Human nongastric H+-K+-ATPase: transport properties of ATP1al1 assembled with different beta -subunits

Gilles Crambert1, Jean-Daniel Horisberger1, Nikolai N. Modyanov2, and Käthi Geering1

1 Institute Of Pharmacology And Toxicology of The University, CH-1005 Lausanne, Switzerland; and 2 Department Of Pharmacology, Medical College Of Ohio, Toledo, Ohio 43614-5804


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To investigate whether nongastric H+-K+-ATPases transport Na+ in exchange for K+ and whether different beta -isoforms influence their transport properties, we compared the functional properties of the catalytic subunit of human nongastric H+-K+-ATPase, ATP1al1 (AL1), and of the Na+-K+-ATPase alpha 1-subunit (alpha 1) expressed in Xenopus oocytes, with different beta -subunits. Our results show that beta HK and beta 1-NK can produce functional AL1/beta complexes at the oocyte cell surface that, in contrast to alpha 1/beta 1 NK and alpha 1/beta HK complexes, exhibit a similar apparent K+ affinity. Similar to Na+-K+-ATPase, AL1/beta complexes are able to decrease intracellular Na+ concentrations in Na+-loaded oocytes, and their K+ transport depends on intra- and extracellular Na+ concentrations. Finally, controlled trypsinolysis reveals that beta -isoforms influence the protease sensitivity of AL1 and alpha 1 and that AL1/beta complexes, similar to the Na+-K+-ATPase, can undergo distinct K+-Na+- and ouabain-dependent conformational changes. These results provide new evidence that the human nongastric H+-K+-ATPase interacts with and transports Na+ in exchange for K+ and that beta -isoforms have a distinct effect on the overall structural integrity of AL1 but influence its transport properties less than those of the Na+-K+-ATPase alpha -subunit.

X+-K+-ATPases; Na+ transport; Xenopus oocytes; intersubunit interactions


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE CATALYTIC SUBUNIT of the human nongastric H+-K+-ATPase is termed ATP1al1 (AL1) (35, 43, 46). This enzyme is a member of the closely related X+-K+-ATPases of the P-type ATPase family, which transport K+ in exchange for Na+ and/or H+. Based on their structural, functional, and pharmacological properties, animal nongastric H+-K+-ATPases (for recent review and references see Ref. 20) occupy an intermediate position between Na+-K+-ATPase and gastric H+-K+-ATPase. For instance, their alpha -subunits are structurally equally distant from Na+-K+-ATPase and gastric H+-K+-ATPase alpha -subunits, and the nongastric H+-K+- ATPases are moderately sensitive to the Na+-K+- ATPase inhibitor, ouabain, and poorly sensitive to the gastric H+-K+-ATPase inhibitor, SCH-28080 (9, 16, 22, 34). Similar to Na+-K+-ATPase and gastric H+-K+-ATPase, nongastric H+-K+-ATPases are oligomeric P-type ATPases that need a beta -subunit for the structural maturation of the catalytic alpha -subunit (for review see Ref. 13). However, it is presently not known which of the four known X+-K+-ATPase beta -subunits (Na+-K+-ATPase beta 1-, beta 2-, and beta 3-isoforms and gastric H+-K+-ATPase beta -subunit) is associated with the nongastric H+-K+-ATPase alpha -subunit in situ.

In mammals, nongastric H+-K+-ATPase alpha -mRNA is widely distributed, with prominent expression in skin, colon, and kidney (37). At the protein level, the nongastric H+-K+-ATPase alpha -subunit has been identified in apical membranes of surface cells in distal colon (30) and of principal cells in the outer medullary collecting duct of rat kidney (41) or of connecting tubule cells of rabbit kidney (12). In humans, the AL1 protein was detected in intercalated and, to a lesser extent, in principal cells of the renal collecting ducts (28). mRNA and protein abundance is significantly upregulated by dietary Na+ depletion in the rat and mouse colon and by dietary K+ depletion in the rat kidney (23, 31, 41, 45). Although these adaptive, regulatory mechanisms and findings from mice deficient in nongastric H+-K+-ATPase alpha -subunits (33, 45) suggest that nongastric H+-K+-ATPases play a role in the maintenance of K+ homeostasis in vivo under K+- or Na+-deprived conditions, the exact biological function of these ATPases is still unclear, mainly because of the still insufficient characterization of their functional properties and of subunit composition.

