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J Biol Chem, Vol. 274, Issue 28, 19693-19698, July 9, 1999

The Colonic H⁺,K⁺-ATPase Functions as a Na⁺-dependent K⁺(NH₄⁺)-ATPase in Apical Membranes from Rat Distal Colon^*

Juan Codina, Thomas A. Pressley^§, and Thomas D. DuBose Jr.^¶

From the  Division of Renal Diseases and Hypertension Department of Internal Medicine, University of Texas, Houston Medical School, Houston, Texas 77030 and the ^§  Department of Physiology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Recent studies have suggested that the colonic H⁺,K⁺-ATPase (HK₂) can secrete either Na⁺ or H⁺ in exchange for K⁺. If correct, this view would indicate that the transporter could function as either a Na⁺ or a H⁺ pump. To investigate this possibility a series of experiments was performed using apical membranes from rat colon which were enriched in colonic H⁺,K⁺-ATPase protein. An antibody specific for HK₂ was employed to determine whether HK₂ functions under physiological conditions as a Na⁺-dependent or Na⁺-independent K⁺-ATPase in this same membrane fraction. K⁺-ATPase activity was measured as [-³²P]ATP hydrolysis. The Na⁺-dependent K⁺-ATPase accounted for approximately 80% of overall K⁺-ATPase activity and was characterized by insensitivity to Sch-28080 but partial sensitivity to ouabain. The Na⁺-independent K⁺-ATPase activity was insensitive to both Sch-28080 and ouabain. Both types of K⁺-ATPase activity substituted NH₄⁺ for K⁺ in a similar manner. Furthermore, our results demonstrate that when incubated with native distal colon membranes, the blocking antibody inhibited dramatically Na⁺-dependent K⁺-ATPase activity. Therefore, these data demonstrate that HK₂ can function in native distal colon apical membranes as a Na⁺-dependent K⁺-ATPase. Elucidation of the role of the pump as a transporter of Na⁺ versus H⁺ or NH₄⁺ versus K⁺ in vivo will require additional studies.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Both the distal colon and renal medulla participate importantly in K⁺ homeostasis (1). A unique H⁺,K⁺-ATPase (HK₂)¹ was cloned by Crowson and Shull (2) from a rat distal colon library, which was distinct at the amino acid level from both ₁-Na⁺,K⁺-ATPase (73% similarity) and from gastric H⁺,K⁺-ATPase (72% similarity). It has been demonstrated, using the Xenopus laevis oocyte as an expression system, that HK₂ can internalize Rb⁺ (K⁺) in exchange for H⁺ when coexpressed with any known X⁺,K⁺-ATPase -subunit (3-5). Furthermore, HK₂ was insensitive to Sch-28080, a specific inhibitor of the gastric H⁺,K⁺-ATPase, but partially sensitive to ouabain (IC₅₀ ~ 400-600 µM) (3, 4), a specific inhibitor of the Na⁺,K⁺-ATPase.

HK₂ mRNA and protein are expressed in low abundance in the renal medulla (2, 6, 7). However, HK₂ mRNA and protein abundance in the kidney are dramatically augmented by chronic dietary K⁺-depletion (6, 8-10). This finding indicates that HK₂ may play a major role in renal K⁺ conservation. Furthermore, the site of this regulatory response to chronic hypokalemia has been shown to be the renal medulla (10). Based on data obtained using heterologous expression systems it has been predicted that the increase in K⁺-reabsorption in the collecting tubule during chronic hypokalemia would be insensitive to Sch-28080 in vivo. Nevertheless, several laboratories which have evaluated HCO₃ reabsorption during chronic hypokalemia have defined H⁺,K⁺-ATPase function by its sensitivity to Sch-28080. For example, Wall et al. (11) and Nakamura et al. (12) have demonstrated that the increase in bicarbonate absorption (J_tCO2) observed in the medullary collecting duct during chronic hypokalemia is inhibited by Sch-28080 in low concentration (10 µM). Sensitivity to low concentrations of Sch-28080 is characteristic of the gastric H⁺,K⁺-ATPase (13). Nevertheless, it has been suggested that in the inner medullary collecting tubule HK₂ may become sensitive to low concentrations of Sch-28080 during chronic hypokalemia (12).

