Chimeric Domain Analysis of the Compatibility between H+,K+-ATPase and Na+,K+-ATPase β-Subunits for the Functional Expression of Gastric H+,K+-ATPase*

Gastric H+,K+-ATPase consists of α-subunit with 10 transmembrane domains and β-subunit with a single transmembrane domain. We constructed cDNAs encoding chimeric β-subunits between the gastric H+,K+-ATPase and Na+,K+-ATPase β-subunits and co-transfected them with the H+,K+-ATPase α-subunit cDNA in HEK-293 cells. A chimeric β-subunit that consists of the cytoplasmic plus transmembrane domains of Na+,K+-ATPase β-subunit and the ectodomain of H+,K+-ATPase β-subunit assembled with the H+,K+-ATPase α-subunit and expressed the K+-ATPase activity. Therefore, the whole cytoplasmic and transmembrane domains of H+,K+-ATPase β-subunit were replaced by those of Na+,K+-ATPase β-subunit without losing the enzyme activity. However, most parts of the ectodomain of H+,K+-ATPase β-subunit were not replaced by the corresponding domains of Na+,K+-ATPase β-subunit. Interestingly, the extracellular segment between Cys152 and Cys178, which contains the second disulfide bond, was exchangeable between H+,K+-ATPase and Na+,K+-ATPase, preserving the K+-ATPase activity intact. Furthermore, the K+-ATPase activity was preserved when the N-terminal first 4 amino acids (67DPYT70) in the ectodomain of H+,K+-ATPase β-subunit were replaced by the corresponding amino acids (63SDFE66) of Na+,K+-ATPase β-subunit. The ATPase activity was abolished, however, when 4 amino acids (76QLKS79) in the ectodomain of H+,K+-ATPase β-subunit were replaced by the counterpart (72RVAP75) of Na+,K+-ATPase β-subunit, indicating that this region is the most N-terminal one that discriminates the H+,K+-ATPase β-subunit from that of Na+,K+-ATPase.


EXPERIMENTAL PROCEDURES
Materials-HEK-293 cells (human embryonic kidney cell line) were a kind gift from Dr. Jonathan Lytton (University of Calgary, Calgary, Canada). pcDNA3 vector was obtained from Invitrogen Co. (San Diego, CA). Pfu DNA polymerase was from Stratagene. Restriction enzymes and other DNA and RNA modifying enzymes were from Toyobo (Osaka, Japan) and New England Biolabs (Beverly, MA). Endoglycosidase H (Endo H) 1 and N-glycosidase F (PNGase F) were obtained from Roche Molecular Biochemicals (Tokyo, Japan). Anti-gastric H ϩ ,K ϩ -ATPase ␤-subunit monoclonal antibody, 2B6, was obtained from Molecular Biological Laboratories (Nagoya, Japan). SCH 28080 was obtained from Schering Co. (Keniworth, NJ). All other reagents were of molecular biology grade or the highest grade of purity available.
Site-directed Mutagenesis-Introduction of site-directed mutations between SnaBI and EcoRV sites of the H ϩ ,K ϩ -ATPase ␤-subunit was carried out by sequential polymerase chain reaction (PCR) steps as described elsewhere (6). Two kinds of flanking sequence primers were prepared, one is the 5Ј-flanking sense primer, 5Ј-GCAATTAACCCT-CACTAAAGG-3Ј (sequence in pBluescript II vector), and the other is the 3Ј-flanking antisense primer, 5Ј-CGTGCTGTCAGACACGTTG-3Ј (close to the EcoRV site of the H ϩ ,K ϩ -ATPase ␤-subunit cDNA). Additionally, sense and antisense oligonucleotides, each 21 bases long containing mutated bases near the center, were designed (referred as the sense mutating primer and antisense mutating primer). In the first PCR amplification step, the NsH chimeric ␤-subunit cDNA or H ϩ ,K ϩ -ATPase ␤-subunit cDNA was used as a template DNA. Two fragments were prepared in this step: one between the 5Ј-flanking sense primer and the antisense mutating primer, and the other between the sense mutating primer and the 3Ј-flanking antisense primer. Each amplified fragment was purified by gel electrophoresis, combined, and incubated with the 5Ј-flanking sense primer and the 3Ј-flanking antisense primer in the second PCR amplification. The amplified fragment was purified by gel electrophoresis, subcloned in pCR-Script Amp SK(ϩ) vector (Stratagene), and sequenced. PCR was routinely carried out in the presence of 200 M each dNTP, 500 nM primers, 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 20 mM Tris-HCl, pH 8.8, 0.1% Triton X-100, 100 g/ml bovine serum albumin, and 2.5 units of Pfu DNA polymerase for 30 cycles. After sequencing, the fragment amplified in the second PCR was digested with AatII and EcoRI and ligated back into the relevant position of the wild-type H ϩ ,K ϩ -ATPase ␤-subunit or the NsH chimeric ␤-subunit construct.
DNA Sequencing-DNA sequencing was done by the dideoxy chain termination method using an Autoread DNA sequencing kit and an ALFexpress DNA sequencer (Amersham Pharmacia Biotech).
Cell Culture, Transfection, and Preparation of Membrane Fractions-Cell culture of HEK-293 was carried out as described previously (7). ␣and ␤-subunit cDNA transfection was performed by the calcium phosphate method with 10 g of cesium chloride-purified DNA/10-cm dish. Cells were harvested 2 days after the DNA transfection. Membrane fractions of HEK cells were prepared as described previously (7).
