Functional expression of putative H+-K+-ATPase from guinea pig distal colon

Shinji Asano1, Satomi Hoshina2, Yumi Nakaie2, Toshiyuki Watanabe3, Michihiko Sato4, Yuichi Suzuki5, and Noriaki Takeguchi2

1 Molecular Genetics Research Center and 2 Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama 930-01; 3 Department of Physiology and 4 Central Laboratory for Research and Education, Yamagata University School of Medicine, Yamagata 990-23; and 5 Laboratory of Physiology, School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka 422, Japan

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

A guinea pig cDNA encoding the putative colonic H+-K+-ATPase alpha -subunit (T. Watanabe, M. Sato, K. Kaneko, T. Suzuki, T. Yoshida, and Y. Suzuki; GenBank accession no. D21854) was functionally expressed in HEK-293, a human kidney cell line. The cDNA for the putative colonic H+-K+-ATPase was cotransfected with cDNA for either rabbit gastric H+-K+-ATPase or Torpedo Na+-K+-ATPase beta -subunit. In both expressions, Na+-independent, K+-dependent ATPase (K+-ATPase) activity was detected in the membrane fraction of the cells, with a Michaelis-Menten constant for K+ of 0.68 mM. The expressed K+-ATPase activity was inhibited by ouabain, with its IC50 value being 52 µM. However, the activity was resistant to Sch-28080, an inhibitor specific for gastric H+-K+-ATPase. The ATPase was not functionally expressed in the absence of the beta -subunits. Therefore, it is concluded that the cDNA encodes the catalytic subunit (alpha -subunit) of the colonic H+-K+-ATPase. Although the beta -subunit of the colonic H+-K+-ATPase has not been identified yet, both gastric H+-K+-ATPase and Na+-K+-ATPase beta -subunits were found to act as a surrogate for the colonic beta -subunit for the functional expression of the ATPase. The present colonic H+-K+-ATPase first expressed in mammalian cells showed the highest ouabain sensitivity in expressed colonic H+-K+-ATPases so far reported (rat colonic in Xenopus oocytes had an IC50 = 0.4-1 mM; rat colonic in Sf9 cells had no ouabain sensitivity).

colonic proton-potassium-adenosinetriphosphatase; ouabain; proton pump inhibitor

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE TRANSEPITHELIAL K+ transport in the colon is involved in systemic K+ homeostasis. It was proposed that K+ absorption in the distal colon is an active process. Evidence is accumulating for the presence of a colonic H+-K+-ATPase, which is localized in distal colon and mediates K+ absorption and, possibly, acid secretion in rat, rabbit, and guinea pig colons (1, 7, 20-24).

Suzuki and Kaneko (19, 20) reported that molecular mechanisms responsible for ouabain-sensitive K+ absorption and acid secretion are localized in the middle and distal parts of the guinea pig colon. Watanabe et al. (24) found ouabain-sensitive K+-ATPase activity in the guinea pig distal but not in the proximal colon. The activity was stimulated by K+ and inhibited by vanadate, responses similar to those of Na+-K+-ATPase and gastric H+-K+-ATPase. However, the colonic K+-ATPase activity is not sensitive to Na+, unlike Na+-K+-ATPase activity. Furthermore, the colonic K+-ATPase is inhibited by ouabain but not by omeprazole or 2-methyl-8-(phenylmethoxy)imidazo[1,2-alpha ]pyridine-3-acetonitrile (Sch-28080), whereas gastric H+-K+-ATPase is inhibited by omeprazole and Sch-28080 but not by ouabain (23, 24). Similarly, an ouabain-sensitive K+-ATPase activity was observed in the membrane fraction of rat colonic mucosae (7, 22).