Cougnon et al. (14) demonstrated that HK₂ can also function to secrete Na⁺ in exchange for K⁺. These findings were supported by studies from Grishin and Caplan (15), which demonstrated that HEK-293 cells co-transfected with human ATP1AL1 (90% similar to HK₂) and the rabbit -subunit of the gastric H⁺,K⁺-ATPase, grow in the presence of ouabain. This observation suggests that in transfected cells, during inhibition of the native Na⁺ pump by ouabain, ATP1AL1 which is relatively insensitive to ouabain (16, 17), functions as a Na⁺ pump. A possible physiological role for the rat HK₂ or the human ATP1AL1, as apical Na⁺ pumps, is difficult to envision. Moreover, that these findings were obtained using heterologous expression systems, raises concern regarding the possibility that a similar function might not exist in native membranes.

High levels of expression of HK₂ have been reported in apical membranes from rat distal colon (10) where the protein has also been identified by immunolocalization (9). Taking advantage of this observation, we prepared apical membranes from rat distal colon to determine: (a) if under physiological conditions HK₂ is sensitive to low concentrations of Sch-28080, and (b) if HK₂ function, here defined as K⁺-ATPase enzymatic activity, is Na⁺-dependent or Na⁺- independent.

Our results demonstrate that these membranes contain both Na⁺-dependent and Na⁺-independent ATPase activities. Using an inhibitory antibody specific for HK₂, we demonstrate that this pump is responsible for Na⁺-dependent activity. This finding indicates that the colonic H⁺,K⁺-ATPase, which has been shown to function as a proton pump, may also function as a sodium pump under certain physiological conditions. Furthermore, we also demonstrate that both Na⁺-dependent and Na⁺-independent activities can substitute readily NH₄⁺ for K⁺.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Preparation of Plasma Membranes (Method I)

To prepare plasma membranes (18, 19) rat distal colon (1 g of tissue) was homogenized using a Brinkmann Polytron, Model PT 10/35, followed by homogenization in a Dounce homogenizer using pestle A (4-5 strokes). The homogenization was performed in 10 ml of buffer A (10 mM Tris-HCl, pH 8.0, 1 mM EDTA-Tris, 1 mM phenylmethylsulfonyl fluoride, 3 mM benzamidine, and 1 µg/ml soybean trypsin inhibitor) containing 27% sucrose (w/v). Nuclei were removed by centrifugation at 2000 × g for 4 min at 4 °C, the supernatant was applied to the top of 45% (w/w) sucrose in buffer A and centrifuged at 200,000 × g for 45 min at 4 °C. The membranes in the interphase 27/45% sucrose were diluted in buffer A and collected by centrifugation at 25,000 × g, resuspended in buffer A, and stored in aliquots at 70 °C. The final protein concentration was measured using the method of Lowry et al. (20).

Preparation of Apical Membranes (Method II)

Apical membranes from distal colon were prepared as described by Aronson (21). Distal colon (1 g in 10 ml of buffer B: 300 mM mannitol, 1 mM Tris-HCl, pH 7.2, 1 mM phenylmethylsulfonyl fluoride, 3 mM benzamidine, and 1 µg/ml soybean trypsin inhibitor) was homogenized with a Polytron, followed by 4-5 strokes with pestle A of a Dounce homogenizer. The particulate matter was removed by centrifugation for 2 min at 200 × g. To the supernatant, 1 M MgSO₄ was added to a final concentration of 10 mM MgSO₄. The sample was placed on ice for 15 min and was shaken intermittently. The aggregated material was removed by centrifugation for 12 min at 2500 × g at 4 °C, and apical membranes from the supernatant were collected by centrifugation at 27,000 × g for 20 min at 4 °C. The pellet was resuspended in 5 ml of buffer B containing 10 mM MgSO₄, and homogenized with pestle B of a Dounce homogenizer. After removal of the aggregated material at 3100 × g for 12 min at 4 °C, membranes were collected by centrifugation at 27,000 × g for 20 min at 4 °C. The final membranes were resuspended in 10 mM Tris-HCl, pH 7.2, 1 mM EDTA-Tris, 1 mM phenylmethylsulfonyl fluoride, 3 mM benzamidine, and 1 µg/ml soybean trypsin inhibitor and stored in aliquots at 70 °C. The protein concentration was measured using the method of Lowry et al. (20).