SDS-Polyacrylamide Gel Electrophoresis and Western Blot-SDSpolyacrylamide gel electrophoresis was carried out as described elsewhere (25). Membrane preparations (30 g of protein) were incubated in a sample buffer containing 2% SDS, 2% ␤-mercaptoethanol, 10% glycerol, and 10 mM Tris-HCl, pH 6.8, at room temperature for 2 min and applied to the SDS-polyacrylamide gel. Western blot was carried out as described previously (7).
Glycosidase Treatment-30 g of membrane fraction was treated with Endo H or PNGase F following the manufacturer's instructions. For EndoH digestion, 30 g of membrane fraction was treated with 10 milliunits of Endo H in a solution containing 0.1% SDS, 1 M 2-mercaptoehanol, 0.5 mM phenylmethylsulfonyl fluoride, and 50 mM sodium phosphate, pH 6.0, at 37°C overnight. For PNGase F digestion, 30 g of membrane fraction was treated with 1 unit of PNGase F in a solution containing 0.1% SDS, 1% n-octylglucoside, 1 M 2-mercaptoehanol, 30 mM EDTA, and 50 mM sodium phosphate, pH 6.0, at 37°C overnight.
Immunoprecipitation-Membrane fractions (1 mg) of HEK cells expressing the ␣-␤ complex was incubated in 1 ml of lysis buffer containing 1% Nonidet P-40, 150 mM NaCl, 0.5 mM EDTA, and 50 mM Tris-HCl, pH 7.4, at 4°C for 30 min. After centrifugation at 16,000 ϫ g for 20 min, the supernatant was incubated with an anti-␣-subunit antibody, Ab1024, at a 1:100 dilution, and 10 l of ImmunoPure immobilized protein A (Pierce) at 4°C for 12 h. After centrifugation, the pellet was washed four times with the lysis buffer followed by two washes in 0.1% Nonidet P-40, 150 mM NaCl, 0.5 mM EDTA, and 50 mM Tris-HCl, pH 7.4. The pellet was solubilized in the sample buffer for SDS-polyacrylamide gel electrophoresis and incubated at room temperature for 10 min. The proteins were separated on SDS-polyacylamide gel and blotted. The ␤-subunit in the blot was detected by an anti-␤-subunit antibody, 2B6, in combination with a peroxidase-conjugated anti-mouse antibody, which was preabsorbed with rabbit serum. When indicated, the precipitated proteins were deglycosylated, that is, immunoprecipitated samples were treated with PNGase F as described above, solubilized in the sample buffer for SDS-polyacrylamide gel electrophoresis, and blotted.
Assay of H ϩ ,K ϩ -ATPase Activity-H ϩ ,K ϩ -ATPase activity was measured from the decrease in the amount of NADH coupled with regeneration of ATP from ADP ("coupled-enzyme assay") in 1.2 ml of a reaction mixture containing 50 g of membrane protein, 3 mM MgCl 2 , 800 M ATP, 160 M NADH, 0.8 mM phosphoenolpyruvate, 3 units/ml pyruvate kinase, 2.75 units/ml lactate dehydrogenase, 5 mM NaN 3 , 1 mM ouabain, 15 mM KCl, and 40 mM Tris-HCl, pH 7.4. The decrease in the amount of NADH was measured at 37°C from the absorbance at 340 nm by a Beckman spectrophotometer as described elsewhere (27). H ϩ ,K ϩ -ATPase activity, defined as the SCH 28080-sensitive K ϩ -ATPase, was calculated as the difference between the K ϩ -ATPase activities in the presence and absence of 50 M SCH 28080.
When the K ϩ -ATPase activity was measured as a function of K ϩ concentrations, the ATPase activity was measured from the measurement of inorganic phosphate released from ATP. K ϩ -ATPase activity was measured in 1 ml of solution containing 50 g of membrane protein, 3 mM MgSO 4 , 1 mM ATP, 5 mM NaN 3 , 2 mM ouabain, and 40 mM Tris-HCl, pH 6.8, in the presence and absence of various concentrations of KCl. After incubation at 37°C for 30 min, the reaction was terminated by the addition of ice-cold stop solution containing 12% perchloric acid and 3.6% ammonium molybdate. Inorganic phosphate released was measured from the absorbance at the wavelength of 320 nm as described elsewhere (28). The K ϩ -ATPase activity was calculated as the difference between activities in the presence and absence of KCl. The K ϩ -ATPase activity was sensitive to 50 M SCH 28080. Inorganic phosphate released in the enzyme reaction with the wild-type H ϩ ,K ϩ -ATPase was 4 -5 times higher than the background level of inorganic phosphate released in the absence of enzyme. Values of K ϩ -ATPase activity measured from colorimetric assay of released inorganic phosphate were comparable with those measured in coupled enzyme assay. Protein was measured using the BCA protein assay kit from Pierce with bovine serum albumin as a standard.

