleted rats compared with normal rats. HKcbeta message expression was normalized to glyceraldehyde-3-phosphate dehydrogenase expression and quantitated by densitometric analysis. K depletion resulted in a 3.5-fold increase in HKcbeta message expression compared with controls (Fig. 3B). In contrast, HKcbeta mRNA abundance in Na-depleted rats was not significantly altered (Fig. 3), although both dietary K depletion and Na depletion induce comparable increases in active K absorption in the rat distal colon (11, 12). In parallel studies with mRNA isolated from proximal colon, an organ in which active K absorption is not present (11), dietary K depletion did not increase HKcbeta mRNA abundance (Fig. 4).


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Fig. 3.   Expression of HKcbeta mRNA in rat distal colon of normal, dietary Na-depleted, and dietary K-depleted rats. A: poly(A)+ RNA from distal and proximal colon was analyzed by Northern blot, as described in METHODS. Lanes 1-3, normal; lanes 4-6, Na depletion; lanes 7-9, K depletion. Each mRNA sample was obtained from an individual animal. HKcbeta message is marked by arrow. Blot was stripped and reprobed with GAPDH to indicate approximately equal loading of samples. B: densitometric analysis of HKcbeta -to-GAPDH mRNA abundance is expressed in arbitrary units.


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Fig. 4.   Expression of HKcbeta mRNA in proximal colon of normal and dietary K-depleted rats. A: poly(A)+ RNA from proximal colon was analyzed by Northern blot, as described in METHODS. Lanes 1-3, normal; lanes 4-6, K depletion. Each mRNA sample was obtained from an individual animal. HKcbeta message is marked by arrow. Blot was stripped and reprobed with GAPDH to indicate approximately equal loading of samples. B: densitometric analysis of HKcbeta -to-GAPDH mRNA abundance is expressed in arbitrary units.

As shown in Fig. 2, HKcbeta mRNA expression was highest in testis and lung. Therefore, additional Northern blot analyses were performed to determine whether dietary K depletion increased HKcbeta mRNA abundance in these two organs. Figure 5 demonstrates that dietary K depletion did not alter HKcbeta mRNA expression in either lung or testis. In both tissues, two distinct bands with equal intensity were identified; the smaller bands may represent alternatively spliced products of HKcbeta mRNA. It should be noted that HKcbeta cDNA probe hybridizes with slightly smaller mRNA species in testis than in lung. Therefore, the selective increase in HKcbeta mRNA expression in distal colon of K-depleted rats (Fig. 3) suggests that this beta -subunit may be associated with the colonic H-K-ATPase alpha -subunit.


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Fig. 5.   Expression of HKcbeta mRNA in testes and lung of normal and K-depleted rats. Poly(A)+ RNA from lung and testis were analyzed by Northern blot, as described in METHODS. Lane 1, normal lung; lane 2, K-depleted lung; lane 3, normal testis; lane 4, K-depleted testis. Blot was stripped and reprobed with GAPDH to indicate approximately equal loading of samples.

To assess the significance of the increase in HKcbeta mRNA by K depletion, the expression of NaKbeta 1 mRNA abundance in distal and proximal colon of normal, Na-depleted, and K-depleted rats was also analyzed by Northern blot; these results are presented in Fig. 6. Expression of NaKbeta 1 mRNA in distal colon of K-depleted rats did not change compared with control animals. In contrast, NaKbeta 1 mRNA abundance was modestly increased in Na-depleted rats, an observation consistent with the stimulation of active Na absorption in the distal colon by Na depletion (37). Northern blot analyses were performed to determine the presence of other beta -subunit mRNAs in rat distal colon. Neither HKgbeta nor NaKbeta 2 cDNA probes hybridized with mRNA prepared from rat distal colon (data not shown).


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Fig. 6.   Expression of NaKbeta 1 mRNA in rat colon. Poly(A)+ RNA from distal and proximal colon of normal, Na-depleted, and K-depleted rats was analyzed by Northern blot, as described in METHODS. Lane 1, normal distal colon; lane 2, Na-depleted distal colon; lane 3, K-depleted distal colon; lane 4, normal proximal colon; lane 5, Na-depleted proximal colon; lane 6, K-depleted proximal colon. NaKbeta 1 is shown by arrow. * An additional mRNA was also hybridized by NaKbeta 1 probe. Blot was stripped and reprobed with GAPDH to indicate approximately equal loading of samples.