Preparation of Vesicle Membranes (Method III)

Distal colon was homogenized with a Polytron in 3 volumes of iced 0.25 M sucrose, 1 mM EDTA, as described previously in our laboratory (22). The homogenate was filtered through 500-µm pore size mesh nylon and centrifuged at 8,000 × g. The supernatant was retained and centrifuged at 200,000 × g. The pellet was resuspended in 250 mM sucrose, 6 mM histidine, pH 7.0, and recentrifuged at 200,000 × g for 30 min at 4 °C. The new pellet was resuspended in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA and stored at 70 °C. The protein concentration was measured using the method of Lowry et al. (20) and immunoblots were performed using a specific antibody against HK₂ (10).

ATPase Assays

All ATPase assays were performed for 30 min at 37 °C in a final volume of 200 µl containing 30 mM Tris-HCl, pH 7.2, 1 mM EDTA-Tris, 0.1 mM EGTA-Tris, 4 mM MgCl₂, 3 mM ATP-Tris containing 1-10 × 10⁶ cpm of [-³²P]ATP (Amersham Pharmacia Biotech, catalog number AA0068), 1 mM N-ethylmaleimide, 10 µg/ml oligomycin, 1 mM phenylmethylsulfonyl fluoride, 3 mM benzamidine, 1 µg/ml soybean trypsin inhibitor and, when necessary, ouabain, Sch-28080, NaCl, and/or KCl were added (see figure legends for the concentrations of ouabain, Sch-28080, NaCl, and KCl). The reaction was started by addition of rat distal colon apical membranes (5-15 µg) diluted in 10 mM Tris-HCl, pH 7.2. The reaction was stopped by addition of activated charcoal (1 ml, 50% slurry) (Fisher Scientific, catalog number C170-500) in 10 mM Na⁺-phosphate, pH 7.5. The samples were vortexed, cooled in ice, centrifuged, and the supernatant (450 µl) was used to quantify the ³²P released during the incubation.

K⁺-ATPase Activity in the Presence or Absence of Anti-HK₂ Antibody

Group A-- Serum (15 µl) containing the anti-HK₂ antibody was mixed with the immunizing peptide (10 µl) (1.5 mM) to inactivate the antibody (19), the antibody/synthetic peptide mixture was incubated for 1 h at 4 °C. After incubation, distal colon apical membranes (100-200 µg) were added to the mixture and incubated for 1 h at 4 °C with occasional vortexing. The membranes were diluted with 10 mM Tris-HCl, pH 7.2, to a final concentration of 0.5-1.5 µg of protein/µl and 20 µl of the diluted membranes were used in the ATPase assays.

Group B-- H₂O (10 µl) was mixed with serum (15 µl) containing the anti-HK₂ antibody. After 1 h incubation at 4 °C, rat distal colon apical membranes (100-200 µg) were added and incubated for 1 additional hour at 4 °C. The membranes were then diluted with 10 mM Tris-HCl, pH 7.2, to a final concentration of 0.5-1.5 µg of protein/µl. Finally, immunizing peptide (10 µl) was added. A graphic representation of this assay is shown in the top panel of Fig. 5. A volume of 20 µl of diluted membranes was used in the ATPase assays.

Other Reagents

The characterization of the antibody against HK₂ has been described recently by our laboratory (10, 19). Immunoblots were performed as reported previously by our laboratory (10, 19). Harlan Sprague-Dawley male rats (150-200 g) were used in all experiments. Ouabain was purchased from Sigma. Sch-28080 was a gift from Dr. Kaminski at Schering-Plow Research Institute. The anti-NASE and anti-LEAVE antibodies were characterized previously (23). The amino acid alignment between different -subunits was performed with the "Bestfit" program of the Genetics Computer Group (Madison, WI).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Distal Colon Apical Membranes Are Enriched in HK₂-- As displayed in Fig. 1, plasma (method I), apical (method II), and vesicle (method III) membranes (20 µg/each) were applied to a 10% SDS-PAGE, transferred to a nitrocellulose membrane and incubated with the anti-HK₂ antibody (dilution 1:1000). As expected (10), of the three methods to prepare membranes the apical membrane method yielded membranes which were most enriched in HK₂ protein.

View larger version (45K): [in this window] [in a new window]   Fig. 1.   Distal colon apical membranes are enriched in HK2. Membranes prepared according to Methods I, II, or III (20 µg) were resolved on a 10% SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with anti-HK2 (dilution 1:1000). The presence of the protein was determined using an ECL system.