RESULTS
Construction of Chimeric ␤-Subunits between H ϩ ,K ϩ -ATPase and Na ϩ ,K ϩ -ATPase-First, we used two series of chimeric ␤-subunits that were constructed using ␤-subunits of hog gastric H ϩ ,K ϩ -ATPase and T. californica Na ϩ ,K ϩ -ATPase as shown in Fig. 1. One set of chimeras (NxH series) was prepared by successively exchanging 5Ј-portion of the H ϩ ,K ϩ -ATPase ␤-subunit cDNA with the corresponding portion of the Na ϩ ,K ϩ -ATPase ␤-subunit cDNA (Fig. 1A). The other set of chimeras (HxNyH series) was prepared by replacing a middle portion of the H ϩ ,K ϩ -ATPase ␤-subunit (between adjacent unique restriction sites) with the corresponding portion of the Na ϩ ,K ϩ -ATPase ␤-subunit (Fig. 1B).
Expression of ␣and ␤-Subunits- Fig. 2 shows Western blot patterns of the membrane fractions of the transfectants, detected by using an anti-gastric H ϩ ,K ϩ -ATPase ␣-subunit antibody. When the cells were transfected with the wild-type ␣-sub-unit cDNA in the absence of the ␤-subunit cDNA, a single faint band was detected around 95 kDa, which represents the expression of the H ϩ ,K ϩ -ATPase ␣-subunit (lane 8 in both panels A and B in Fig. 2). The expression of the ␣-subunit increased when the cells were co-transfected with the wild-type H ϩ ,K ϩ -ATPase ␤-subunit cDNA (lane 1 in both panels A and B in Fig.  2). A similar increase in expression of the ␣-subunit was also observed when the cells were co-transfected with the NsH chimeric ␤-subunit cDNA (lane 2 in both panels A and B in Fig.  2) or HpNeH chimeric ␤-subunit cDNA (lane 6 in Fig. 2B). However, there was no increased expression of the ␣-subunit when co-expressed with other chimeric ␤-subunits including NvH, NmH, NpH, NeH ( Fig. 2A), HsNvH, HvNmH, HmNpH, and HeN chimeras (Fig. 2B) and the wild-type Na ϩ ,K ϩ -ATPase ␤-subunit (lane 7 in Fig. 2A). Fig. 3 shows Western blot patterns of the membrane fractions of the transfectants, detected by using an anti-gastric H ϩ ,K ϩ -ATPase ␤-subunit antibody, 2B6 (23). The epitope of H ϩ ,K ϩ -ATPase and Na ϩ ,K ϩ -ATPase, respectively. Lowercase letters show the restriction sites that are used as the joining points for chimeric construction; s, v, m, p, and e represent SnaBI, EcoRV, MunI, SphI, and EcoT22I sites, respectively. H ϩ ,K ϩ -ATPase (closed bar) and Na ϩ ,K ϩ -ATPase (open bar) and their transmembrane domains (hatched and cross-hatched bars) are shown schematically. The locations of disulfide bonds in the ectodomain are shown with brackets. The numeric numbers show the connecting points between the two ␤-subunits and refer to the hog H ϩ ,K ϩ -ATPase ␤-subunit. The total numbers of amino acids in these chimeras are shown on the right. Glycosylation sites are shown with sugar chain symbols.
. These cell membrane fractions (30 g) were applied on the gel and blotted with Ab1024, which is an anti-H ϩ ,K ϩ -ATPase ␣-subunit antibody. Bands representing H ϩ ,K ϩ -ATPase ␣-subunit are shown by a bold arrow. B, HEK-293 cells were co-transfected with the wild-type H ϩ ,K ϩ -ATPase ␣-subunit cDNA plus wild-type H ϩ ,K ϩ - this monoclonal antibody is located on the C-terminal of the ␤-subunit. Therefore, this antibody reacted neither with the wild-type Na ϩ ,K ϩ -ATPase ␤-subunit (lane 7 in Fig. 3A) nor HeN chimera (lane 7 in Fig. 3B). When the cells were cotransfected with both the wild-type H ϩ ,K ϩ -ATPase ␣-subunit and ␤-subunit cDNAs, dense doublet bands with a lower molecular mass (48 -50 kDa) (␤ c ) and a smear band with a higher molecular mass (60 -70 kDa) (␤ m ) were observed (lane 1 in both panels A and B in Fig. 3). Similar patterns were observed when the cells were co-transfected with the ␣-subunit cDNA plus NsH (lane 2 in both panels A and B in Fig. 3), HvNmH (lane 4 in Fig. 3B), or HpNeH chimeric ␤-subunit cDNA (lane 6 in Fig.  3B). The ␤-subunits with higher molecular masses (60 -70 kDa) (␤ m ) were resistant to Endo H, while they were digested with PNGase F. After treatment with PNGase F, molecular mass of the ␤-subunit decreased from 60 -70 kDa to about 30 kDa (protein core of the ␤-subunit) (data not shown). On the other hand, the ␤-subunits with lower molecular masses (40 -48 kDa) (␤ c ) were digested with both Endo H and PNGase F, resulting in the appearance of single bands with about 30 kDa (data not shown). These results indicate that the 60 -70-kDa band represents the ␤-subunit with complex-type (Endo Hresistant) carbohydrate chains and that the bands with lower molecular masses (around 40 -48 kDa) represent the ␤-subunits with high mannose-type (Endo H-sensitive) carbohydrate chains. Therefore, some part of the wild-type, NsH, HvNmH, and HpNeH chimeric ␤-subunits are supposed to leave an endoplasmic reticulum compartment. However, no smear band with a higher molecular mass (␤ m ) was observed when the cells were co-transfected with the ␣-subunit cDNA plus other chimeric ␤-subunit cDNAs such as NvH, NmH, NpH, NeH, HsNvH, and HmNpH cDNAs. The apparent molecular masses of the bands with smaller size (␤ c ) was variable in the chimeric ␤-subunits. There are six putative N-glycosylation sites (Asn-Xaa-(Ser/Thr)) in the wild-type H ϩ ,K ϩ -ATPase ␤-subunit and three in the wild-type Torpedo Na ϩ ,K ϩ -ATPase ␤-subunit as shown in Fig. 1. All of the putative N-glycosylation sites have been shown to contain carbohydrate chains in both ATPases (29 -31). The present chimeric ␤-subunits contain different numbers of putative N-glycosylation sites (Fig. 1). This gave different apparent molecular masses for ␤ c as shown on the gels (Fig. 3).
