Colonic H-K-ATPase alpha - and beta -subunits express ouabain-insensitive H-K-ATPase

Pitchai Sangan, Sundararajah Thevananther, Sheela Sangan, Vazhaikkurichi M. Rajendran, and Henry J. Binder

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Active K absorption in the rat distal colon is energized by an apical H-K-ATPase, a member of the gene family of P-type ATPases. The H-K-ATPase alpha -subunit (HKcalpha ) has been cloned and characterized (together with the beta -subunit of either Na-K-ATPase or gastric H-K-ATPase) in Xenopus oocytes as ouabain-sensitive 86Rb uptake. In contrast, HKcalpha , when expressed in Sf9 cells without a beta -subunit, yielded evidence of ouabain-insensitive H-K-ATPase. Because a beta -subunit (HKcbeta ) has recently been cloned from rat colon, this present study was initiated to determine whether H-K-ATPase and its sensitivity to ouabain are expressed when these two subunits (HKcalpha and HKcbeta ) are transfected into a mammalian cell expression system. Transfection of HEK-293 cells with HKcalpha and HKcbeta cDNAs resulted in the expression of HKcalpha and HKcbeta proteins and their delivery to plasma membranes. H-K-ATPase activity was identified in crude plasma membranes prepared from transfected cells and was 1) saturable as a function of increasing K concentration with a Km for K of 0.63 mM; 2) inhibited by orthovanadate; and 3) insensitive to both ouabain and Sch-28080. In parallel transfection studies with HKcalpha and Na-K-ATPase beta 1 cDNAs and with HKcalpha cDNA alone, there was expression of ouabain-insensitive H-K-ATPase activity that was 60% and 21% of that in HKcalpha /HKcbeta cDNA transfected cells, respectively. Ouabain-insensitive 86Rb uptake was also identified in cells transfected with HKcalpha and HKcbeta cDNAs. These studies establish that HKcalpha cDNA with HKcbeta cDNA express ouabain-insensitive H-K-ATPase similar to that identified in rat distal colon.

potassium absorption; epithelial transport; rat colon; rubidium-86 uptake; hydrogen-potassium-adenosinetriphosphatase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ACTIVE POTASSIUM ABSORPTION in mammalian distal colon is mediated by one or more H-K-ATPases of the P-type gene family of ion transport ATPases (2, 10, 16, 30). P2-type ATPases are heterodimers that consist of alpha - and beta -subunits, whereas P1-type ATPases only consist of alpha -subunits. Colonic H-K-ATPase alpha -subunit (HKcalpha ) (8) is localized in apical membranes of surface epithelial cells (15, 24), and its mRNA and protein expression are increased threefold in the distal colons of dietary Na-depleted rats (24). Functional expression of HKcalpha cDNA without beta -subunit in Sf9 cells revealed H-K-ATPase activity that was insensitive to ouabain (18). In contrast, HKcalpha cRNA when coexpressed with the beta -subunit of Na-K-ATPase (NaKbeta 1) or gastric H-K-ATPase (HKgbeta ) either in Xenopus oocytes or in human embryonic kidney 293 cells (HEK-293 cells) demonstrated ouabain-sensitive 86Rb uptake (5, 7). Neither of these studies reported the expression of K-dependent ATP hydrolysis (i.e., H-K-ATPase activity) (5, 7).

Recently, we have isolated and identified a beta -subunit from rat distal colon that we refer to as HKcbeta and that is expressed in both apical and basolateral membranes of rat distal colon (23). We have also demonstrated by coimmunoprecipitation the physical association of this HKcbeta protein with HKcalpha protein and its upregulation in apical membrane by dietary K depletion (23). As a result, we proposed that this is the beta -subunit for the colonic H-K-ATPase. This present study was, therefore, designed to determine whether HKcalpha , when coexpressed with HKcbeta cDNA in mammalian cells, exhibited ATPase activity. In this present communication, we report that both HKcbeta and NaKbeta 1 proteins formed a functional enzyme complex with HKcalpha protein in HEK-293 cells and yielded the expression of both ouabain-insensitive H-K-ATPase activity and 86Rb uptake.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

HEK-293 cells were a gift from Dr. B. Forbush (Yale University). pcDNA 3.1+ vector was purchased from Invitrogen (Carlsbad, CA); restriction enzymes were from New England Biolabs (Beverly, MA). All other reagents were of molecular biology or analytical grade.