K⁺-ATPase Activities in Distal Colon Apical Membranes-- In heterologous expression systems, the activity of rat HK₂ has been reported to be K⁺-dependent (IC₅₀ < 1 mM), Sch-28080 insensitive, and only partially sensitive to ouabain (IC₅₀ ~ 400-600 µM) (3, 4). To test which of the different K⁺-ATPases observed in apical membranes of distal colon best fits this pharmacological profile, we measured Na⁺-dependent and Na⁺-independent K⁺-ATPase activity in preparations of rat distal colon apical membranes in the presence of KCl (10 mM). The data displayed in Fig. 2 (left panel) demonstrate that the predominant K⁺-ATPase in distal colon apical membranes was Na⁺-dependent and was abolished by ouabain (2 mM), but was insensitive to Sch-28080 (100 µM). This same figure also demonstrates that distal colon apical membranes contain a Na⁺-independent K⁺-ATPase. The Na⁺-independent K⁺-ATPase represents only 20% of the total Na⁺-dependent K⁺-ATPase activity (at 10 mM KCl), and is insensitive to both ouabain (2 mM) and Sch-28080 (100 µM). Treatment of the membrane preparations with CHAPS or Triton X-100 did not alter the response to Sch-28080 or ouabain for either Na⁺-dependent or Na⁺-independent K⁺-ATPase fractions. Since Sch-28080 did not inhibit either K⁺-ATPase activity in apical membranes from distal colon, we tested if the Na⁺-independent K⁺-ATPase ("H⁺,K⁺-ATPase") in apical membranes of rat stomach which are enriched in HK₁ is sensitive to Sch-28080. The right panel of Fig. 2 demonstrates that concentrations of Sch-28080 as low as 10 µM inhibit 80-90% of the Na⁺-independent K⁺-ATPase activity in the presence of K⁺ (5 mM).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Left panel, rat distal colon apical membranes contain both Na+-dependent and Na+-independent K+-ATPases. The ATPase assay was performed as described under "Experimental Procedures." Right panel, rat stomach apical membrane. In contrast to the Sch-28080 insensitivity of both K+-ATPases in distal colon, Sch-28080 has high affinity for rat gastric H+,K+-ATPase (HK1), as expected. The assay was performed as described under "Experimental Procedures" with the only exception that all Na+ salts were avoided. 1, 1-subunit of the Na+,K+-ATPase.

Based on the observation that many transporters of K⁺, including the ₁-Na⁺,K⁺-ATPase, can transport either K⁺ or NH₄⁺ with similar affinity (24-26), we characterized the ion dependence of the K⁺-ATPases present in our preparation of distal colon apical membranes. In Fig. 3 (left panel) apical membranes were incubated in the presence of NaCl (20 mM) and with increasing concentrations of KCl (closed diamonds) or NH₄Cl (closed squares). Na⁺-dependent K⁺-ATPase activity increased with increasing concentrations of K⁺ and reached saturation at concentrations near 5 mM. Substitution of K⁺ by NH₄ also induced an increase in Na⁺-dependent NH₄⁺-ATPase in a concentration-dependent manner. The V_m for K⁺ and NH₄⁺ are similar. However, the enzyme had a higher affinity for K⁺ (IC₅₀ ~ 0.2 mM) than for NH₄⁺ (IC₅₀ ~ 2 mM). The affinity for Na⁺ was identical (IC₅₀ ~ 5 mM) whether K⁺ or NH₄⁺ was used in the ATPase assay (Fig. 3, middle panel). Finally we determined if K⁺ can be replaced by NH₄⁺ in the Na⁺-independent K⁺-ATPase fraction. The results of a representative experiment are displayed in Fig. 3 (right panel). The Na⁺-independent K⁺-ATPase activity displayed a low affinity for K⁺ (activity was not detectable until [K⁺] was >2.5 mM, IC₅₀ > 10 mM, and did not reach saturation until 40 mM [K⁺]). Similar results were obtained when K⁺ was replaced by NH₄⁺.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Left panel, the Na+-dependent K+-ATPase has high affinity for both K+ and NH4+. The assay was performed in the presence of NaCl (20 mM) with increasing concentrations of KCl or NH4Cl. Center panel, HK2 has similar affinity for Na+ in the presence of either K+ or NH4+. The assay was performed in the presence of KCl (5 mM) or NH4Cl (5 mM) with increasing concentrations of NaCl. Right panel, the Na+-independent K+-ATPase fraction can employ either K+ or NH4+ but at relatively low affinities. The assay was performed in the absence of Na+.