Immunoprecipitation of ␣and ␤-Subunits- Fig. 4 (A and B) shows Western blot patterns of the samples immunoprecipitated with the anti-␣-subunit antibody and then detected with the anti-␤-subunit antibody, 2B6. In Fig. 4 (A and B), every lane shows a nonspecific band with a molecular mass of 60 kDa. This band was observed even when the immunoprecipitation reaction was carried out in the absence of membrane fraction or when the cells were transfected only with the H ϩ ,K ϩ -ATPase ␣-subunit cDNA (data not shown). Although the anti-mouse secondary antibody used in the Western blot had been preabsorbed with rabbit serum, the antibody was likely to crossreact with the rabbit antibody.
The anti-␣-antibody co-precipitated proteins with molecular masses of 70 kDa (␤ m in panels A and B in Figs. 4) and 48 kDa (␤ c in panels A and B in Fig. 4) when the cells were co-transfected with the H ϩ ,K ϩ -ATPase ␣-subunit cDNA plus wild-type H ϩ ,K ϩ -ATPase ␤-subunit (lane 1 in both panels A and B in Fig.  4), chimeric ␤-subunit NsH (lane 2 in Fig. 4A), or HpNeH cDNA (lane 5 in Fig. 4B). This pattern was similar to that observed in the Western blot of the ␤-subunits in the membrane fractions (Fig. 3). On the other hand, no ␤-subunit-related band was observed when the cells were transfected only with the H ϩ ,K ϩ -ATPase ␤-subunit cDNA (data not shown). When the cells were co-transfected with the H ϩ ,K ϩ -ATPase ␣-subunit cDNA plus chimeric ␤-subunit NvH, NmH, NpH, NeH (Fig. 4A), HsNvH, HvNmH, or HmNpH (Fig. 4B) cDNAs, a single band or doublet bands with molecular mass of 40 -48 kDa (␤ c ) were observed. The amount of these bands was much smaller than those observed in the samples with the wild-type H ϩ ,K ϩ -ATPase ␤-subunit (lane 1 in both panels A and B in Fig. 4), NsH chimera (lane 2 in Fig. 4A), or HpNeH chimera (lane 5 in Fig. 4B). When the precipitated samples were treated with PNGase F, the molecular mass of the bands shifted to 30 -35 kDa on the blot (CP in panels C and D in Fig. 4), indicating that the precipitated proteins detected with 2B6 were the wild-type H ϩ ,K ϩ -ATPase ␤-subunit or its chimeric ␤-subunits. The bands with molecular masses of 70 kDa represent the ␤-subunits with complex-type carbohydrate chains (␤ m ), and those around 48 kDa represent the ␤-subunits with high mannose-type carbohydrate chains (␤ c ). These results indicate that all the chimeric ␤-subunits as well as the wild-type H ϩ ,K ϩ -ATPase ␤-subunit assembled with H ϩ ,K ϩ -ATPase ␣-subunit.
Detailed Chimeric and Mutational Analysis of the N-terminal Domain of the ␤-Subunit-As shown in Fig. 2, the NsH chimeric ␤-subunit increased the expression of the ␣-subunit in the membrane, whereas the NvH chimeric ␤-subunit did not. Therefore, it is likely that some structure that is important for the stabilization of the ␣-subunit is located in the portion between Tyr 45 (SnaBI site) and Ile 96 (EcoRV site) of the ␤-subunit. Because Tyr 45 is in the transmembrane domain (from Trp 37 to Ile 66 ) located about one quarter of the domain length from the cytoplasmic/membrane boundary, it is not clear whether the whole transmembrane domain of the H ϩ ,K ϩ -ATPase ␤-subunit is replaceable with the corresponding domain of the Na ϩ ,K ϩ -ATPase ␤-subunit, preserving its ability to stabilize the ␣-subunit in the membrane. Here, we constructed a new chimera termed NNH (Fig. 1A) by replacing the whole cytoplasmic plus transmembrane domains of H ϩ ,K ϩ -ATPase ␤-subunit with the corresponding domains of Na ϩ ,K ϩ -ATPase ␤-subunits. As shown in Fig. 5, the expression level of the ␣-subunit was similar between the cells expressing the wildtype H ϩ ,K ϩ -ATPase ␣-␤ complex (lane 1) and ␣-NNH complex (lane 2). Therefore, the chimeric H ϩ ,K ϩ -ATPase ␤-subunit, in which the whole cytoplasmic plus transmembrane domains were replaced with the corresponding domains of the Na ϩ ,K ϩ -ATPase ␤-subunit, stabilized the ␣-subunit in the membrane. The anti-␣-antibody also co-precipitated ␤-subunit proteins with molecular masses of 70 kDa (␤ m in Fig. 6A) and 48 kDa (␤ c in Fig. 6A) when the cells were co-transfected with the H ϩ ,K ϩ -ATPase ␣-subunit cDNA plus NNH chimera (lane 1 in Fig. 6).