Crowson and Shull (6) isolated from a rat colon library a novel cDNA encoding a putative colonic H+-K+-ATPase, which is similar to gastric H+-K+-ATPase and Na+-K+-ATPase (60-65% amino acid identity). Recently, it was functionally expressed in Sf9 cells (16) and Xenopus laevis oocytes (5), indicating that the putative rat colonic H+-K+-ATPase encodes the catalytic subunit of colonic H+-K+-ATPase. Watanabe and colleagues cloned a novel cDNA (4242 bp) that encodes a 114-kDa product having 63% amino acid identity with Na+-K+-ATPase and 64% amino acid identity with gastric H+-K+-ATPase alpha -subunits.1 It is likely that this is a homologue of rat colonic H+-K+-ATPase alpha -subunit because of the 88% amino acid identity between them. However, it has not been determined whether the molecular product of this guinea pig cDNA shows K+-ATPase activity. Here, we try to functionally express the putative colonic H+-K+-ATPase cDNA in a HEK cell line as was previously done for gastric H+-K+-ATPase (2, 3) and find that properties of the expressed ATPase were similar to those reported for the membrane fraction of guinea pig distal colon, that is, stimulated by K+, sensitive to ouabain, and insensitive to Sch-28080.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. HEK-293 cells (a human embryonic kidney cell line) were a kind gift from Dr. Jonathan Lytton (Brigham and Women's Hospital, Harvard Medical School, Boston, MA). pCIS2 vector was obtained from Genentech (San Diego, CA). Restriction enzymes and other DNA and RNA modifying enzymes were from Toyobo (Osaka, Japan), New England Biolabs, Life Technologies, or Pharmacia Biotechnology (Tokyo, Japan). Sch-28080 was obtained from Schering (Bloomfield, NJ). All other reagents were of molecular biology grade or the highest grade of purity available.

cDNAs. A cDNA encoding the putative colonic H+-K+-ATPase was cloned from guinea pig colonic cDNA library and subcloned in pBluescript II SK(-). The sequence was registered in European Molecular Biology Laboratories (EMBL) database (GenBank accession no. D21854). cDNAs of gastric H+-K+-ATPase alpha - and beta -subunits were prepared from rabbit stomach as described elsewhere (3). A cDNA of Torpedo Na+-K+-ATPase beta -subunit was a kind gift from Dr. Kawamura (University of Occupational and Environmental Health, Kitakyushu, Japan). The cDNA of the colonic H+-K+-ATPase was digested with Cla I and Not I. The obtained fragment was ligated into pCIS2 vector treated with Cla I and Not I.

Removal of 5'-noncoding sequence of the colonic H+-K+-ATPase cDNA. The colonic H+-K+-ATPase cDNA between nucleotides -36 and 842 was amplified by PCR. The PCR primers were 5'-GGCTCGAGCCCCGAGCCGCCCTCCAG-3' and 5'-CCAAGCTTGAAGCTATGCGTCCAATGATGG-3'. The 880-bp fragment was purified on the gel and digested with Xho I and BstE II. The cDNA cassette between Xho I and BstE II of the colonic H+-K+-ATPase cDNA was replaced by the PCR-derived fragment to remove the cDNA sequence in its 5'-noncoding region (nucleotides -144 to -37).

Removal of 3'-noncoding sequence of the colonic H+-K+-ATPase cDNA. The colonic H+-K+-ATPase cDNA was digested with Xho I and Dra I. The 3,300-bp fragment was purified on the gel and ligated with pBluescript SK(-) digested with Xho I and EcoR V to remove the cDNA sequence in its 3'-noncoding region (nucleotides 3364-4101).

DNA sequencing. For DNA sequencing, an Autoread DNA sequencing kit and an ALF-II DNA sequencer (Pharmacia) were used.

Cell culture, transfection, and preparation of membrane fractions. Cell culture of HEK-293 was carried out as described previously (3). cDNA transfection was performed by the calcium phosphate method with 10 µg of cesium chloride-purified DNA per 10-cm dish. Cells were harvested 2 days after the DNA transfection. Membrane fractions of HEK cells were prepared as described previously (3). Briefly, cells in a 10-cm petri dish were washed with PBS and incubated in 2 ml of low ionic salt buffer (0.5 mM MgCl2 and 10 mM Tris · HCl, pH 7.4) at 0°C for 10 min. After the addition of phenylmethylsulfonyl fluoride (1 mM) and aprotinin (0.09 U/ml), the cells were homogenized in a Dounce homogenizer, and the homogenate was diluted with an equal volume of a solution containing 500 mM sucrose and 10 mM Tris · HCl, pH 7.4. The homogenized suspension was centrifuged at 800 g for 10 min. The supernatant was centrifuged at 100,000 g for 90 min, and the pellet was suspended in a solution containing 250 mM sucrose and 5 mM Tris · HCl, pH 7.4.