Plasmid construction. The full-length cDNAs encoding the rat colonic HKcalpha (8), the colonic HKcbeta (23), and the rat NaKbeta 1 (20) were modified by PCR both at the 5'end and the 3'end with BamH I and EcoR I, respectively. The HKcalpha , HKcbeta , and NaKbeta 1 cDNAs were digested separately with BamH I and EcoR I enzymes and ligated using T4 DNA ligase independently into pcDNA 3.1+ that had been previously digested with BamH I and EcoR I and dephosphorylated with calf intestinal phosphatase. The ligation mixture was transformed into XL1-blue Escherichia coli cells, and the plasmid DNAs were prepared according to the method described by Morelle (21). The plasmid DNAs were digested with BamH I and EcoR I enzymes to release the insert, and the plasmids containing expected size inserts were sequenced by an automated fluorescence sequencer (William Keck Sequencing Facility, Yale University). Plasmids containing full-length cDNAs (hereafter referred to as pHKcalpha , pHKcbeta , and pNaKbeta 1) with the correct sequences were used for the expression studies.

Cell culture and transfection. COS-7 cells were grown (13) in high-glucose DMEM containing 10% fetal bovine serum, 50 U/ml penicillin, 50 µg/ml streptomycin, and 2 mM L-glutamine (complete DMEM). Transfections were carried out in subconfluent COS-7 cells with vector alone, pHKcalpha alone, pHKcbeta alone, pNaKbeta 1 alone, pHKcalpha /pHKcbeta , or pHKcalpha /pNaKbeta 1 in independent transfections using superfect transfection reagent (Qiagen, Chatsworth, CA) according to the manufacturer's recommendations. In brief, plasmid DNA was mixed with serum, antibiotic-free DMEM, and the recommended amount of superfect reagent and incubated at room temperature for 30 min. After incubation, 2 ml of serum and antibiotic-free DMEM were added. The entire mixture was added to the cells that previously had been washed three times with serum and antibiotic-free medium. The plates were incubated at 37°C in a humidified CO2 incubator for 2 h. The medium was removed and washed with 5 ml medium. Finally, 6 ml of medium were added, and the cells were placed in the CO2 incubator at 37°C for 60 h. After 60 h, the medium was removed, the cells were washed with 1× PBS three times, 1× PBS was added, and the cells were scraped and harvested by centrifugation.

HEK-293 cells were grown (13) and the transfections were performed similar to that for COS-7 cells, with the exception that the cells were grown in low-glucose DMEM. To prepare stable cell lines, after 60 h of transfections, the cells were split in serial dilution (1:10, 1:50, 1:250) in the complete DMEM containing G418 (900 µg/ml; GIBCO BRL, Gaithersburg, MD). Fresh complete DMEM containing G418 was replaced every 72 h. Six weeks after transfections, colonies grown in the presence of G418 (32 colonies for each transfection) were screened for the expression of HKcalpha protein, HKcbeta protein, or NaKbeta 1 protein in cells transfected with pHKcalpha alone, pHKcbeta alone, pHKcalpha /pHKcbeta , and pHKcalpha /pNaKbeta 1 by Western blot analysis with their respective antibodies.

Plasma membrane preparation. Cells were harvested and washed twice with 1× PBS. Cells were then resuspended in sonication buffer containing 50 mM Tris · HCl (pH 7.4), 1 mM EDTA, 250 mM sucrose, and 1 mM phenylmethylsufonyl fluoride. The cells were then sonicated for 15 s each time (3 times) with 30-s intervals between them. The lysate was centrifuged at 30,000 g for 30 min at 4°C (Sorvall RC5B, SS34 rotor), and the membrane pellet was resuspended in a buffer containing 50 mM Tris · HCl (pH 7.4) and 250 mM sucrose. These crude plasma membranes were used for enzyme assay and Western blot analysis. Protein was estimated by the Bradford method using bovine gamma -globulin as standard (3).

Assay of ATPases. H-K-ATPase activity was assayed in 0.5 ml of reaction volume containing 50 µg of membrane protein, 40 mM Tris · HCl (pH 7.4), 3 mM MgCl2, and 10 µM ouabain in the presence and absence of 5 mM KCl. The reaction was initiated by the addition of 3 mM ATP (Tris salt) and incubated at 37°C for 1 h; inorganic phosphate released was measured, as described previously (9). H-K-ATPase activity was calculated as the difference between activities in the presence and absence of KCl. Na-K-ATPase activity was also assayed using the above reaction condition but also included 100 mM NaCl and was calculated as the difference between activities in the presence and absence of Na. The specific activity of the enzyme is expressed as nanomoles of phosphorus liberated per milligram of protein per minute.