The capacity of different, putative nongastric H+-K+-ATPases to transport K+, H+, and NH<UP><SUB>4</SUB><SUP>+</SUP></UP> has been established in various experimental setups (2, 9, 11, 16, 22, 27, 34, 44). Whereas the H+ transport is dependent on extracellular K+, the K+ transport appears not to be modulated by intracellular pH (pHi) (16). Moreover, the K+ influx is at least 10-fold higher than the H+ efflux (16), but the ion transport activity is electroneutral (4). One reason for this apparent discrepancy may be that nongastric H+-K+-ATPases can mediate Na+/K+ exchange, as suggested by the finding that intracellular Na+ concentration ([Na+]i) decreases in Xenopus oocytes (10) or in HEK-293 cells (17) expressing rat colonic H+-K+-ATPase or AL1/beta complexes, respectively. The Na+ transport capacity of nongastric H+-K+-ATPase is still ill defined. For instance, nothing is known on the Na+ dependence of the K+ transport and the actual affinity for intracellular Na+.

For their functional expression, nongastric H+-K+-ATPase alpha -subunits depend on the presence of a beta -subunit (1, 2, 16, 22, 34, 39). Both Na+-K+-ATPase beta 1-subunit (6, 29) and beta 3-subunit (40) have been proposed as the natural partner subunit of nongastric H+-K+-ATPase alpha -subunits in kidney and colon. In expression systems, several beta -subunits, including the Na+-K+-ATPase beta 1-subunit (2, 7, 9), the Na+-K+-ATPase beta 2-like Bufo bladder beta -subunit (11), a Torpedo beta -subunit (2), as well as the gastric H+-K+-ATPase beta -subunit (1, 7, 16, 21, 27, 34) can support the functional expression of nongastric H+-K+-ATPases. On the other hand, in HEK-293 cells, nongastric H+-K+-ATPases cannot assemble with endogenous, probably beta 1-subunits (2, 39), and in Xenopus oocytes only gastric H+-K+-ATPase beta -subunits and Na+-K+-ATPase beta 2 and beta 2-like Bufo bladder beta -subunits can promote efficient maturation of alpha -subunits of nongastric H+-K+-ATPase such as AL1 (15). Altogether, these apparently contradictory results reflect the uncertainty about the real, physiologically significant subunit composition of nongastric H+-K+-ATPase. This may be of relevance for the interpretation of functional properties of nongastric H+-K+- ATPases studied in expression systems, in terms of their physiological importance. Indeed, beta -subunits are not only specific chaperones necessary for the maturation of alpha -subunits of X+-K+-ATPases, but they were also shown to modulate the transport properties and, in particular, the K+ activation of Na+-K+-ATPase and gastric H+-K+-ATPase (for review see Ref. 13).

The present study aimed at a further characterization of the functional properties of the human nongastric H+-K+-ATPase, in particular of its K+/Na+ transport capacity, and at a better understanding of the influence of different beta -subunits on various functional parameters. For this purpose, we expressed AL1 or Na+-K+-ATPase alpha -subunits together with Na+-K+-ATPase beta 1-subunit, gastric H+-K+-ATPase beta -subunit, or Bufo bladder beta -subunit in Xenopus oocytes and compared the Na+ and K+ dependence of the transport activity of the various pumps and the ligand-dependent conformational changes of the beta -associated alpha -subunits, as reflected by controlled proteolysis. Our results provide further evidence for a Na+/K+ exchange mode of AL1. Moreover, our findings indicate that beta -isoforms indeed influence the overall structural integrity of AL1 but, compared with the Na+-K+-ATPase, have less effects on the cation transport properties of the human nongastric H+-K+-ATPase.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of expression constructs and cRNAs. The expression constructs of AL1 in the pSD3 vector and of human Na+-K+-ATPase alpha 1-subunits (alpha 1) in the pNKS2 vector have been prepared as described previously (34). Human Na+- K+-ATPase beta 1-subunits (beta 1) (26) (kindly provided by K. Kawakami), rabbit gastric H+-K+-ATPase alpha -subunit (alpha HK), and beta -subunit (beta HK; kindly provided by G. Sachs), Bufo Na+-K+-ATPase alpha 1-subunits (Bufo alpha 1) (21), Bufo bladder beta -subunits (beta  bl; kindly provided by F. Jaisser) (22), and alpha -, beta -, and gamma -subunits of the rat epithelial Na+ channel (rENaC) (5) were subcloned in the pSD5 vector. cRNAs were prepared by in vitro transcription (32).