Expression of HKcbeta in apical and basolateral membranes. The results of the Northern blot analyses (Fig. 3) are consistent with the thesis that HKcbeta is the beta -subunit required for colonic H-K-ATPase function. HKcbeta has significant (80%) homology to a recently identified human beta -subunit that was designated NaKbeta 3 (18). This assignment of NaKbeta 3 was based on chromosomal localization and sequence relatedness. As a consequence, additional studies were performed with HKcbeta to establish whether HKcbeta is present in apical and/or basolateral membranes and whether HKcbeta is associated with HKcalpha .

Several studies were performed that established the specificity of HKcbeta antibody to HKcbeta protein without evidence of cross-reactivity to NaKbeta 1 protein. 1) The specificity of HKcbeta antibody was initially confirmed by Western blot analysis against the GST-HKcbeta fusion protein (data not shown). 2) Immunodepletion experiments were performed in which GST-HKcbeta fusion protein and HKcbeta antibody complex mixture was used as an antibody source for Western blot analysis. Figure 7A demonstrates that no protein was identified in apical (lane 1) or in basolateral (lane 2) membranes, but the immunoglobulin band (lane 3) that was present in the original blot (Fig. 8B, lane 3) was identified. 3) Expression studies of HKcbeta cDNA in COS-7 cells demonstrated that HKcbeta protein was identified by the HKcbeta antibody only in cells transfected with HKcbeta cDNA (Fig. 7B, lane 3). HKcbeta antibody did not cross-react with NaKbeta 1 protein in cells transfected with NaKbeta 1 cDNA (Fig. 7B, lane 4). 4) Highly purified Na-K-ATPase protein from rabbit renal medulla that consists of NaKalpha 1 and NaKbeta 1 proteins was not identified by HKcbeta antibody (Fig. 7C) but was identified by NaKbeta 1 antibody (Fig. 7D). These several observations establish the specificity of HKcbeta antibody to HKcbeta protein.


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Fig. 7.   Specificity of HKcbeta antibody. A: immunodepletion experiment with glutathione S-transferase-HKcbeta protein complex as an antibody source. Lane 1, apical membrane; lane 2, basolateral membrane; lane 3, apical membrane immunoprecipitated by HKcalpha antibody. B: COS-7 cell membranes following transfection with HKcbeta cDNA and NaKbeta 1 cDNA. Lane 1, nontransfected cells; lane 2, cells transfected only with vector; lane 3, cells transfected with plasmid carrying HKcbeta cDNA; lane 4, cells transfected with plasmid carrying NaKbeta 1 cDNA. C and D: Western blot of purified Na-K-ATPase with HKcbeta antibody (C) and NaKbeta 1 antibody (D) (35).


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Fig. 8.   Western blot and coimmunoprecipitation analyses: apical and basolateral membranes were used for Western blot analysis. A: Western blot analysis using HKcalpha antibody (20). Lane 1, apical membrane; lane 2, basolateral membrane. HKcalpha protein band is marked by arrow. B: Western blot analysis using HKcbeta antibody. Lane 1, apical membrane; lane 2, basolateral membrane; lane 3, a Western blot analysis was performed with HKcbeta antibody for apical membrane proteins immunoprecipitated with HKcalpha antibody. HKcbeta protein band is shown by arrow at left. * Immunoglobulin band that was bound to protein A-Sepharose during immunoprecipitation experiments. Additional band seen in lane 1 may represent a nonglycosylated form of HKcbeta protein or a degradation product of HKcbeta . C: Western blot analysis using NaKbeta 1 antibody (35). Blot from B was stripped and used to perform Western blot analysis using NaKbeta 1 antibody. D: Western blot analysis using preimmune serum. Blot from C was stripped to perform Western blot analysis using preimmune serum. Specific protein bands that had been previously identified by HKcbeta and NaKbeta 1 antibodies in B and C, respectively, were not identified by preimmune serum, except for immunoglobulin band in lane 3.