SDS-PAGE and Western blot analysis. SDS-PAGE was carried out, as previously described (17). Fifty micrograms of membrane protein were incubated in a sample buffer containing 10 mM Tris · HCl (pH 6.8), 2% SDS, 2% mercaptoethanol, and 10% glycerol at room temperature for 5 min and loaded on an SDS-polyacrylamide gel. Western blot analyses were performed using HKcalpha , HKcbeta , and NaKbeta 1 antibodies, as previously described (23).

Immunofluorescence studies. Stably transfected HEK-293 cells that express HKcalpha protein alone and express both HKcalpha and HKcbeta proteins were grown on 22 × 22 mm glass coverslips for 48 h. The cells were washed with 1× PBS, fixed for 20 min in 4% paraformaldehyde prepared in 1× PBS, and processed as described previously (12). The fixed cells were incubated with appropriate antibodies for 1 h at room temperature at a dilution of 1:100. CY3-conjugated anti-rabbit IgG (Amersham) secondary antibodies were used at a dilution of 1:2,000 at room temperature for 1 h. Immunofluorescence images were visualized using a Zeiss-Axiophot microscope.

86Rb uptake studies. Tissue culture plates (24 wells) were treated with 0.1% (wt/vol) poly-L-lysine (200 µl/well) for 5 min in a tissue culture Laminar flow hood. The plates were then washed with sterilized deionized water and were allowed to dry for 5 min. For the untransfected cells, 1 × 105 cells/well were plated and grown in growth media (low-glucose DMEM containing 10% fetal bovine serum and 50 units penicillin/streptomycin solution). The transfected cell lines were grown in the same growth media but with the addition of 900 µg/ml G418. The cells grew at a slower rate but did not wash off during washing while performing 86Rb uptake studies in the poly-L-lysine-treated plates compared with those grown in the untreated plates.

The cells were grown for 4-5 days and fresh media were changed every 48 h. After the cells had grown to complete confluency (about 5 days), the media were aspirated and the cells were washed six times with 500 µl uptake buffer containing 145 mM NaCl, 1 mM KCl, 10 mM glucose, 1.2 mM MgCl2, 1.0 mM CaCl2, 2 mM NaH2PO4, 32 mM HEPES, and 200 µM bumetanide, pH 7.4. Bumetanide was added to block any 86Rb uptake by Na-K-2Cl cotransporter. Preliminary studies had shown that 86Rb uptake was linear for up to 30 min in both the untransfected and the transfected cell lines; therefore, all uptake studies were performed for 10 min. Additional preliminary studies in the untransfected cell line demonstrated that 10 µM ouabain reduced 86Rb uptake by ~95%, which was attributed to uptake via Na-K-ATPase; therefore, all studies were performed in the presence of 10 µM ouabain. The cells were initially incubated for 20 min at 37°C in uptake buffer solution. The uptake buffer solution was then replaced by 200 µl uptake buffer containing 86Rb (4 µCi/ml) in the presence or absence of either 1 mM ouabain or 100 µM vanadate, and the cells were incubated for 10 min at 37°C. After incubation and removal of buffer, the cells were washed six times with 500 µl ice-cold stop buffer (10 mM HEPES-Tris, 100 mM MgCl2, pH 7.4). The cells were solubilized in 500 µl of 2% SDS/0.1 N NaOH, and 10 µl of the solubilized cell lysate were used to estimate the protein concentration by the method of Bradford (3). The remaining cell lysate was mixed with 3.5 ml scintillation solution, and radioactivity was measured in a scintillation photometer. 86Rb uptake was expressed as nanomoles per milligrams of protein per 10 min.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