The activity of Na⁺-dependent K⁺-ATPase in apical membranes was inhibited by high concentrations of ouabain (IC₅₀ ~ 200-300 µM). Replacement of K⁺ by NH₄⁺ did not alter this inhibitory profile (Fig. 4). Furthermore, Sch-28080 did not inhibit K⁺-ATPase activity when K⁺ was replaced by NH₄⁺ (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Na+-dependent K+-ATPase in the apical membrane has a low affinity for ouabain. The effect of ouabain was measured in the presence of KCl (10 mM) or NH4Cl (10 mM) plus NaCl (20 mM).

The studies shown above (Figs. 2-4) demonstrate that apical membranes, which are enriched in HK₂ protein, display pharmacological properties which are virtually indistinguishable from ₁-Na⁺,K⁺-ATPase (27, 28). To exclude the possibility that we were studying ₁-Na⁺,K⁺-ATPase activity (which are contaminating the membranes preparation) rather than the activity of HK₂, we took advantage of two observations made by our laboratory: (a) our anti-HK₂ antibody can immunoprecipitate the HK₂/₁-Na⁺,K⁺-ATPase complex in both the renal medulla and distal colon (19) and, (b) this same anti-HK₂ antibody does not cross-react or immunoprecipitate ₁-Na⁺-K⁺-ATPase or any other X⁺,K⁺-ATPase characterized thus far (19). We asked whether the anti-HK₂ antibody could block Na⁺-dependent K⁺-ATPase activity. To answer this question, the anti-HK₂ antibody (15 µl) was incubated in the presence (group A) or absence of immunizing peptide (500 µM) (group B) for 1 h at 4 °C (10). Incubation was followed by addition of membranes (100 µg) to each group. The mixture was then incubated for 1 h at 4 °C. Membranes were diluted to 0.5 µg/µl in the presence of 10 mM Tris-HCl, pH 7.2, and incubated at 37 °C for 30 min as described under "Experimental Procedures." In group B the immunizing peptide was added after dilution of the membranes. This approach was taken to correct for possible interference of the peptide with the ATPase assay. A schematic representation of this protocol is displayed in Fig. 5 (top panel). Fig. 5 (middle panel), demonstrates that incubation of apical membranes with the anti-HK₂ antibody markedly decreased Na⁺-dependent K⁺-ATPase activity (closed bar). A similar experiment was performed by substituting K⁺ for NH₄⁺ (Fig. 5, lower panel). Similar Na⁺-dependent K⁺(NH₄⁺)-ATPase activity was observed in the presence of either K⁺ or NH₄⁺ (5 mM) under control conditions. Na⁺-dependent NH₄⁺-ATPase activity was blocked by the anti-HK₂ antibody, exactly as observed in the presence of K⁺. As a control we repeated the experiment described in Fig. 5, but with plasma membranes isolated from rat renal cortex. These membranes, in contrast to distal colon membranes, are depleted of HK₂ but are enriched in ₁-Na⁺,K⁺-ATPase. The result of such an experiment is displayed in Fig. 6. This finding demonstrates that Na⁺,K⁺-ATPase activity in the renal cortex is not inhibited by the anti-HK₂ antibody. Thus, the anti-HK₂ antibody does not cross-react with a membrane fraction enriched in ₁-Na⁺,K⁺-ATPase.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Top panel, schematic representation of the protocol to determine if the Na+-dependent K+-ATPase activity in distal colon apical membranes represented HK2. For differences in two groups (A and B) see "Experimental Procedures." In group A (controls) the immunizing peptide was added prior addition of the anti-HK2 antibody and membranes to block recognition of HK2 protein in the membrane by the antibody. Therefore, only the group B protocol allowed exposure of the native HK2 to the anti-HK2 to the anti-HK2 antibody. Middle panel, the Na+-dependent K+-ATPase of HK2 was blocked by the anti-HK2 antibody. Bottom panel, the Na+-dependent K+-ATPase was blocked by the anti-HK2 antibody when K+ was replaced by NH4+.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   The anti-HK2 antibody does not block Na+,K+-ATPase activity in plasma membranes from renal cortex. The assay was performed exactly as described in the legend to Fig. 5.