Figure 8 presents a Western blot analysis of apical and basolateral membranes using antibodies to HKcalpha , HKcbeta , and NaKbeta 1 and preimmune serum. HKcalpha was predominantly expressed in apical membranes (Fig. 8A, lane 1), confirming previous studies of the localization of HKcalpha protein to the apical membrane of surface cells of rat distal colon (20). The very low expression of HKcalpha protein in basolateral membrane (Fig. 8A, lane 2) probably reflects minimal contamination of the basolateral membrane preparation by apical membranes. Figure 8B demonstrates that HKcbeta protein was expressed in both apical (lane 1) and basolateral (lane 2) membranes in approximately equal amounts. In contrast, NaKbeta 1 protein was expressed only in basolateral membranes (Fig. 8C, lane 2). The presence of HKcbeta and not NaKbeta 1 in apical membrane supports a possible role of HKcbeta as the beta -subunit for HKcalpha . The specificity of HKcbeta protein expression in rat colonic membranes was provided by the immunodepletion experiment shown in Fig. 7A. This observation confirms that the protein band at 45 kDa in apical and basolateral membranes is specific to HKcbeta antibody.

Additional Western blot analyses were performed on apical and basolateral membranes from normal and K-depleted rats using HKcbeta antibody. Figure 9 presents the results of two of the three separate preparations of apical and basolateral membranes. Each preparation was prepared from at least six normal or K-depleted rats. HKcbeta protein expression was significantly increased by 216% in apical membranes but was reduced by 30% in basolateral membranes (Fig. 9). The results presented in Figs. 3 and 9 suggest that K depletion regulates HKcbeta mRNA and protein.


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Fig. 9.   Western blot analysis of HKcbeta protein in apical and basolateral membranes of normal and K-depleted rats. A: equal amounts (50 µg) of membrane proteins were used to perform Western blot analysis using HKcbeta antibody. Western blot analyses were performed on 3 different apical and basolateral membrane preparations. Each membrane sample was prepared from 6-8 rats. Lanes 1 and 2, normal rats; lanes 3 and 4, K-depleted rats. B: densitometric analysis of HKcbeta protein in apical (left) and basolateral (right) membranes expressed in arbitrary units.

Identification of physical interaction of HKcbeta and HKcalpha . Experiments were designed to establish whether HKcbeta protein was associated with HKcalpha protein in the apical membrane of distal colon. Therefore, apical membrane proteins from rat distal colon were immunoprecipitated with HKcalpha antibody (see Coimmunoprecipitation). The resulting antigen-antibody complex was analyzed by Western blot using HKcbeta or NaKbeta 1 antibodies. Figure 8B (lane 3) demonstrates the presence of HKcbeta protein in the immunoprecipitate, demonstrating the physical interaction of HKcalpha and HKcbeta proteins. In contrast, NaKbeta 1 was not identified in the immunoprecipitate (Fig. 8C, lane 3). The specificity of the bands of the immunoprecipitate was confirmed by performing a Western blot analysis of the protein blot in Fig. 8C using preimmune serum following stripping. The preimmune serum identified only the immunoglobulin, as shown in Fig. 8D, lane 3. In addition, the immunodepletion experiments (Fig. 7A) established the specificity of HKcbeta antibody to HKcbeta protein. These observations are consistent with HKcbeta functioning as the beta -subunit for the apical membrane localized alpha -subunit of colonic H-K-ATPase.


    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Active K absorption and an apical membrane H-K-ATPase have been studied extensively in the rat large intestine. Active K absorption is restricted to the distal colon and is upregulated both by dietary K depletion and by aldosterone and Na depletion (11, 12). It is generally accepted that active K absorption is energized by apical membrane H-K-ATPase, the alpha -subunit of which (HKcalpha ) was recently cloned (7, 14) and has been studied extensively (5, 6, 15, 20, 33). HKcalpha message is restricted to surface (and the upper 20% of the crypt) cells of the distal colon, whereas its protein is localized to the apical membrane of surface cells of the rat distal colon (14, 20). Although several beta -subunit isoforms of both Na-K-ATPase and noncolonic H-K-ATPase have been isolated, a colonic H-K-ATPase beta -subunit had not previously been identified.