To establish that all the constructs expressed the expected size proteins, we first transiently transfected COS-7 cells using the plasmid containing full-length HKcalpha , HKcbeta , and NaKbeta 1 cDNAs as either individual plasmid constructs (pHKcalpha , pHKcbeta , pNaKbeta 1) or as a combination of two plasmids (pHKcalpha /pHKcbeta or pHKcalpha /pNaKbeta 1). The results of these expression studies revealed that untransfected COS-7 cells or the cells transfected with vector alone did not express either HKcalpha or HKcbeta proteins (Fig. 1, A and B, lanes 1-3). Cells transfected with pHKcalpha alone, pHKcalpha /pHKcbeta , or pHKcalpha /pNaKbeta 1 expressed HKcalpha protein, as detected by HKcalpha antibody (Fig. 1A, lanes 4, 7, and 8, respectively). Cells transfected with pHKcbeta alone or pHKcalpha /pHKcbeta expressed HKcbeta protein, as detected by HKcbeta antibody (Fig. 1B, lanes 5 and 7). Cotransfection of pHKcalpha /pHKcbeta resulted in the expression of less HKcalpha protein compared with that identified in cells transfected with pHKcalpha alone. In contrast, HKcbeta protein was expressed in higher amounts than HKcalpha protein whether pHKcbeta was transfected alone or cotransfected with pHKcalpha . Several attempts were made to alter the ratio of the alpha /beta constructs for transfections to result in the expression of approximately equal amounts of HKcalpha and HKcbeta proteins. None of these approaches resulted in the expression of H-K-ATPase activity in cell membranes from these transfected cells. Therefore, we proceeded to establish stable cell lines that would express HKcalpha and HKcbeta protein in approximately equal amounts.


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Fig. 1.   Expression of H-K-ATPase alpha -subunit (HKcalpha ) and H-K-ATPase beta -subunit (HKcbeta ) protein in untransfected and transfected COS-7 cells. A: HKcalpha antibody was used for Western blot analysis with crude plasma membranes prepared from untransfected cells (lane 1), vector pcDNA 3.1+ (1 µg) transfected cells (lane 2), vector pcDNA 3.1+ (2 µg) transfected cells (lane 3), pHKcalpha transfected cells (lane 4), pHKcbeta transfected cells (lane 5), pNaKbeta 1 transfected cells (lane 6), pHKcalpha /pHKcbeta transfected cells (lane 7), and pHKcalpha /pNaKbeta 1 transfected cells (lane 8). Arrow, HKcalpha protein detected by HKcalpha -specific antibody. B: Western blot analysis using HKcbeta antibody. Lane description is same as that for A. Arrow, HKcbeta protein detected by HKcbeta -specific antibody.

Several stable HEK-293 cell lines were developed after transfection with the following cDNAs: 1) both HKcalpha and HKcbeta , 2) HKcalpha and NaKbeta 1, 3) HKcalpha alone, and 4) HKcbeta alone. Western blot analyses were performed using the crude membranes from the stable cell lines (Fig. 2). The untransfected HEK-293 cells did not express either HKcalpha protein or HKcbeta protein. Cell lines stably transfected with pHKcalpha alone, pHKcalpha /pHKcbeta , or pHKcalpha /pNaKbeta 1 expressed HKcalpha protein, as detected by HKcalpha antibody (Fig. 2A, lanes 3, 5, and 6, respectively). Cell lines stably transfected with pHKcbeta alone or with pHKcalpha /pHKcbeta expressed HKcbeta protein, as detected by HKcbeta antibody (Fig. 2B, lanes 4 and 5), whereas those cell lines transfected with pHKcalpha /pNaKbeta 1 expressed NaKbeta 1 as protein, as detected by NaKbeta 1 antibody (Fig. 2C, lane 6).


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Fig. 2.   Expression of HKcalpha and HKcbeta proteins from untransfected and transfected HEK-293 cells. A: HKcalpha antibody was used for Western blot analysis with crude plasma membranes prepared from untransfected cells (lane 1), vector pcDNA 3.1+ transiently transfected cells (lane 2), pHKcalpha stably transfected cells (lane 3), pHKcbeta stably transfected cells (lane 4), pHKcalpha /pHKcbeta stably transfected cells (lane 5), and pHKcalpha /pNaKbeta 1 stably transfected cells (lane 6). Arrowhead, HKcalpha protein detected by HKcalpha antibody. B: Western blot analysis using HKcbeta antibody. Lane description is same as that for A. Arrowhead, HKcbeta protein detected by HKcbeta antibody. C: Western blot analysis using Na-K-ATPase beta 1-subunit (NaKbeta 1) antibody. Lane description is same as that for A. Arrowhead, NaKbeta 1 protein detected by NaKbeta 1 antibody.

To establish whether the expressed HKcalpha or HKcbeta proteins were transported to plasma membranes, immunofluorescence studies were performed. The results of such immunofluorescence studies are presented in Fig. 3. The stable cell line expressing both HKcalpha and HKcbeta proteins stained with HKcalpha antibody (Fig. 3C) and stained with HKcbeta antibody (Fig. 3D) demonstrate that both HKcalpha and HKcbeta proteins are transported to and localized in the plasma membranes. Staining (Fig. 3A) was not identified in the same stable cell line expressing both HKcalpha and HKcbeta proteins when stained with preimmune serum, indicating that the staining produced by HKcalpha and HKcbeta antibodies is specific (Fig. 3A). HKcalpha protein in the pHKcalpha alone stably transfected cell line was not efficiently localized to the plasma membrane (Fig. 3B). Therefore, HKcalpha protein requires a beta -subunit to be transported and localized in the plasma membrane.