To confirm further that HK₂ functions as a Na⁺-dependent K⁺-ATPase in apical membranes of distal colon origin we performed an additional experiment described in Fig. 7. In this study Na⁺-dependent K⁺-ATPase activity was measured in plasma membranes from renal cortex as well as apical membranes from distal colon. We reasoned that if the HK₂ functions in distal colon as a Na⁺-dependent K⁺-ATPase, more ₁-subunit from the renal cortex would be required to reach the same level of Na⁺-dependent K⁺-ATPase activity present in the distal colon apical membranes. Fig. 7 (left panel) demonstrates that both membrane fractions contain similar specific activities (expressed as nanomole of ATP hydrolyzed/hour) at any given concentration of total protein. However, the absolute level of ₁-Na⁺,K⁺-ATPase was much greater in renal cortex compared with distal colon (right panel of Fig. 7). This study was performed with two different antibodies, anti-NASE which recognizes only ₁-Na⁺,K⁺-ATPase and anti-LEAVE which should recognize ₁-, ₂-, and ₃-Na⁺,K⁺-ATPase (23). The observation that the immunoblots for both the distal colon and renal cortex membranes using either the anti-NASE and anti-LEAVE antibodies were indistinguishable, demonstrates that neither ₂- nor ₃-Na⁺,K⁺-ATPase accounts for the Na⁺-independent K⁺-ATPase activity in distal colon apical membranes. These findings provide additional evidence that the distal colon apical membrane fraction is enriched in HK₂ protein and functions as a Na⁺-dependent K⁺-ATPase which is distinct from ₁-Na⁺,K⁺-ATPase activity.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   The Na+-dependent K+-ATPase activity in apical membranes from distal colon cannot be accounted for by 1-Na+,K+-ATPase. Left panel, Na+-dependent K+-ATPase activity was similar in both membranes at any concentration of membranes used. The assay was performed in the presence of KCl (10 mM) and in the presence or absence of NaCl (50 mM). Right panel, abundance of -subunits of Na+,K+-ATPases in renal cortex exceeds abundance in distal colon.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Our studies demonstrate that plasma membranes from distal colon, which are enriched severalfold in HK₂ protein, contain a K⁺-ATPase activity which is Na⁺-dependent, Sch 28080-insensitive, and partially ouabain-sensitive. To conclude that this activity represents functionally the -subunit of the colonic H⁺,K⁺-ATPase, is contingent on the specificity of our anti-HK₂ antibody. Previous studies by our laboratory have demonstrated that the antibody used in the present study does not cross-react with any of the known X⁺,K⁺-ATPases (10, 19). Nevertheless, alternative explanations for this activity which were evaluated in the course of this study include all known members of the X⁺,K⁺-ATPase superfamily. The antibody used in these studies was raised against a synthetic peptide designed after the rat colonic H⁺,K⁺-ATPase the epitope of which (amino acids 686-698) is not found on any other known rat X⁺,K⁺-ATPases. Furthermore, there is no evidence that another H⁺,K⁺-ATPase or Na⁺,K⁺-ATPase, except HK₂ or ₁-Na⁺,K⁺-ATPase, exists in distal colon (Fig. 7). Moreover, in this, and in a previous study (10, 19), we have demonstrated that this antibody does not cross-react or immunoprecipitate other known X⁺,K⁺-ATPases, including the ₁-Na⁺,K⁺-ATPase. In addition, we now demonstrate that our anti-HK₂ antibody blocks Na⁺-dependent K⁺-ATPase activity (Fig. 5), that Na⁺,K⁺-ATPase activity in the renal cortex is not inhibited by the specific blocking antibody (Fig. 6), and finally that neither ₂ nor ₃-Na⁺,K⁺-ATPase could account for the activity in the distal colon apical membrane fraction (Fig. 7). Therefore, we conclude that HK₂ can function in distal colon as a Na⁺-dependent K⁺-ATPase.