Recent studies have established that dietary Na depletion upregulates HKcalpha message and protein abundance and apical membrane H-K-ATPase activity in the rat distal colon but does not alter HKcalpha message and protein expression in the kidney (15, 33). In contrast to the effect of Na depletion on HKcalpha expression in the colon, dietary K depletion did not affect HKcalpha message and protein expression in the rat distal colon (33). Of potential importance, HKcalpha protein expression was enhanced in the principal cell in the kidney of dietary K-depleted rats compared with control rats. Thus the mechanism by which dietary K depletion increases active K absorption in the rat distal colon is not known. Lescale-Matys et al. (21) demonstrated in LLC-PK1 cells that a decrease in extracellular K concentration was associated with an increase in NaKbeta 1 message and protein but not in NaKalpha 1 message and protein. Their study concluded that regulation of Na-K-ATPase activity by K concentration was pretranslational and that beta -subunit synthesis was rate limiting. These observations with LLC-PK1 cells parallel the present results with rat distal colon. Dietary K depletion enhances active K absorption but is associated with an increase in HKcbeta (Fig. 3) but not HKcalpha mRNA abundance (33). As a result, we speculate that the mechanism of regulation of H-K-ATPase function by dietary K depletion is mediated via HKcbeta and not via HKcalpha .

The data presented in Fig. 3 are consistent with the role of HKcbeta mRNA in the regulation of H-K-ATPase by dietary K depletion. These Northern blot analyses using the newly cloned colon-derived beta -subunit establish that HKcbeta mRNA abundance was enhanced in dietary K-depleted rats. Specificity of this observation is provided by four important experiments. First, dietary K depletion did not increase the mRNA abundance of the NaKbeta 1 subunit (Fig. 6), indicating that dietary K depletion does not result in a nonspecific increase in message abundance of all beta -subunits. Second, HKcbeta mRNA abundance was not increased in Na-depleted rats (Fig. 3), indicating that the increase in HKcbeta message was specific and was not merely secondary to an increase in active K absorption. Third, although HKcbeta message is present in the proximal colon (Fig. 4), a tissue in which active K absorption is not present (11), HKcbeta mRNA abundance was not increased in dietary K-depleted animals in proximal colon. Fourth, expression of HKcbeta mRNA abundance was not altered in lung and testis of K-depleted rats compared with normal rats (Fig. 5). Thus the effect of K depletion on HKcbeta mRNA is tissue specific, as HKcbeta mRNA is upregulated in the distal but not in the proximal colon, testis, or lung of K-depleted rats. These observations provide compelling evidence that HKcbeta is closely linked to the stimulation of active K absorption by dietary K depletion in the rat distal colon.

Controversy exists regarding the role of a beta -subunit in the functional expression of HKcalpha . Although HKcalpha was expressed as ouabain-insensitive H-K-ATPase in Sf9 cells without the apparent presence of a beta -subunit (20), the expression of HKcalpha in oocytes requires the coinjection of cRNAs of a Bufo marinus urinary bladder beta -subunit (6) or of HKgbeta or NaKbeta 1 (5). It is of interest that the amphibian beta -subunit (17) that induced 86Rb uptake in oocytes when coinjected with HKcalpha (6) has significant homology (52.3%) to HKcbeta . The demonstration that dietary K depletion resulted in an increase in HKcbeta mRNA abundance but not an enhancement of HKcalpha mRNA abundance strongly suggests that the regulation of active K absorption by dietary K depletion is mediated by this beta -subunit and not by HKcalpha . Because Na depletion stimulates HKcalpha but not HKcbeta message abundance in the rat distal colon (Refs. 15, 33 and Fig. 3), differential regulation of HKcalpha and HKcbeta subunits plays a significant role in the stimulation of active K absorption in rat distal colon by K depletion and Na depletion. In addition, because dietary K depletion increased the abundance of HKcbeta message but not that of NaKbeta 1, these observations suggest that HKcbeta may be the specific beta -subunit required for optimal H-K-ATPase function in the distal colon.