SDS-PAGE and immunoblot. SDS-PAGE was carried out as described elsewhere (15). Membrane preparations (30 µg of protein) were incubated in a sample buffer containing 2% SDS, 2% beta -mercaptoethanol, 10% glycerol, and 10 mM Tris · HCl (pH 6.8) at room temperature for 2 min and applied to the SDS-polyacrylamide gel. Immunoblot was carried out as described previously (3).

Antibody. Antibody CHK-N was raised against the amino-terminal peptide (residues 10-24) of the guinea pig colonic H+-K+-ATPase alpha -subunit (TKDTKQLGQEEGKKC). The amino acid sequence of the colonic H+-K+-ATPase alpha -subunit in this segment is different from those of gastric H+-K+-ATPase and Na+-K+-ATPase alpha -subunits. The peptide was coupled with a carrier protein keyhole limpet hemocyanin. The carrier- conjugated peptide was emulsified with Freund's complete adjuvant, and the emulsion was injected subcutaneously into Japanese White rabbits five times at intervals of 14-28 days.

Assay of K+-ATPase activity. K+-ATPase activity was assayed in 1 ml of solution containing 50 µg of membrane protein, 3 mM MgCl2, 3 mM ATP (Tris salt), 5 µM oligomycin, and 40 mM Tris · HCl (pH 7.4) in the presence and absence of 15 mM KCl. After incubation at 37°C for 30 min, the inorganic phosphate released was measured as described elsewhere (25). The K+-ATPase activity was calculated as the difference between activities in the presence and absence of KCl.

Protein was measured using a bicinchoninic acid protein assay kit from Pierce (Rockford, IL), with BSA as a standard.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The protein deduced from the cDNA for the putative guinea pig colonic H+-K+-ATPase consists of 1,033 amino acids and has a molecular weight of 114,365. It contains the putative phosphorylation site (Asp-385) and a 5'-FITC binding site (Lys-517), which are conserved in the members of P-type ATPases, and shows ~60% homology with gastric H+-K+-ATPase and Na+-K+-ATPase alpha -subunits. Therefore, it is likely that this protein is the alpha -subunit of colonic H+-K+-ATPase. Here, we transfected this putative guinea pig colonic H+-K+-ATPase cDNA into HEK-293 cells after introduction into a mammalian expression vector, pCIS2 (Genentech). The cDNA cloning of colonic H+-K+-ATPase beta -subunit has not been reported. Therefore, this putative colonic H+-K+-ATPase cDNA was transfected without beta -subunit cDNA or with beta -subunit cDNA of either rabbit gastric H+-K+-ATPase or Torpedo Na+-K+-ATPase. Figure 1 shows the Western blot patterns of the HEK membrane fractions transfected with the gastric H+-K+-ATPase beta -subunit cDNA alone or the colonic H+-K+-ATPase cDNA alone (full alpha ) and fractions cotransfected with the colonic H+-K+-ATPase cDNA plus the gastric H+-K+-ATPase beta -subunit cDNA or Torpedo Na+-K+-ATPase beta -subunit cDNA (detected with antibody CHK-N raised against the amino-terminal synthetic peptide of guinea pig colonic H+-K+-ATPase). When the cells were transfected with the colonic H+-K+-ATPase cDNA alone, a faint band was detected around 100 kDa. The intensity of this band, that is, the expression of the colonic H+-K+-ATPase, significantly increased when the cells were transfected together with the gastric H+-K+-ATPase or Na+-K+-ATPase beta -subunit cDNA.


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Fig. 1.   Immunoblotting with CHK-N antibody of the membrane fraction of HEK cells transfected with putative colonic H+-K+-ATPase cDNA. Thirty micrograms of HEK cell membrane fractions were applied to the gel and blotted with antibody CHK-N directed against the putative colonic H+-K+-ATPase. Lane 1, molecular weight standards; lane 2, mock-transfected cells. Cells were transfected with rabbit gastric H+-K+-ATPase beta -subunit cDNA (HKbeta , lane 3), putative colonic H+-K+-ATPase cDNA (full alpha , lane 4), putative colonic H+-K+-ATPase cDNA + rabbit gastric H+-K+-ATPase beta -subunit cDNA (lane 5), and putative colonic H+-K+-ATPase cDNA + Torpedo Na+-K+-ATPase beta -subunit cDNA (NaKbeta , lane 6).