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Fig. 3.   Immunofluorescence studies of stable HEK-293 cell lines with HKcalpha and HKcbeta antibodies. A: stable HEK-293 cell lines expressing HKcalpha and HKcbeta proteins stained with preimmune serum. B: stable HEK-293 cell lines expressing HKcalpha protein stained with HKcalpha antibody. C: stable HEK-293 cell lines expressing HKcalpha and HKcbeta proteins stained with HKcalpha antibody. D: stable HEK-293 cell lines expressing HKcalpha and HKcbeta proteins stained with HKcbeta antibody. CY3-conjugated anti-rabbit IgG was used as secondary antibody and slides were visualized and photographed in a fluorescent microscope (×400 magnification).

ATPase activity was determined in the untransfected HEK-293 cell membranes before establishing the assay conditions for the transfected cells. Only Na-K-ATPase activity was identified in the untransfected HEK-293 cell membranes (15.8 ± 1.3 nmol Pi liberated · mg protein-1 · min-1). This endogenous Na-K-ATPase activity was completely inhibited by 10 µM ouabain (data not presented). Similar levels of Na-K-ATPase activities (16.2 ± 0.9 and 16.1 ± 0.8 nmol Pi liberated · mg protein-1 · min-1) were identified in the two doubly transfected cell lines, pHKcalpha /pHKcbeta and pHKcalpha /NaKbeta 1, respectively. Although Na-K-ATPase activity was present endogenously in the untransfected cell line, increased levels of Na-K-ATPase were not expressed in the cell lines transfected with both alpha - and beta -subunits. Thus all subsequent assays included 10 µM ouabain in the ATPase assay in membranes from transfected cells.

To determine whether H-K-ATPase is expressed in the stable cell lines, H-K-ATPase assay was determined in membranes prepared from the different cell lines. The maximal H-K-ATPase activity was observed in the pHKcalpha /pHKcbeta stably transfected cell line (Fig. 4). In contrast, H-K-ATPase activity in the pHKcalpha /pNaKbeta 1 stably transfected cell line was 60% of that in the pHKcalpha /pHKcbeta stably transfected cell line. Cells that had been stably transfected with pHKcalpha alone expressed H-K-ATPase activity that was 21% of maximal activity. Minimal H-K-ATPase activity was noted both in cells that had been stably transfected with pHKcbeta alone and in the untransfected cell lines.


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Fig. 4.   H-K-ATPase activities expressed in crude membranes prepared from several different transfected HEK-293 cell lines. Cell lines included untransfected HEK-293 cells and stable cell lines after transfection with pHKcalpha alone, pHKcbeta alone, pHKcalpha /pHKcbeta , and pHKcalpha /pNaKbeta 1. H-K-ATPase activity is presented as the difference in activity between that in the presence of and absence of K in the assay mixture, which contained 10 µM ouabain to eliminate endogenous Na-K-ATPase activity. Values are means ± SE of 3 experiments.

The effect of K+ concentrations on the H-K-ATPase activity in the crude plasma membranes prepared from the cell line stably transfected with pHKcalpha /pHKcbeta was also studied to determine whether the in vitro expressed H-K-ATPAse activity in plasma membranes of HEK-293 cells had similar or different properties from those of H-K-ATPase activity in native colonic apical membranes. Increasing K concentrations in the incubation medium stimulated and saturated H-K-ATPase activity (Fig. 5). Analysis of this data with a Linweaver-Burk plot yielded a Km for K of 0.63 mM. This kinetic constant is comparable to those previously reported for H-K-ATPase in native rat colonic apical membranes (Km = 0.75 mM) (9), the rat colonic H-K-ATPase expressed as 86Rb uptake in Xenopus oocytes (Km = 0.73 mM) (7), and the Km calculated for rat H-K-ATPase alpha -subunit expressed in Sf9 cells (Km = 1.2 mM) (18).


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Fig. 5.   Effect of K concentrations. H-K-ATPase activity was measured in presence of different K concentrations (0.25-20 mM) in crude membranes prepared from stable cell line expressing HKcalpha and HKcbeta proteins. Typical values presented represent means of triplicate assays from 3 different membrane preparations. The best-fit curve was drawn using the Michaelis-Menten equation.