Protein expression in Xenopus oocytes, controlled proteolysis, and immunopreciptations. Oocytes were obtained from Xenopus laevis females as described before (14). Routinely, 8 ng of alpha -cRNA and 1 ng of beta -cRNA were injected, and the oocytes were incubated in the presence of 0.8 mCi/ml [35S]Easytag express protein labeling mix (New England Nuclear) and in the absence or presence of 5 µg/ml brefeldin A, which retains proteins in the endoplasmic reticulum (14). After a 24-h pulse period at 19°C, microsomal fractions were prepared as described (14) in the absence of protease inhibitors, and the pelleted membranes were taken up in a solution containing 30 mM DL-histidine, 5 mM EDTA, and 18 mM Tris, pH 7.4. Aliquots of 12 µg protein were incubated in the presence of 4 mM MgCl2, 50 mM NaCl, 15 mM KCl, 1 or 4 mM Tris-ATP (Sigma Chemical), 1.5 mM ouabain, and 7.5 mM Tris-Pi, alone or in combination, as indicated in figure legends. Osmolarity was adjusted to 59 mM with choline chloride. Samples were preincubated for 30 min at 25°C before addition of diphenyl carbamyl chloride-treated trypsin (Sigma), at a trypsin to protein ratio of 0.05. Samples were incubated for 1 h at 25°C before addition of a fivefold excess over trypsin (wt/wt) of soybean trypsin inhibitor (Sigma). After 10 min at 25°C, SDS was added to a final concentration of 3.7% and the samples were heated at 56°C for 7 min. Immunoprecipitations of alpha - and beta -subunits were performed with specific antibodies under denaturing conditions as previously described (14). In some instances, samples were treated with endoglycosidase H (Endo H; Biolab) (14), to remove core sugars from beta -subunits, and subjected to SDS-PAGE without immunoprecipitation. Immunoprecipitated and nonimmunoprecipitated alpha - and beta -subunits were detected by fluorography. Quantification of immunoprecipitated alpha -subunits was performed with a laser densitometer (LKB Ultroscan 2202). Statistical analysis was performed with unpaired Student's t-test.

Measurement of [Na+]i. Oocytes were injected with 0.3 ng each of alpha -, beta -, and gamma -subunit cRNAs of rENaC and 1 ng of beta 1- or beta HK cRNA with or without 8 ng of Bufo alpha 1, AL1, alpha HK, or alpha 1-cRNA and incubated in modified Barth's solution containing 90 mM NaCl. Determinations of [Na+]i were performed 3 days after injection using the two-electrode voltage-clamp technique as described before (18). The day before the measurements, oocytes expressing the moderately ouabain-resistant Bufo alpha 1/beta , AL1/beta , and the ouabain-resistant alpha HK/beta complexes were incubated with 1 µM ouabain to inhibit the endogenous oocyte Na+-K+-ATPase. [Na+]i was calculated from the reversal potential of the amiloride-sensitive current mediated by the coexpressed rENaC and deduced from current-voltage curves recorded in the absence or presence of 20 µM amiloride in a solution containing 5 mM sodium gluconate, 0.5 mM MgCl2, 2.5 mM BaCl2, 95 mM N-methyl-D-glucamine (NMDG)-Cl, and 10 mM NMDG-HEPES, pH 7.4.

Determination of the apparent affinity for intracellular Na+ of AL1/beta complexes. Immediately after injection with X+-K+-ATPase and rENaC subunit cRNAs, oocytes were incubated in the absence of K+ in modified Barth's solution containing 0, 5, 20, and 100 mM sodium gluconate. In each solution, the osmolarity was adjusted to 100 mM with choline chloride. After 3 days of incubation, [Na+]i was determined as described above. Measurement of 86Rb uptake was carried out as described before (34) by transferring oocytes into a solution containing 90 mM NaCl, 2 mM CaCl2, 5 mM BaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.4, 5 µM amiloride, 10 µM bumetamide, 1 µM ouabain, and 5 µCi/ml 86Rb (Amersham) for 12 min at room temperature before washing three times in a solution containing 90 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES, pH 7.4. Individual oocytes were dissolved in 0.5% SDS and counted.

Measurements of pHi. pHi measurements were carried out by using a double-barreled pH-sensitive microelectrode containing ion exchanger H-ionophore II cocktail A (Fluka) as previously described (19). These electrodes had a resistance of 2-10 GOmega and were calibrated in HEPES buffer solutions (pH 6.5 and 7.5) immediately before and after each pHi measurement. The pH electrodes were used only if they showed a pH response >52 mV/pH unit. pHi was calculated from the voltage read with the pH barrel minus the membrane voltage read from the reference barrel filled with 3 M KCl.