Next, we measured the K+-ATPase activity of the membrane fractions of the transfected cells. K+-ATPase activity was measured in the absence of Na+ to eliminate the possible effect of endogenous Na+-K+-ATPase present in the fraction. A small amount of K+-ATPase activity was found in the cells transfected with the colonic H+-K+-ATPase cDNA alone (full alpha ), which was slightly higher than that observed in the cells transfected with the gastric H+-K+-ATPase beta -subunit cDNA alone. However, the cells cotransfected with the colonic H+-K+-ATPase cDNA plus the gastric H+-K+-ATPase or Na+-K+-ATPase beta -subunit cDNA showed K+-ATPase activity significantly higher than that found in the cells transfected with the colonic H+-K+-ATPase cDNA alone (Fig. 2). This property of the colonic H+-K+-ATPase was different from that of gastric H+-K+-ATPase. Gastric H+-K+-ATPase activity was found in the membrane fraction when the H+-K+-ATPase alpha -subunit cDNA was cotransfected with the H+-K+-ATPase beta -subunit cDNA but not with the Na+-K+-ATPase beta -subunit cDNA (Fig. 3).


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Fig. 2.   K+-ATPase activities expressed in transfected cells. K+-ATPase activities of membrane fractions of cells transfected with rabbit gastric H+-K+-ATPase beta -subunit cDNA, putative colonic H+-K+-ATPase cDNA, putative colonic H+-K+-ATPase cDNA + rabbit gastric H+-K+-ATPase beta -subunit cDNA, and putative colonic H+-K+-ATPase cDNA + Torpedo Na+-K+-ATPase beta -subunit cDNA were measured as described in MATERIALS AND METHODS. Values are means ± SE of 3 observations in 3 transfections.


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Fig. 3.   K+-ATPase activities expressed in transfected cells. K+-ATPase activities of membrane fractions of cells transfected with rabbit gastric H+-K+-ATPase alpha -subunit cDNA + Torpedo Na+-K+-ATPase beta -subunit cDNA (rab. H,Kalpha  + Na,Kbeta ), rabbit gastric H+-K+-ATPase alpha -subunit plus beta -subunit cDNAs (rab. H,Kalpha  + rab. H,Kbeta ), and rabbit gastric H+-K+-ATPase alpha -subunit cDNA in the absence of beta -subunit cDNA were measured as described in MATERIALS AND METHODS. Values are means ± SE of 3 observations in 3 transfections.

Furthermore, we modified the structures of the 5'- and 3'-untranslated regions of the colonic H+-K+-ATPase cDNA. Here, we prepared two different kinds of cDNA constructs of the colonic H+-K+-ATPase: a cDNA truncated to its 5'-untranslated region (5'-alpha ) and cDNA truncated to its 3'-untranslated region (3'-alpha ) (as shown in MATERIALS AND METHODS), and HEK cells were transfected with each of these cDNAs with the gastric H+-K+-ATPase beta -subunit cDNA. The expression level of H+-K+-ATPase was higher in the membrane fraction of the cells transfected with 5'-alpha colonic H+-K+-ATPase plus gastric H+-K+-ATPase beta -subunit cDNAs than that transfected with full-length colonic H+-K+-ATPase (full alpha ) plus gastric H+-K+-ATPase beta -subunit cDNAs (Fig. 4). The expressed K+-ATPase activity was also significantly (~30%) higher in the cells transfected with 5'-alpha colonic H+-K+-ATPase plus gastric H+-K+-ATPase beta -subunit cDNAs (Fig. 5). It is likely that some element suppresses the expression in the 5'-untranslated region of the colonic H+-K+-ATPase cDNA. It is noteworthy that similar findings were reported in the expressions of urinary bladder H+-K+-ATPase and gastric H+-K+-ATPase (3, 12). Although it is difficult to estimate quantitative correlation between the apparent level of expression and the measured level of expressed K+-ATPase activity, because Western analysis is semiquantitative, we observed as reproducible experimental results in a qualitative manner that the denser the ATPase band the higher the activity. Hereafter, we used the 5'-alpha construct as the colonic H+-K+-ATPase cDNA preparation and cotransfected this with the gastric H+-K+-ATPase beta -subunit cDNA.