To establish the functional properties of the H-K-ATPase activity identified in membranes from cell lines stably transfected with pHKcalpha /pHKcbeta or pHKcalpha /pNaKbeta 1, the effect of different ATPase inhibitors on H-K-ATPase activity was determined. One millimolar orthovanadate inhibited H-K-ATPase activity by ~75%. In contrast, neither 1 mM ouabain nor 0.5 mM Sch-28080 altered H-K-ATPase activity (Fig. 6).


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Fig. 6.   Effect of different ATPase inhibitors on H-K-ATPase activity expressed in HEK-293 cells. H-K-ATPase activity was determined in crude membranes prepared from HEK-293 cells that had been stably transfected with HKcalpha and HKcbeta cDNAs or with HKcalpha and NaKbeta 1 cDNAs. Membranes were prepared from either HKcalpha and HKcbeta transfected cell lines labeled as pHKcalpha /pHKcbeta or from HKcalpha and NaKbeta 1 transfected cell lines labeled as pHKcalpha /pNaKbeta 1. H-K-ATPase activity was measured in the absence and presence of 1 mM vanadate, 1 mM ouabain, and 0.5 µM Sch-28080. All assay mixtures also contained 10 µM ouabain to eliminate endogenous Na-K-ATPase activity. Values are means ± SE of 3 experiments.

86Rb uptake was performed in untransfected, in pHKcalpha /pHKcbeta transfected, and in pHKcalpha /pNaKbeta 1 transfected HEK-293 cell lines (Fig. 7.) Preliminary studies had shown that 86Rb uptake was linear for up to 30 min in both the untransfected and the transfected cell lines; therefore, all uptake studies were performed for 10 min. Additional preliminary studies in the untransfected cell line demonstrated that 10 µM ouabain reduced 86Rb uptake by ~95%, which was attributed to uptake via endogenous Na-K-ATPase and is consistent with the studies of endogenous Na-K-ATPase in this cell line. As a result, all 86Rb studies were performed in the presence of 10 µM ouabain.


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Fig. 7.   86Rb uptake in untransfected and the 2 doubly transfected HEK-293 cell lines. 86Rb uptake was performed in untransfected and in pHKcalpha /pHKcbeta and pHKcalpha /pNaKbeta 1 transfected cell lines, as described in MATERIALS AND METHODS. All uptake studies were performed in the presence of 10 µM ouabain and in the presence or absence of 100 µM vanadate or 1 mM ouabain. Values are means ± SE of 3 experiments.

The results of these 86Rb uptake studies demonstrate that 86Rb uptake in pHKcalpha /pHKcbeta transfected cells was 66-fold greater than that in untransfected cells (118.8 ± 4.0 vs. 1.8 ± 0.3 nmol · mg protein-1 · 10 min-1). 86Rb uptake in pHKcalpha /pNaKbeta 1 transfected cells was also substantially greater (52-fold) than that in untransfected cells (Fig. 7). Similar to the observations of H-K-ATPase in the doubly transfected cell lines, uptake in pHKcalpha /pHKcbeta was 26% greater than that in the pHKcalpha /pNaKbeta 1 cell line. One hundred micromolar vanadate markedly inhibited 86Rb uptake in both doubly transfected cell lines by ~90%, indicating that 86Rb uptake is mediated by an ATPase. In contrast, 1 mM ouabain did not significantly alter 86Rb uptake, establishing that 86Rb uptake in both doubly transfected cell lines is ouabain insensitive. These data establish that HKcalpha , when coexpressed with a beta -subunit, expresses a ouabain-insensitive H-K-ATPase function.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In addition to the kidney, the mammalian large intestine contributes to the regulation of overall K balance via both absorptive and secretory processes (11, 27, 28). Active K absorption that is energized by one (or more) apical membrane H-K-ATPases has been identified in the distal colon of rat, guinea pig, and rabbit and has been the focus of several investigations during the past decade (1, 9, 26, 29). HKcalpha has been cloned and is a member of the gene family of P-type ATPases (8). HKcalpha mRNA and protein are present exclusively in surface (and ~20% of the upper crypt) epithelial cells, and its enzymatic activity is localized to the apical membrane (15, 18, 24). Considerable controversy exists regarding whether this HKcalpha cDNA encodes a ouabain-sensitive or ouabain-insensitive H-K-ATPase and whether a colon-specific beta -subunit is required for maximal function of the colonic H-K-ATPase (5, 7, 18).