The close identity between HKcbeta and the recently reported human putative NaKbeta 3 (2, 22) requires comment. The latter cDNA sequence was identified from the human-expressed sequence tag data bank and was designated NaKbeta 3 based solely on chromosomal localization and sequence relatedness, but without demonstration of membrane localization or physical association with an alpha -subunit. In addition, this human putative NaKbeta 3 has an amino acid sequence with a 55% identity to a recently cloned beta -subunit from amphibian bladder (17), which, when coexpressed in Xenopus oocytes with an amphibian bladder H-K-ATPase alpha -subunit, stimulated H/K and not Na/K function (16). Because HKcbeta protein has 52% identity to the amphibian bladder beta -subunit, HKcbeta may be the rat homologue of this amphibian bladder beta -subunit. Thus it is possible that the human putative NaKbeta 3 may also manifest H-K-ATPase function.

The identification of HKcbeta protein in basolateral membranes (see Fig. 8B, lane 2) raised the possibility that HKcbeta might also function with alpha -subunits of Na-K-ATPase and that therefore the upregulation of HKcbeta mRNA by dietary K depletion (Fig. 3) was causally related to an increase in Na-K-ATPase activity by dietary K depletion. Such a possibility would be consistent with the previous observations that dietary K depletion and incubation of LLC-PK1 cells in a low-K medium are associated with increases in NaKbeta 1 subunit and/or Na-K-ATPase activity (21). Therefore, additional experiments were performed to determine whether dietary K depletion was associated with an increase in Na-K-ATPase activity in rat distal colon. These studies demonstrated that Na-K-ATPase activity in basolateral membranes isolated from rat distal colon was not altered by dietary K depletion (unpublished observations). Thus the observed increase in HKcbeta mRNA abundance in dietary K depletion cannot be responsible for an increase in Na-K-ATPase. Therefore, HKcbeta is uniquely regulated by dietary K depletion in the distal colon and likely is the beta -subunit required for H-K-ATPase in the rat distal colon.

H-K-ATPase is localized to the apical membrane in the distal colon (8), in contrast to the localization of Na-K-ATPase to the basolateral membrane. The Western blot studies presented in Fig. 8 present three important observations that indicate HKcbeta is the beta -subunit for the colonic H-K-ATPase. First, HKcbeta protein was expressed in apical membrane (Fig. 8B, lane 1), the site at which HKcalpha protein is selectively expressed (see Fig. 8A, lane 1). Second, although HKcbeta protein was identified in both apical and basolateral membranes (Fig. 8B, lanes 1 and 2), K depletion resulted in an increase in HKcbeta protein expression only in apical membranes (Fig. 9). The selective increase in HKcbeta protein in the apical membrane requires comment. It is possible that the protein recognized by the HKcbeta antibody in the basolateral membrane is not HKcbeta protein but a closely related beta -subunit that was recognized by the HKcbeta antibody but not regulated by K depletion. The mechanism for the 30% decrease of HKcbeta protein in basolateral membrane of K-depleted rats is not known. Third, coimmunoprecipitation experiments demonstrated the physical association between HKcbeta and HKcalpha proteins in the apical membrane (Fig. 8B, lane 3). In addition, the preimmune serum (Fig. 8D, lane 3) and immunodepletion experiment (Fig. 7A, lane 3) did not identify any protein bands except immunoglobulins. These observations confirm that HKcbeta protein bands are specific, and the two additional low-molecular weight protein bands in the immunoprecipitate may be a HKcbeta degradation product. In contrast, an antibody to NaKbeta 1 identified protein in basolateral but not in apical membranes (Fig. 8C, lane 2). As a result, it is unlikely that NaKbeta 1 protein is associated with HKcalpha protein or is the beta -subunit for H-K-ATPase. The demonstration of the selective increase in HKcbeta protein expression in apical membrane of rat distal colon and the association of HKcbeta protein with HKcalpha protein in apical membrane (Fig. 8B, lane 3) strongly support the possibility that HKcbeta is most likely the beta -subunit for colonic H-K-ATPase alpha -subunit.