From the above findings, it is likely that the segment of H ϩ ,K ϩ -ATPase ␤-subunit from Asp 67 to Ile 96 is important for the stabilization of the ␣-subunit. To study this point further, we prepared two additional ␤-subunit mutants. One is the mutant in which only the first four amino acids ( 67 DPYT 70 ) in the beginning of the ectodomain of the H ϩ ,K ϩ -ATPase ␤-subunit were replaced by the corresponding amino acids of the H ϩ ,K ϩ -ATPase ␤-subunit (lanes 1), chimeric ␤-subunit NsH (lanes 2), NvH (lanes 3), NmH (lanes 4), NpH (lanes 5), or NeH (lanes 6). B and D, HEK-293 cells were co-transfected with the wild-type H ϩ ,K ϩ -ATPase ␣-subunit cDNA plus wild-type H ϩ ,K ϩ -ATPase ␤-subunit (lanes 1), chimeric ␤-subunit HsNvH (lanes 2), HvNmH (lanes 3), HmNpH (lanes 4), or HpNeH (lanes 5). The solubilized membrane fractions were incubated with an anti-␣-subunit antibody, Ab1024, and protein A-coated beads. The precipitated preparations were treated with (C and D) or without PNGase F (A and B), separated on SDS-polyacrylamide gel and blotted with anti-␤-subunit antibody, 2B6. ␤ m , ␤ C , and CP represent the ␤-subunit with complex-type (mature) carbohydrate chains and that with high mannose type (core) carbohydrate chains and core protein of the ␤-subunit, respectively. Na ϩ ,K ϩ -ATPase ␤-subunit, 63 SDFE 66 (termed SDFE mutant). The other is the one in which only four amino acids 76 QLKS 79 in the ectodomain of H ϩ ,K ϩ -ATPase ␤-subunit were replaced by the counterpart of Na ϩ ,K ϩ -ATPase ␤-subunit, 72 RVAP 75 (termed RVAP mutant). As shown in Fig. 7, these eight amino acids of H ϩ ,K ϩ -ATPase differ from those of Na ϩ ,K ϩ -ATPase. As shown in Fig. 5, the expression level of the ␣-subunit was similar between the cells expressing the wild-type H ϩ ,K ϩ -ATPase ␣-␤ complex (lane 1) and ␣-SDFE complex (lane 3). However, the expression level of the ␣-subunit for ␣-RVAP complex (lane 4) was significantly lower than that for the wild-type ␣-␤ complex (lane 1) and similar to that expressed in the absence of ␤-subunit (lane 5).
The anti-␣-antibody co-precipitated ␤-subunit proteins with molecular masses of 70 kDa (␤ m in Fig. 6A) and 48 kDa (␤ c in Fig. 6A) when the cells were co-transfected with the H ϩ ,K ϩ -ATPase ␣-subunit cDNA plus SDFE mutant (lane 2). When the precipitated samples were treated with PNGase F, the molecular mass of these bands shifted to 30 -35 kDa on the blot (CP in Fig. 6B). Therefore, the SDFE ␤-subunit assembled with the ␣-subunit to form a stable ␣-␤ complex. On the other hand, RVAP mutant ␤-subunit did not form a stable ␣-␤ complex (Fig.  5) although weakly assembled with the ␣-subunit (Fig. 6, A and  B). Fig. 8 shows the H ϩ ,K ϩ -ATPase activity in the membrane fractions expressing chimeric ␣-␤ complex and the wild-type H ϩ ,K ϩ -ATPase. As the wild-type complex, we used a heterocomplex of rabbit gastric H ϩ ,K ϩ -ATPase ␣-subunit and hog gastric H ϩ ,K ϩ -ATPase ␤-subunit. The H ϩ ,K ϩ -ATPase activity found in the membrane fraction was sensitive to a gastric proton pump inhibitor, SCH 28080 (data not shown). There is no significant difference between the activities of this rabbit-hog ␣-␤ heterocomplex and rabbit H ϩ ,K ϩ -ATPase ␣-␤ homocomplex (data not shown), suggesting that rabbit gastric H ϩ ,K ϩ -ATPase ␣-subunit assembled with hog gastric H ϩ ,K ϩ -ATPase ␤-subunit and formed a functional H ϩ ,K ϩ -ATPase complex despite the species difference. In the NxH series (x indicates s, v, m, p, or e), only the ␣-NsH complex showed the H ϩ ,K ϩ -ATPase activity (Fig. 8A). This ␣-NsH complex exhibited a 40% higher H ϩ ,K ϩ -ATPase activity than that of the wild-type enzyme. From these results, it became clear that the segment from the N terminus in the cytoplasm to Tyr 45 (SnaBI site) in the transmembrane domain was replaceable with the corresponding segment of the Na ϩ ,K ϩ -ATPase ␤-subunit for the functional expression of H ϩ ,K ϩ -ATPase. However, the segment from Tyr 45 (SnaBI site) to Ile 96 (EcoRV site), which is located in the transmembrane domain and ectodomain of the H ϩ ,K ϩ -ATPase ␤-subunit, was not replaceable. This result is comparable with the finding that the corresponding segment of the Na ϩ ,K ϩ -ATPase ␤-subunit is not replaceable with that of the H ϩ ,K ϩ -ATPase ␤-subunit for stable complex formation with the Na ϩ ,K ϩ -ATPase ␣-subunit and for the functional expression of Na ϩ ,K ϩ -ATPase (22).