Studies of the coexpression of HKcalpha with either HKgbeta or NaKbeta 1 cRNAs in Xenopus oocytes yielded evidence of ouabain-sensitive 86Rb uptake that was consistent with H-K-ATPase function (5, 7). In contrast, expression of HKcalpha cDNA in Sf9 cells without any exogenous beta -subunit resulted in ouabain-insensitive H-K-ATPase activity (18). It should be noted that in these present studies with HEK-293 cells, H-K-ATPase activity in cells stably transfected with pHKcalpha alone (Fig. 4) was 21% of its activity in cells stably transfected with pHKcalpha /pHKcbeta . It is not known whether the H-K-ATPase activity in the absence of a transfected beta -subunit reflects the presence of an endogenous beta -subunit in both Sf9 and HEK-293 cells or the ability of HKcalpha protein to manifest partial H-K-ATPase activity in the absence of any beta -subunit.

Recent studies have established the distribution of H-K-ATPase activity in apical membranes of both surface and crypt cells (22). Although H-K-ATPase activity in surface cell apical membranes was both ouabain sensitive and ouabain insensitive, H-K-ATPase activity in apical membranes of crypt cells was exclusively ouabain sensitive. As previously noted, HKcalpha mRNA and protein are primarily present in surface and not in crypt cells (15, 18, 24). Therefore, if HKcalpha cDNA encodes a ouabain-sensitive H-K-ATPase, it would be necessary to postulate the presence of three distinct H-K-ATPases in the rat distal colon, two that are ouabain sensitive and one that is ouabain insensitive. Alternatively, however, the presence of only two H-K-ATPases would be required if HKcalpha cDNA encoded the ouabain-insensitive H-K-ATPase. At the present time, there is evidence for at least two H-K-ATPase isoforms in the rat distal colon.

A beta -subunit, HKcbeta , was recently cloned and identified from rat colon (23). A closely related isoform has been designated by others as NaKbeta 3 (19). Although HKcbeta mRNA is present in high abundance in testis and lung, several lines of evidence have been presented that provide the basis for the suggestion that HKcbeta is the beta -subunit for the colonic H-K-ATPase (23): 1) HKcbeta protein is present in both apical and basolateral membranes of rat distal colon; 2) HKcbeta protein was coprecipitated with HKcalpha protein from apical membranes; 3) HKcbeta mRNA and its apical membrane protein were increased in distal colon of K-depleted rats (23). Thus it is likely that the HKcbeta is the beta -subunit for H-K-ATPase in native tissue. The present study sought to establish whether HKcbeta cDNA, when cotransfected with HKcalpha cDNA in a mammalian cell line, expressed both H-K-ATPase activity and 86Rb uptake that were inhibited by vanadate but not by ouabain.

These present studies were designed to express HKcalpha cDNA in a mammalian expression system in view of the conflicting observations previously reported in the expression of HKcalpha cDNA in nonmammalian cells. The initial studies with COS-7 cells did not result in the expression of H-K-ATPase activity but did provide important information that was critical in the design of the subsequent experiments with HEK-293 cells. Such cells had been successfully used for the expression of other P-type ATPases, e.g., ATP1AL1 (13).

Immunofluorescence studies confirmed the presence of both HKcalpha and HKcbeta proteins in plasma membranes (Fig. 3). H-K-ATPase activity was minimal in the nontransfected HEK-293 cells and those transfected with only the HKcbeta cDNA. In contrast, transfection of HKcalpha cDNA resulted in H-K-ATPase activity both in the absence or presence of one of the beta -subunits. Maximal H-K-ATPase activity was observed in those cells stably transfected with pHKcalpha /pHKcbeta and was 60% greater than that determined in the cells stably transfected with pHKcalpha /pNaKbeta 1 (Fig. 4). Because equal amounts of HKcalpha protein were present in the crude plasma membranes of pHKcalpha /pHKcbeta and pHKcalpha /pNaKbeta 1 transfected cell lines (Fig. 2A, lanes 5 and 6), the observed differences in H-K-ATPase activities in these doubly transfected cell lines cannot be attributed to differences in HKcalpha protein expression. Thus the higher rate of H-K-ATPase activity observed when HKcalpha cDNA was cotransfected with HKcbeta than with NaKbeta 1 is consistent with the recent suggestion (23) that HKcbeta is the beta -subunit for colonic H-K-ATPase.