Although dietary K depletion unequivocally stimulates active K absorption in the large intestine of both rat (12) and mouse (25), enhancement of H-K-ATPase in dietary K depletion has been inconstant. It is generally believed that active K absorption in the distal colon is a result of an H/K exchange energized by an apical membrane H-K-ATPase (2), but the effect of dietary K depletion on colonic active K absorption and H-K-ATPase activity is complex. Confounding variables of the presumed stimulation of H-K-ATPase by dietary K depletion include its duration and the presence of two H-K-ATPase isoforms and two components of active K absorption. The two H-K-ATPases have different sensitivities to ouabain and spatial distributions (8, 31), and the two active K absorptive processes also have different sensitivities to ouabain, as well as different responsiveness to aldosterone (28). Additionally, direct demonstration of an H/K exchange in colonic apical membranes has not as yet been established, and Feldman and Ickes (10) presented evidence of a dissociation between active K absorption and protein secretion.

In conclusion, we have identified a beta -subunit from rat distal colon, and its mRNA is upregulated in a tissue-specific manner in the distal colon of K-depleted but not of Na-depleted rats. This beta -subunit protein localized in the apical membrane, physically associated with HKcalpha protein. Its protein expression is selectively increased in the apical membrane of K-depleted rats. These observations are all consistent with the probability that HKcbeta is the putative beta -subunit for colonic H-K-ATPase.


    ACKNOWLEDGEMENTS

We thank Mary Guidone for excellent secretarial assistance and Andrea Mann for excellent technical assistance in the preparation of the HKcbeta antibody. The beta 3-subunit cDNA from an astrocytoma cell line (38) was kindly provided by Drs. T. Watanabe and Y. Suzuki. NaKbeta 1 antibody was kindly provided by Dr. Michael Caplan.


    FOOTNOTES

This research was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-18777. S. S. Kolla is a trainee of NIDDK Grant DK-07017.

Address for reprint requests: H. J. Binder, Yale University School of Medicine, Department of Internal Medicine, Section of Digestive Diseases, 333 Cedar St., 89 LMP, New Haven, CT 06520-8019.

Received 21 November 1997; accepted in final form 26 October 1998.


    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Arystarkhora, E., and K. J. Sweadner. Tissue-specific expression of the Na,K-ATPase beta 3 subunit. J. Biol. Chem. 272: 22405-22408, 1997[Abstract/Free Full Text].

2.   Binder, H. J., and G. I. Sandle. Electrolyte transport in the mammalian colon. In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by L. R. Johnson. New York: Raven, 1994, vol. 1, p. 2133-2172.

3.   Blanco, G., A. W. DeTomaso, J. Koster, Z. J. Xie, and R. W. Mercer. The alpha  subunit of the Na,K-ATPase has catalytic activity independent of the beta  subunit. J. Biol. Chem. 271: 23420-23425, 1994.

4.   Chow, D. C., and J. G. Forte. Functional significance of the beta  subunit for heterodimeric P-type ATPases. J. Exp. Biol. 198: 1-17, 1995[Abstract/Free Full Text].

5.   Codina, J., B. C. Kone, J. T. Delmas-mata, and T. D. DuBose, Jr. Functional expression of the colonic H+,K+-ATPase alpha  subunit. J. Biol. Chem. 271: 29759-29763, 1996[Abstract/Free Full Text].

6.   Cougnon, M., G. Planelles, M. S. Crowson, G. E. Shull, B. C. Rossier, and F. Jaisser. The rat distal colon P-ATPase alpha  subunit encodes ouabain-sensitive H+,K+-ATPase. J. Biol. Chem. 271: 7277-7280, 1996[Abstract/Free Full Text].

7.   Crowson, M. S., and G. E. Shull. Isolation and characterization of a cDNA encoding the putative distal colon H+,K+-ATPase. J. Biol. Chem. 267: 13740-13748, 1992[Abstract/Free Full Text].