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Fig. 4.   Immunoblotting with CHK-N antibody of membrane fraction of HEK cells cotransfected with modified colonic H+-K+-ATPase and gastric H+-K+-ATPase beta -subunit cDNAs. Thirty micrograms of HEK cell membrane fractions were applied to the gel and blotted with antibody CHK-N directed against the putative colonic H+-K+-ATPase. Lane 1, molecular mass standards; lane 2, mock-transfected cells. Cells were cotransfected with full-length colonic H+-K+-ATPase cDNA (full alpha ) plus rabbit gastric H+-K+-ATPase beta -subunit cDNA (full alpha  + HKbeta , lane 3), with colonic H+-K+-ATPase cDNA truncated in the 5'-untranslated region (5'-alpha ) + rabbit gastric H+-K+-ATPase beta -subunit cDNA (lane 4), and with colonic H+-K+-ATPase cDNA truncated in the 3'-untranslated region (3'-alpha ) + rabbit gastric H+-K+-ATPase beta -subunit cDNA (lane 5).


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Fig. 5.   K+-ATPase activities expressed in transfected cells. K+-ATPase activities of membrane fractions of cells transfected with rabbit gastric H+-K+-ATPase beta -subunit cDNA alone or with full-length colonic H+-K+-ATPase cDNA (full alpha ) alone or cotransfected with modified colonic H+-K+-ATPase (full alpha , 5'-alpha , and 3'-alpha ) and gastric H+-K+-ATPase beta -subunit cDNAs were measured as described in MATERIALS AND METHODS. Values are means ± SE of 3 observations in 3 transfections.

The K+-ATPase activity was almost completely inhibited by 2 mM ouabain but not inhibited by 100 µM Sch-28080 (only 8% inhibition with 100 µM Sch-28080), which is an inhibitor specific to gastric H+-K+-ATPase (18). These properties are qualitatively similar to those of K+-ATPase found in guinea pig distal colon (23, 24). The inhibitory effects of ouabain on the K+-ATPase expressed in HEK cells are quantitatively shown in Fig. 6. Ouabain inhibited the K+-ATPase in a concentration-dependent manner, with an IC50 of ~52 µM. This value was smaller than that reported for the rat colonic H+-K+-ATPase expressed in Xenopus oocytes [inhibition constant (Ki) value of 970 µM] (5) but higher than that reported for guinea pig colonic epithelial cells (Ki value of 3.2 µM) (24). Figure 7 shows the effects of K+ concentrations on the K+-ATPase expressed in the membrane fraction of cells transfected with the colonic H+-K+-ATPase and gastric H+-K+-ATPase beta -subunit cDNAs. The ATPase activity was stimulated with K+ in a concentration-dependent manner; the Michaelis-Menten constant (Km) value was 0.68 mM. This value was close to the value reported for the rat colonic H+-K+-ATPase expressed in Xenopus oocytes (Km value of 730 µM) (5) but larger than that reported for the gastric H+-K+-ATPase expressed in HEK cells (Km value of 0.2 mM) (3) and much higher than that reported for guinea pig colonic epithelial cells (Km value of 55 µM) (24).


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Fig. 6.   Effects of ouabain concentrations on expressed colonic K+-ATPase activity. K+-ATPase activities of the membrane fraction of the HEK cells cotransfected with the putative colonic H+-K+-ATPase alpha -subunit (5'-alpha ) and rabbit gastric H+-K+-ATPase beta -subunit cDNAs were measured as a function of ouabain concentrations. K+-ATPase activities are expressed as percentage of control values measured in the absence of ouabain. Values are means ± SE of 3 observations in 3 transfections. K+-ATPase activity in the absence of ouabain was 0.62 ± 0.09 µmol · mg-1 · h-1.