HKcbeta protein is not the only beta -subunit to combine with HKcalpha protein and to manifest H-K-ATPase activity. Codina et al. (4) recently demonstrated that HKcalpha assembles with NaKbeta 1 protein in the rat distal colon and kidney medulla. In addition, in the present studies the coexpression of HKcalpha with NaKbeta 1 resulted in H-K-ATPase function, indicating that NaKbeta 1 protein could act as a surrogate beta -subunit for HKcalpha .

Our previous observations in Sf9 cells (18) are similar to the results shown in Fig. 4, in which H-K-ATPase activity was identified in the cell line stably transfected with HKcalpha cDNA alone. Although the experiment shown in Fig. 4 revealed evidence of H-K-ATPase activity in the absence of an obvious beta -subunit, the coexpression of HKcalpha cDNA with HKcbeta cDNA resulted in an almost fivefold higher level of H-K-ATPase expression. The expression of H-K-ATPase in the absence of a beta -subunit would be consistent either with an endogenous NaKbeta 1 in the HEK-293 cells functioning as a surrogate or promiscuous beta -subunit or with the presence of a previously unidentified beta -subunit in the HEK-293 cells.

These present results demonstrate that both the expressed H-K-ATPase activity and the expressed 86Rb uptake in the cell lines stably transfected with pHKcalpha /pHKcbeta or with pHKcalpha /pNaKbeta 1 are ouabain insensitive (Figs. 6 and 7) and conflict with prior observations in Xenopus oocytes that HKcalpha cDNA encodes a ouabain-sensitive function (5, 7). It should be noted that all prior demonstrations of the ouabain-sensitive HKcalpha cDNA expression used 86Rb uptake as the parameter of HKcalpha protein expression (5, 7). In contrast, these present studies directly determined both H-K-ATPase activity and 86Rb uptake as a surrogate marker of K uptake. An adequate explanation for this difference is not evident; however, it is possible that in the nonmammalian Xenopus oocyte expression system, HKcalpha cDNA activated an endogenous ouabain-sensitive function.

K-dependent proton secretion has been described in the guinea pig colon and has been attributed to an H-K-ATPase (26). Recently, a cDNA from guinea pig distal colon (25), which has significant (88%) sequence homology at the protein level to rat HKcalpha , was expressed in HEK-293 cells with Torpedo Na-K-ATPase beta -subunit cDNA and was associated with ouabain-sensitive H-K-ATPase activity. The rat and guinea pig colonic H-K-ATPase alpha -subunit cDNAs share significant sequence homology; however, it would be important to know whether the guinea pig H-K-ATPase alpha -subunit is present in surface or crypt cells. Physiological studies in the rat indicate the presence of two distinct H-K-ATPases, one that is ouabain sensitive and present in crypt cells and the other that is ouabain insensitive and present in surface cells (22). The encoded proteins from rat and guinea pig respond differently to ouabain and, therefore, these two proteins likely represent different H-K-ATPase isoforms.

Two nongastric H-K-ATPase (colonic H-K-ATPase and ATP1AL1) alpha -subunits with a beta -subunit have recently been shown to manifest Na/K exchange function (6, 14) in addition to their H/K exchange activity. Neither study assessed whether the expressed proteins had ATPase activity. Although this present study identified H-K-ATPase activity in the stably transfected HEK-293 cell lines, transfection with these alpha - and beta - subunits did not result in an increase of the expression of Na-K-ATPase activity above that in untransfected cells. It is possible that these different results reflect the different beta -subunits used in these studies.

In conclusion, this study demonstrated that HKcalpha and HKcbeta cDNAs, when expressed in a mammalian cell system, manifest ouabain-insensitive H-K-ATPase functions, both enzymatic ATPase activity and 86Rb uptake.


    ACKNOWLEDGEMENTS

We thank Dr. John P. Hayslett and Lawrence J. Macala for allowing us to use their tissue culture facility. HEK-293 cells were kindly provided by Dr. Bliss Forbush. NaKbeta 1 cDNA and NaKbeta 1 antibody were kindly provided by Dr. Michael Caplan.


    FOOTNOTES

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-18777-24.

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.

Address for reprint requests and other correspondence: H. J. Binder, Section of Digestive Diseases, Dept. of Internal Medicine, Yale Univ. School of Medicine, 333 Cedar St., 89 LMP, New Haven, CT 06520-8019 (E-mail: binder{at}biomed.med.yale.edu).

Received 23 March 1999; accepted in final form 20 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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