8.   Del Castillo, J. R., V. M. Rajendran, and H. J. Binder. Apical membrane localization of ouabain-sensitive K+-activated ATPase activities in rat distal colon. Am. J. Physiol. 261 (Gastrointest. Liver Physiol. 24): G1005-G1011, 1991[Abstract/Free Full Text].

9.   Devreux, J., P. Haeberli, and R. Smithies. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12: 387-395, 1984[Abstract].

10.   Feldman, G. M., and J. W. Ickes, Jr. Net H+ and K+ fluxes across the apical surface of rat distal colon. Am. J. Physiol. 272 (Gastrointest. Liver Physiol. 35): G54-G62, 1997[Abstract/Free Full Text].

11.   Foster, E. S., J. P. Hayslett, and H. J. Binder. Mechanism of active potassium absorption and secretion in the rat colon. Am. J. Physiol. 246 (Gastrointest. Liver Physiol. 9): G611-G617, 1984[Abstract/Free Full Text].

12.   Foster, E. S., G. I. Sandle, J. P. Hayslett, and H. J. Binder. Dietary potassium modulates active potassium absorption and secretion in rat distal colon. Am. J. Physiol. 251 (Gastrointest. Liver Physiol. 14): G619-G626, 1986[Medline].

13.   Grishin, A. V., M. O. Bevensee, N. N. Modyanov, V. Rajendran, W. F. Boron, and M. J. Caplan. Functional expression of the cDNA encoded by the human ATP1AL1 gene. Am. J. Physiol. 271 (Renal Fluid Electrolyte Physiol. 40): F539-F551, 1996[Abstract/Free Full Text].

14.   Jaisser, F., N. Coutry, N. Farman, H. J. Binder, and B. C. Rossier. A putative H-K-ATPase is selectively expressed in surface epithelial cells of rat distal colon. Am. J. Physiol. 265 (Cell Physiol. 34): C1080-C1089, 1993[Abstract/Free Full Text].

15.   Jaisser, F., B. Escoubet, N. Coutry, E. Eugene, J. Bonvalet, and N. Farman. Differential regulation of putative K+-ATPase by low-K+ diet and corticosteroids in rat distal colon and kidney. Am. J. Physiol. 270 (Cell Physiol. 39): C679-C687, 1996[Abstract/Free Full Text].

16.   Jaisser, F., J. D. Horisberger, K. Geering, and B. C. Rossier. Mechanisms of urinary K+ and H+ excretion: primary structure and functional expression of novel H,K-ATPase. J. Cell Biol. 123: 1421-1429, 1993[Abstract].

17.   Jaisser, F., J. D. Horisberger, and B. C. Rossier. Primary sequence and functional expression of a novel beta  subunit of the P-ATPase gene family. Pflügers Arch. 425: 446-452, 1993[Medline].

18.   Jorgensen, P. L. Purification and characterization of (Na+-K+)-ATPase. III. Purification from the outer medulla of mammalian kidney after selective removal of membrane components by sodium dodecylsulphate. Biochim. Biophys. Acta 356: 36-52, 1974[Medline].

19.   Kyte, J., and R. F. Doolittle. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157: 105-132, 1982[Medline].

20.   Lee, J., V. M. Rajendran, A. S. Mann, M. Kashgarian, and H. J. Binder. Functional expression and segmental localization of rat colonic K-ATPase. J. Clin. Invest. 96: 2002-2008, 1995[Medline].

21.   Lescale-Matys, L., C. B. Hensley, R. Crnkovic-Markovic, D. S. Putnam, and A. A. McDonough. Low K+ increases Na,K-ATPase abundance in LLC-PK1/Cl4 cells by differentially increasing beta , and not alpha , subunit mRNA. J. Biol. Chem. 265: 17935-17940, 1990[Abstract/Free Full Text].

22.   Malik, N., V. A. Canfield, M. Beckers, P. Gros, and R. Levenson. Identification of the mammalian Na,K-ATPase beta 3 subunit. J. Biol. Chem. 271: 22754-22758, 1996[Abstract/Free Full Text].