Precedent exists for Na⁺ secretion by the HK₂. Cougnon et al. (14) has demonstrated recently that HK₂ can secrete Na⁺ in exchange for K⁺ in X. laevis oocytes. In this study it was reported that Na⁺/K⁺ exchange, which was sensitive to high concentrations of ouabain, and was totally insensitive to Sch-28080, was dependent on co-expression with a -subunit. Moreover, Kone and Higham (29) reported recently that a splice variant of HK₂ (HK_2b), which is truncated by the initial 103 amino acids, supported the growth of HEK-293 cells in the presence of low concentrations of ouabain. In addition, Grishin et al. (17), expressed the human ATP1AL1 (90% similar to HK₂) in HEK-293 cells, and observed that the ratio of H⁺-secretion to K⁺-uptake was approximately 1:10. Based on these observations he postulated that ATP1AL1 did not function solely as a H⁺/K⁺ exchanger. This group also reported that ATP1AL1 supported the growth of HEK-293 cells in the presence of ouabain (15). Since HEK-293 cells transfected with ATP1AL1 or HK_2b grow in the presence of ouabain (15, 29), it is logical to speculate that both the rat HK₂ and the human ATP1AL1 may function as Na⁺ pumps. However, since these results were obtained in heterologous expression systems, questions could be raised regarding the relevance of these results to native colonic apical membranes.

It is appreciated generally that Na⁺,K⁺-ATPases are localized to basolateral membranes (30). In the present study, and from previous studies by our laboratory (10) it has been demonstrated that HK₂ is enriched in apical membrane fractions from distal colon. Sangan et al. (8) have also demonstrated that HK₂ protein localizes to the apical membrane of colonocytes. Moreover, Jaisser et al. (31) have demonstrated by in situ hybridization that HK₂ mRNA localizes to surface epithelial cells of rat distal colon. Assigning a physiological role for a Na⁺-dependent K⁺-ATPase located in the apical membrane of colonocytes is difficult, however.

Del Castillo et al. (32) reported that two types of Na⁺-independent K⁺-ATPases are present in apical membranes from distal colon: one which is sensitive to ouabain (1 mM), and another which is insensitive to ouabain (1 mM). These K⁺-ATPases were not present in basolateral membranes. The effect of Sch-28080 (a standard inhibitor of the gastric H⁺,K⁺-ATPase) was not tested. Lee et al. (33) reported that in preparations of "apical" membranes from distal colon, both activities (ouabain-insensitive and ouabain-sensitive K⁺-ATPases) were inhibited by an antibody directed against the amino-terminal of HK₂. Recently, Rajendran et al. (34) reported that Na⁺-independent, K⁺-dependent pH_i recovery by rat colonocytes was ouabain-insensitive (up to 1 mM). Based on this observation, it was concluded that HK₂ functions in colonocytes as a Na⁺-independent, ouabain-insensitive K⁺-ATPase.

In agreement with these findings, both Na⁺-dependent and Na⁺-independent K⁺-ATPases were detected in the present study, the Na⁺-dependent fraction, which predominated under the conditions of the assay, was relatively ouabain-sensitive. In contrast, the less abundant Na⁺-independent fraction was ouabain-insensitive. Based on the present study it is reasonable to conclude that HK₂ may function in native apical membranes not only as a proton pump, but as a Na⁺ pump. Nevertheless, the physiological conditions which might serve to regulate Na⁺/K⁺ as opposed to H⁺/K⁺ exchange have not been defined. Therefore, future studies will be needed to define the relative contributions of these pumps in the distal colon as well as factors which regulate their abundance and function in physiologic and pathophysiologic conditions.

An additional finding in the present study was that the K⁺-ATPase activity in distal colon membranes also had high affinity for NH₄⁺ (Figs. 3-5). Transport of NH₄⁺ by the colonic H⁺,K⁺-ATPase in kidney has been suggested in preliminary studies previously (35, 36). Our findings are the first to suggest that substitution of K⁺ by NH₄⁺ may occur in distal colon apical membranes. Nevertheless, the physiological role of H⁺/NH₄⁺ exchange by the colonic H⁺,K⁺-ATPase in either kidney or distal colon has not yet been defined clearly.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health, National Institute of Diabetes, Digestive and Kidney Diseases, Grant DK-30603 (to T. D. D.).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. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Div. of Renal Diseases and Hypertension, University of Texas-Houston Medical School, 6431 Fannin St., Rm. 4.148, Houston, TX 77030. Tel.: 713-500-6868; Fax: 713-500-6882; E-mail: tdubose@heart.med.uth.tmc.edu.

	ABBREVIATIONS

The abbreviations used are: HK₁, -subunit of the gastric H⁺,K⁺-ATPase; HK₂, -subunit of the colonic H⁺,K⁺-ATPase; ₁, -subunit of the Na⁺,K⁺-ATPase; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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