The ␣-HeN complex showed no H ϩ ,K ϩ -ATPase activity, indicating that the C-terminal 110 amino acids of the H ϩ ,K ϩ -ATPase ␤-subunit were not replaceable with the counterpart of the Na ϩ ,K ϩ -ATPase ␤-subunit (Fig. 8B). In the HxNyH series of chimeras, only the ␣-HpNeH complex retained the H ϩ ,K ϩ -ATPase activity, 75% of the wild-type enzyme activity (Fig. 8B).

FIG. 6. Western blots with the anti-␤-subunit antibody of the membrane fraction of HEK cells immunoprecipitated with the anti-␣-subunit antibody.
HEK-293 cells were co-transfected with the wild-type H ϩ ,K ϩ -ATPase ␣-subunit cDNA plus NNH (lanes 1), SDFE (lanes 2) or RAVP (lanes 3) cDNAs. The solubilized membrane fractions were incubated with an anti-␣-subunit antibody, Ab1024, and protein A-coated beads. The precipitated preparations were treated with (B) or without PNGase F (A), separated on SDS-polyacrylamide gel, and blotted with anti-␤-subunit antibody, 2B6. ␤ m , ␤ C , and CP represent the ␤-subunit with complex-type (mature) carbohydrate chains and that with high mannose type (core) carbohydrate chains and core protein of the ␤-subunit, respectively.  23) is compared with that of T. californica Na ϩ ,K ϩ -ATPase ␤1-subunit (Torp.NaK-B) (24). Vertical lines indicate identity to the corresponding residues between these two ␤-subunits. Numbering starts from the initiation Met as number 1 for the H ϩ ,K ϩ -ATPase and Na ϩ ,K ϩ -ATPase ␤-subunits, respectively. SnaBI restriction site is shown by the vertical arrow. In the preparation of SDFE and RVAP mutants, 67 DPYT 70 (*) and 76 QLKS 79 (ࡗ) in the H ϩ ,K ϩ -ATPase ␤-subunit were replaced by the corresponding amino acids in the Na ϩ ,K ϩ -ATPase ␤-subunit, respectively. ATPase activity, 27% higher than that of the wild type (Fig.  8C). This result indicates that the whole transmembrane domain and the cytoplasmic domain of the H ϩ ,K ϩ -ATPase ␤-subunit are replaceable with the corresponding domains of the Na ϩ ,K ϩ -ATPase ␤-subunit. The ␣-SDFE complex retained the H ϩ ,K ϩ -ATPase activity, 75% of the wild-type enzyme activity, whereas the ␣-RVAP complex almost lost it (Fig. 8C). Therefore, 76 QLKS 79 block in the ectodomain of H ϩ ,K ϩ -ATPase ␤-subunit is important for the expression of H ϩ ,K ϩ -ATPase activity by stabilizing the ␣-subunit. Fig. 9 shows that double-reciprocal plots between the K ϩ -ATPase activity and the K ϩ concentration for the ␣-NNH, ␣-NsH, and ␣-HpNeH complexes. The K m values for K ϩ of the wild type, ␣-NNH, ␣-NsH, and ␣-HpNeH complexes were 0.32, 0.27, 0.30, and 0.40 mM, respectively, indicating that the replacements of the cytoplasmic and transmembrane domains and the short extracellular segment from Cys 152 to Cys 178 with the corresponding ones of Na ϩ ,K ϩ -ATPase did not change the K ϩ affinity of the enzyme. These results may suggest that these domains and the segment are not involved in determining affinity for K ϩ . DISCUSSION H ϩ ,K ϩ -ATPase ␤-subunit shows a number of structural similarities with Na ϩ ,K ϩ -ATPase ␤-subunit. Both ␤-subunits consist of a short N-terminal cytoplasmic domain (about 40 amino acids) and one transmembrane domain followed by a large ectodomain (13). They contain six conserved cysteine residues in the ectodomain, which form three disulfide bonds (32). These disulfide bonds are important for the protein folding for the maintenance of the ATPase function, because the ATPase activities were abolished by reduction with dithiothreitol or 2-mercaptoethanol (33)(34)(35). Na ϩ ,K ϩ -ATPase activity was also abolished when one of the three disulfide bonds was broken by mutation of the conserved cysteine residue(s) (36). From these similarities between H ϩ ,K ϩ -ATPase and Na ϩ ,K ϩ -ATPase ␤-subunits, we may deduce a possibility that H ϩ ,K ϩ -ATPase ␤-subunit acts as a surrogate for the Na ϩ ,K ϩ -ATPase ␤-subunit for the functional expression of Na ϩ ,K ϩ -ATPase and vice versa for the functional expression of H ϩ ,K ϩ -ATPase. In fact, H ϩ ,K ϩ -ATPase ␤-subunit was assembled with Na ϩ ,K ϩ -ATPase ␣-subunit in Xenopus oocyte to form an ␣-␤ complex exhibiting functional Na ϩ ,K ϩ pump, although the affinity of this pump for K ϩ was lower compared with that of the wildtype Na ϩ ,K ϩ pump (15,37). Heterologous expression of H ϩ ,K ϩ -ATPase ␤-subunit together with Na ϩ ,K ϩ -ATPase ␣-subunit in yeast cells resulted in the appearance of high affinity ouabain binding sites in the membrane (16). Na ϩ ,K ϩ -ATPase ␤-subunit, on the contrary, did not support the functional expression of gastric H ϩ ,K ϩ -ATPase in HEK cells (17).