23.   Martin-Vasallo, P., W. Dackowski, J. Emanuel, and R. Levenson. Identification of a putative isoform of the Na,K-ATPase beta  subunit. J. Biol. Chem. 264: 4613-4618, 1989[Abstract/Free Full Text].

24.   Marxer, A., B. Stieger, A. Quaroni, M. Kashgarian, and H.-P. Hauri. (Na+ + K+)-ATPase and plasma membrane polarity of intestinal epithelial cells: presence of a brush border antigen in the distal large intestine that is immunologically related to beta  subunit. J. Cell Biol. 109: 1057-1069, 1989[Abstract].

25.   Meneton, P., P. J. Schultheis, J. Greeb, M. D. Neiman, L. H. Liu, L. Clarke, J. J. Duffy, T. Doetschman, J. N. Lorenz, and G. E. Shull. Increased sensitivity to K+ deprivation in colonic H,K-ATPase-deficient mice. J. Clin. Invest. 101: 536-542, 1998[Abstract/Free Full Text].

26.   Mercer, R. W., J. W. Schneider, A. Savitz, J. Emanuel, E. J. Benz, Jr., and R. Levenson. Rat-brain Na,K-ATPase beta -chain gene: primary structure, tissue-specific expression, and amplification in ouabain-resistant HeLa C+ cells. Mol. Cell. Biol. 6: 3884-3890, 1986[Medline].

27.   Morelle, G. A plasmid extraction procedure on a miniprep scale. Focus 11.1: 7-8, 1990.

28.   Pandiyan, V., V. M. Rajendran, and H. J. Binder. Mucosal ouabain and Na+ inhibit active Rb+(K+) absorption in normal and sodium-depleted rat distal colon. Gastroenterology 102: 1846-1853, 1992[Medline].

29.   Rajendran, V. M., M. Kashgarian, and H. J. Binder. Aldosterone induction of electrogenic sodium transport in the apical membrane vesicles of rat distal colon. J. Biol. Chem. 264: 18638-18644, 1989[Abstract/Free Full Text].

30.   Rajendran, V. M., M. Oesterlin, and H. J. Binder. Sodium uptake across basolateral membrane of rat distal colon: evidence of Na-H exchange and Na-anion cotransport. J. Clin. Invest. 88: 1379-1385, 1991[Medline].

31.   Rajendran, V. M., S. K. Singh, J. Geibel, and H. J. Binder. Differential localization of colonic H+-K+-ATPase isoforms in surface and crypt cells. Am. J. Physiol. 274 (Gastrointest. Liver Physiol. 37): G424-G429, 1998[Abstract/Free Full Text].

32.   Sambrook, J., E. F. Fritsch, and T. Maniatis. Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989.

33.   Sangan, P., V. M. Rajendran, A. S. Mann, M. Kashgarian, and H. J. Binder. Regulation of colonic H-K-ATPase in large intestine and kidney by aldosterone and dietary K depletion. Am. J. Physiol. 272 (Cell Physiol. 41): C685-C696, 1997[Abstract/Free Full Text].

34.   Shull, G. E. cDNA cloning of the beta  subunit of the rat gastric H,K-ATPase. J. Biol. Chem. 265: 12123-12126, 1990[Abstract/Free Full Text].

35.   Shull, G. E., J. Greeb, and J. B. Lingrel. Molecular cloning of three distinct forms of the Na+,K+-ATPase alpha -subunit from rat brain. Biochemistry 25: 8126-8132, 1986.

36.   Shull, G. E., and J. B. Lingrel. Molecular cloning of the rat stomach (H+ + K+)-ATPase. J. Biol. Chem. 261: 16788-16791, 1986[Abstract/Free Full Text].

37.   Turnamian, S. G., and H. J. Binder. Regulation of active sodium and potassium transport in the distal colon of the rat: role of the aldosterone and glucocorticoid receptors. J. Clin. Invest. 84: 1924-1929, 1989[Medline].

38.   Watanabe, T., M. Sato, T. Yoshida, and Y. Suzuki. Isolation of a cDNA encoding the Na+,K+-ATPase beta 3 subunit of C-6 rat glial cell. GenBank no. D84450, 1996.


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