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Fig. 7.   Effects of K+ concentrations on expressed colonic K+-ATPase activity. K+-ATPase activities of the membrane fraction of HEK cells cotransfected with the putative colonic H+-K+-ATPase alpha -subunit (5'-alpha ) and rabbit gastric H+-K+-ATPase beta -subunit cDNAs were measured as a function of K+ concentrations. K+-ATPase activity was calculated as the difference between the ATPase activities in the presence and absence of KCl. Values are means ± SE of transfections.

    DISCUSSION
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The presence of an active K+ transporter and proton pump in the apical membrane of distal colonic cells has been postulated in many species, including rabbit, rat, and guinea pig. Gustin and Goodman (9, 10) first reported that an ouabain-resistant K+-ATPase is present that is inhibited by vanadate and forms a phosphorylated intermediate in rabbit descending colon. Subsequently, a K+-dependent proton pump activity was observed in the membrane vesicles from rabbit distal colon (14). Similar K+-ATPase activities were also observed in the membrane fractions of guinea pig (21, 23, 24) and rat distal colons (7, 22). The former was sensitive to ouabain and insensitive to Sch-28080, and the latter was partly inhibited by ouabain and Sch-28080. These findings suggest that in the distal colon there should be an isoform of gastric H+-K+-ATPase that is involved in acid secretion and K+ absorption. A putative colonic H+-K+-ATPase was first cloned from rat distal colon (6). It has 60-65% amino acid identity with Na+-K+-ATPase and gastric H+-K+-ATPase alpha -subunits. Recently, the rat colonic cDNA without exogenous beta -subunit cDNA of Na+-K+-ATPase or H+-K+-ATPase was functionally expressed in insect Sf9 cells (16). Successively, the rat colonic H+-K+-ATPase was expressed in Xenopus oocytes; oocytes were injected with the rat colonic H+-K+-ATPase cRNA together with the toad bladder beta -subunit H+-K+-ATPase cRNA (13). The oocytes showed Rb+ uptake, intracellular alkalinization, and acidification of the extracellular medium (5). Therefore, it is concluded that the putative rat colonic H+-K+-ATPase cDNA encodes an alpha -subunit of colonic H+-K+-ATPase.

Another cDNA cloned from guinea pig distal colon by Watanabe et al. (GenBank accession no. D21854) may be regarded as a putative colonic H+-K+-ATPase because this cDNA clone shows 88% amino acid identity with the rat colonic H+-K+-ATPase cDNA. In the present study, we expressed the putative alpha -subunit of guinea pig colonic H+-K+-ATPase together with the beta -subunit of rabbit gastric H+-K+-ATPase or Torpedo Na+-K+-ATPase and examined the properties of the expressed ATPases. When the cells were transfected with the putative colonic H+-K+-ATPase cDNA in combination with the rabbit gastric H+-K+-ATPase beta -subunit cDNA, K+-ATPase activity was found in the membrane fraction of the cells. This K+-ATPase activity in HEK cells was inhibited by ouabain but not by Sch-28080. Therefore, it is regarded that this putative colonic H+-K+-ATPase cDNA encodes an alpha -subunit of guinea pig colonic H+-K+-ATPase.

The K+-ATPase activity expressed in HEK cells showed lower sensitivity to ouabain and lower affinity for K+ than that in the membrane fraction of guinea pig colonic mucosae (Ki values for ouabain of 52 µM and 3.2 µM and Km values for K+ of 0.68 mM and 55 µM, respectively). One explanation for this discrepancy is that the gastric beta -subunit associated with the colonic H+-K+-ATPase alpha -subunit may have conferred a different sensitivity to inhibitors and different affinity for cations. The colonic H+-K+-ATPase alpha -subunit is likely to be assembled with its endogenous beta -subunit in the colonic mucosae, resulting in high sensitivity to ouabain and high affinity for K+. However, the beta -subunit associated in vivo with the colonic alpha -subunit has not been cloned yet. Difference in beta -subunits may result in the conformational difference in expressed H+-K+-ATPases. Similar findings were reported for the expression of rat colonic H+-K+-ATPase. The rat colonic H+-K+-ATPase expressed in Xenopus oocytes by coinjection of the colonic alpha -subunit and toad bladder beta -subunit cRNAs was sensitive to ouabain (Ki = 0.97 mM) and insensitive to Sch-28080 (5). Furthermore, when expressed with the beta 1-subunit of Na+-K+-ATPase or gastric H+-K+-ATPase beta -subunit in oocytes, the Rb+ uptake was sensitive to ouabain (IC50 = 0.4-0.6 mM) and insensitive to Sch-28080 (4). These sensitivities of the expressed ATPase to ouabain were lower than that reported for the rat colonic mucosae (Ki = 0.1 mM) (7). In addition, when the same rat colonic H+-K+-ATPase alpha -subunit cDNA was introduced in insect Sf9 cells without beta -subunit cDNAs, the expressed H+-K+-ATPase activity was insensitive to ouabain (1 mM) and slightly inhibited by Sch-28080 (18% inhibition by 100 µM Sch-28080) (16).