In the present study, we showed that a chimeric H ϩ ,K ϩ -ATPase ␤-subunit (NsH chimera), which contains the cytoplas- mic domain and N-terminal part of the transmembrane domain of Na ϩ ,K ϩ -ATPase ␤-subunit, was assembled with the H ϩ ,K ϩ -ATPase ␣-subunit to stabilize the ␣-subunit and exhibited H ϩ ,K ϩ -ATPase activity. The affinity of the ␣-NsH complex for K ϩ was similar to that of the wild-type H ϩ ,K ϩ -ATPase. It was previously reported that a chimeric Na ϩ ,K ϩ -ATPase ␤-subunit (HsN chimera), which consisted of the cytoplasmic domain and N-terminal part of the transmembrane domain of H ϩ ,K ϩ -ATPase ␤-subunit plus the remaining part of the transmembrane domain and the whole ectodomain of Na ϩ ,K ϩ -ATPase ␤-subunit, stably assembled with the Na ϩ ,K ϩ -ATPase ␣-subunit to exhibit the Na ϩ ,K ϩ -ATPase activity in Xenopus oocyte system (22). Furthermore, K ϩ concentration dependence of the Na ϩ ,K ϩ -ATPase activity was similar between the ␣-HsN complex and the wild-type Na ϩ ,K ϩ -ATPase (22). A combination of these and our present results suggests that the cytoplasmic domains of the ␤-subunits are compatible between H ϩ ,K ϩ -ATPase and Na ϩ ,K ϩ -ATPase to form functional ATPases, although amino acid identity of the cytoplasmic domains is around 30% between these ␤-subunits. The roles of the cytoplasmic domain of Na ϩ ,K ϩ -ATPase ␤-subunit are controversial. Renaud et al. (38) showed that the cytoplasmic domain of Na ϩ ,K ϩ -ATPase ␤-subunit was not directly involved in the ␣␤ assembly in Na ϩ ,K ϩ -ATPase by using mutants that lack the cytoplasmic domain of the ␤-subunit. Hasler et al. (39) reported that a mutant Na ϩ ,K ϩ -ATPase ␤-subunit that lacked the whole cytoplasmic domain assembled with the Na ϩ ,K ϩ -ATPase ␣-subunit and exhibited Na ϩ ,K ϩ pump activity. On the other hand, Jaunin et al. (18) and Eakle et al. (19) reported that the cytoplasmic domain was important for the efficient assembly and stability of the ␣-␤ complex by using chimeric ␤-subunits between H ϩ ,K ϩ -ATPase and Na ϩ ,K ϩ -ATPase. In the present study, we did not observe any significant difference in the expression levels of the ␣and ␤-subunits and ␣-␤ assembly between the ␣-NsH complex and the wild-type H ϩ ,K ϩ -ATPase ␣-␤ complex. Our results can be explained in two ways. One is that the cytoplasmic domain is not important for the functional assembly of the ␣-␤ complex. The other is that the role of the cytoplasmic domain of H ϩ ,K ϩ -ATPase ␤-subunit was similar to that of Na ϩ ,K ϩ -ATPase ␤-subunit; therefore, this domain is compatible between H ϩ ,K ϩ -ATPase and Na ϩ ,K ϩ -ATPase despite the low amino acid homology. It should be pointed out that the ␣-NsH complex showed a significantly (40%) higher K ϩ -ATPase activity than the wild-type H ϩ ,K ϩ -ATPase ␣-␤ complex. Because there was no difference in the affinity for K ϩ and the apparent expression level of H ϩ ,K ϩ -ATPase ␣-subunit on the blot between ␣-NsH complex and the wild-type H ϩ ,K ϩ -ATPase ␣-␤ complex, the NsH chimeric ␤-subunit may stabilize the H ϩ ,K ϩ -ATPase ␣-subunit in the membrane more efficiently than the wild-type ␤-subunit, suggesting that the cytoplasmic domain has an assisting role.
A chimeric ␤-subunit (NNH), which consists of the cytoplasmic plus transmembrane domains of Na ϩ ,K ϩ -ATPase and the ectodomain of H ϩ ,K ϩ -ATPase, also formed a stable complex with the H ϩ ,K ϩ -ATPase ␣-subunit to exhibit the H ϩ ,K ϩ -ATPase activity (Figs. 5, 6, and 8). Therefore, the whole transmembrane domain of the ␤-subunit is also compatible between Na ϩ ,K ϩ -ATPase and H ϩ ,K ϩ -ATPase to form a functional H ϩ ,K ϩ -ATPase. However, the stabilization of the ␣-subunit by the ␤-subunit, and H ϩ ,K ϩ -ATPase activity were abolished when the sequence 76 QLKS 79 in the ectodomain of H ϩ ,K ϩ -ATPase ␤-subunit was replaced by the counterpart, 72 RVAP 75 , of Na ϩ ,K ϩ -ATPase ␤-subunit. Therefore, this portion is the first N-terminal amino acid block that is not conserved between the H ϩ ,K ϩ -ATPase and Na ϩ ,K ϩ -ATPase ␤-subunits and that discriminates between the H ϩ ,K ϩ -ATPase and Na ϩ ,K ϩ -ATPase. It is also noteworthy that this portion was modified when restriction sites were introduced in the preparation of chimeras between the H ϩ ,K ϩ -ATPase and Na ϩ ,K ϩ -ATPase ␤-subunits in the previous study done by Jaunin et al. (18).