It is very interesting that the colonic H+-K+-ATPase cDNA product can be functionally assembled with both the cDNA product of gastric H+-K+-ATPase beta -subunit and that of the Na+-K+-ATPase beta -subunit. A similar finding was reported for the functional expression of rat colonic H+-K+-ATPase in Xenopus oocytes, in which rat colonic H+-K+-ATPase alpha -subunit assembled with either the rat gastric H+-K+-ATPase beta -subunit or the rat Na+-K+-ATPase beta -subunit, resulting in 86Rb+ uptake in both cases (4). The Na+-K+-ATPase alpha -subunit can assemble not only with the Na+-K+-ATPase beta -subunit but also with the H+-K+-ATPase beta -subunit, resulting in a functional heteroligomer (11). On the other hand, the functional expression of H+-K+-ATPase using the gastric H+-K+-ATPase alpha -subunit in combination with the Na+-K+-ATPase beta -subunit has not been reported. In our expression system using HEK cells presented here, K+-ATPase activity was not functionally expressed when the cells were cotransfected with gastric H+-K+-ATPase alpha -subunit and Na+-K+-ATPase beta -subunit cDNAs (Fig. 3). These findings suggest that the colonic H+-K+-ATPase alpha -subunit is intermediary between Na+-K+-ATPase and H+-K+-ATPase alpha -subunits.

The protein encoded in the human ATP1AL1 gene is another member of nongastric H+-K+-ATPase that shares 86-89% amino acid identity with rat and guinea pig colonic H+-K+-ATPases. Recently, Modyanov et al. (17) reported the functional expression of ATP1AL1 cRNA product in Xenopus oocytes. Oocytes coinjected with the ATP1AL1 cRNA and that of rabbit gastric H+-K+-ATPase beta -subunit showed 86Rb+ uptake. This uptake was sensitive to ouabain (Ki = 13 µM) and almost insensitive to Sch-28080 (17). Successively, Grishin et al. (8) reported the functional expression of this cDNA in HEK cells. The cells cotransfected with cDNAs for ATP1AL1 and rabbit gastric H+-K+-ATPase beta -subunit also showed 86Rb+ uptake. The uptake was inhibited by ouabain (Ki = 42 µM) and Sch-28080 (Ki = 131 µM). Thus the functional properties of ATP1AL1 gene product are similar to those exhibited by rat and guinea pig colonic H+-K+-ATPases. It is not clear, however, whether ATP1AL1 is expressed in the colon.

In conclusion, the protein encoded by the putative colonic H+-K+-ATPase cDNA exhibited an ouabain-sensitive, Sch-28080-insensitive K+-ATPase in combination with the expression of the gastric H+-K+-ATPase or Na+-K+-ATPase beta -subunits.

    ACKNOWLEDGEMENTS

This study was supported in part by a Grant-in-Aid for Encouragement of Young Scientists (to S. Asano) and Scientific Research on Priority Areas (to S. Asano, Y. Suzuki, and N. Takeguchi) from the Ministry of Education, Sports, Science, and Culture in Japan.

    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

1 T. Watanabe, M. Sato, K. Kaneko, T. Suzuki, T. Yoshida, and Y. Suzuki; GenBank accession no. D21854.

Address for reprint requests: S. Asano, Molecular Genetics Research Center, Toyama Medical and Pharmaceutical Univ., 2630 Sugitani, Toyama, 930-01, Japan.

Received 26 January 1998; accepted in final form 12 May 1998.

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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Cell Physiol 275(3):C669-C674
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