When the parts of the whole ectodomain of H ϩ ,K ϩ -ATPase ␤-subunit were replaced by the counterparts of Na ϩ ,K ϩ -ATPase ␤-subunit in this study, chimeric ␤-subunits such as HsNvH, HvNmH, and HmNpH did not stabilize the ␣-subunit in the membrane, resulting in loss of the H ϩ ,K ϩ -ATPase activity. However, one small extracellular segment located between Cys 152 (SphI site) and Cys 178 (EcoT22I site) was replaceable. This segment contains the second S-S loop (Cys 162 and Cys 178 ). The amino acid identity of this segment is around 53% between H ϩ ,K ϩ -ATPase and Na ϩ ,K ϩ -ATPase, higher than the overall amino acid identity. It is likely that this segment is compatible between the H ϩ ,K ϩ -ATPase and Na ϩ ,K ϩ -ATPase because the structure of this segment is relatively conserved between two ATPases, although it is not completely excluded that this segment is not directly involved in the stabilization of the ␣-subunit or the function of the enzyme.
Hamrick et al. (40) prepared chimeric proteins between the Na ϩ ,K ϩ -ATPase ␤-subunit and dipeptidyl peptidase IV and found that the ectodomain of the Na ϩ ,K ϩ -ATPase ␤-subunit was sufficient for assembly with the Na ϩ ,K ϩ -ATPase ␣-subunit. They also prepared deletion mutants that lack extracellular C-terminal portions of the Na ϩ ,K ϩ -ATPase ␤-subunit and reported that deletions of up to 146 extracellular amino acids from the C terminus of the ␤-subunit allow reduced assembly with the Na ϩ ,K ϩ -ATPase ␣-subunit. This deletion mutant had the first S-S loop but lacked the second and third S-S loops, and the C terminus was Asn 159 , which was close to SphI site used in the present work. Recently, Colonna et al. (41) reported that the segment from Glu 63 (Pro 68 in the present work) to Asp 125 (Asn 130 , MunI site in the present work) of the Na ϩ ,K ϩ -ATPase ␤-subunit was critical in ␣␤ assembly of the Na ϩ ,K ϩ -ATPase using two-hybrid assay system in yeast. It is noteworthy that the tryptic cleavage site between Arg 134 and Gly 135 (located on the first disulfide loop) of the ectodomain of Na ϩ ,K ϩ -ATPase ␤-subunit was hidden in the presence of Rb ϩ and exposed in the presence of Mg 2ϩ /P i , suggesting that this region is close to the site of interaction between ␣/␤-subunits and the K ϩ binding pocket (42). The sequence (Arg-Gly) was not conserved in H ϩ ,K ϩ -ATPase ␤-subunit. Melle-Milovanovic et al. (43) identified two different segments in the ectodomain of H ϩ ,K ϩ -ATPase ␤-subunit, from Glu 64 to Asn 130 and from Ala 156 to Arg 188 , as possibly associated with the ␣-subunit from the yeast two-hybrid analysis. The former segment includes 72 RVAP 75 sequence reported in the present work, and the latter partly overlaps the segment found in this study to be replaceable between the H ϩ ,K ϩ -ATPase and Na ϩ ,K ϩ -ATPase ␤-subunits. Fig. 10 shows the segments in H ϩ ,K ϩ -ATPase ␤-subunit replaceable with those of Na ϩ ,K ϩ -ATPase ␤-subunit.
Recently, rat colonic H ϩ ,K ϩ -ATPase cRNA and guinea pig colonic H ϩ ,K ϩ -ATPase cDNA were functionally expressed in Xenopus oocyte (44) and HEK-293 cells (17), respectively. In these studies, 86 Rb uptake and K ϩ -ATPase activity were observed under co-expression of the colonic H ϩ ,K ϩ -ATPase catalytic subunit (␣-subunit) with either Na ϩ ,K ϩ -ATPase ␤-subunit or gastric H ϩ ,K ϩ -ATPase ␤-subunit. It is of interest that both rat and guinea pig colonic H ϩ ,K ϩ -ATPase ␣-subunits assembled with either ␤-subunit to functionally express the ATPase.
In conclusion, we have shown that the whole cytoplasmic and transmembrane domains of H ϩ ,K ϩ -ATPase ␤-subunit can be replaced by those of Na ϩ ,K ϩ -ATPase ␤-subunit, retaining the ␣␤ assembly capacity, H ϩ ,K ϩ -ATPase activity and affinity for K ϩ . The ATPase activity was almost abolished when 4 amino acids ( 76 QLKS 79 ) in the early N-terminal part of the ectodomain of H ϩ ,K ϩ -ATPase ␤-subunit were replaced by the counterpart ( 72 RVAP 75 ) of Na ϩ ,K ϩ -ATPase ␤-subunit. The segment between Cys 152 and Cys 178 of H ϩ ,K ϩ -ATPase ␤-subunit was also replaceable with the corresponding segment of Na ϩ ,K ϩ -ATPase ␤-subunit, preserving ␣␤ assembly and H ϩ ,K ϩ -ATPase activity almost intact.