Determination of the apparent affinity for K + and of the effect of extracellular Na+ on the K+ transport of AL1/beta complexes. 86Rb uptake was measured 3 days after injection of oocytes with AL1 together with beta 1 or beta HK cRNAs. To determine the K+ activation constant of AL1/beta 1 and AL1/beta HK complexes, oocytes were loaded with Na+ as described before (24), and the 86Rb uptake was measured in the presence of various K+ concentrations (0.2, 0.3, 0.5, 1, 3, and 5 mM) in a solution containing 90 mM NaCl, 2 mM CaCl2, 5 mM BaCl2, 1 mM MgCl2, 10 µM bumetamide, 1 µM ouabain, and 5 µCi/ml 86Rb as described above. To determine the effect of extracellular Na+ on the K+ transport of AL1/beta 1 and AL1/beta HK complexes, oocytes were loaded with Na+, and 86Rb uptake was measured in a solution containing 0.5 mM KCl and either 90 mM NMDG-Cl or 90 mM NaCl.

Electrophysiological measurements of Na+-K+ pumps. Na+-K+ pump currents were measured as the K+-induced outward current using the two-electrode voltage-clamp technique as described before (24). Briefly, current measurements were performed 3 days after injection of oocytes with alpha 1 cRNAs together with beta 1 or beta HK. To determine the maximal K+-induced current, injected and noninjected oocytes were loaded with Na+ in a K+-free solution 1 day before measurements. The currents induced by addition of 10 mM K+ were recorded at -50 mV in a Na+-containing solution (80 mM sodium gluconate, 0.82 mM MgCl2, 0.41 mM CaCl2, 5 mM BaCl2, 10 mM tetraethylammonium chloride, and 10 mM NMDG-HEPES, pH 7.4). To determine the K+ activation of Na+-K+ pumps, the pump current was measured in the presence of 0.3, 1, 3, and 10 mM K+. The Hill equation, IK = Imax/{1 + (K1/2 K+/[K])nH}, was fitted to the current (IK) induced at different K+ concentrations ([K]) to yield least-square estimates of the maximal current (Imax) and of the half-activation constant for K+ (K1/2 K+). A Hill coefficient (nH) of 1.6 was used as previously described (24). The K+ activation of pump currents mediated by Na+-K+ pumps expressed in oocytes was determined after subtracting the currents due to endogenous oocyte Na+-K+ pumps.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Functional cell surface expression of AL1 or Na+-K+-ATPase alpha 1 associated with different beta -subunits. By using the Xenopus oocyte as an expression system, we have previously established (for review see Ref. 13) that all beta -subunits can act as chaperones for the structural maturation of Na+-K+-ATPase alpha 1-subunits (alpha 1). On the other hand, only the gastric H+-K+-ATPase beta -subunit (beta HK) and the Na+-K+-ATPase beta 2-like Bufo bladder beta -subunit (beta  bl), but not the Na+-K+-ATPase beta 1-subunit (beta 1), can efficiently associate with AL1 and form stable AL1/beta complexes (15). In the present study, we have tested the cell surface expression and transport properties of AL1 and alpha 1 associated with different beta -subunits. As revealed by electrophysiological measurements, coexpression of beta HK with alpha 1 led to a reduced Na+-K+ pump activity compared with that produced after coexpression with beta 1 (Fig. 1A, compare lanes 1 and 2), which, as shown below, is likely due to the lower apparent K+ affinity of alpha 1/beta HK complexes. Similar to results obtained with nongastric Bufo bladder H+-K+-ATPase (4), the transport activity of AL1/beta HK complexes could not be revealed by electrophysiological measurements (data not shown) due to lack of electrogenicity. On the other hand, 86Rb uptake measurements showed that, despite its moderate association efficiency, beta 1 produces a number of K+-transporting AL1 complexes at the cell surface similar to beta HK or beta  bl (Fig. 1B, lanes 1, 3, and 4). As previously described (34), 1 mM ouabain partially inhibited the transport function of AL1/beta complexes (lane 2).


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Fig. 1.   Functional cell surface expression of the catalytic subunit of human nongastric H+-K+-ATPase ATP1al1 (AL1) and of the Na+-K+-ATPase alpha 1-subunit (alpha 1) coexpressed with different beta -subunits in Xenopus oocytes. A: Na+-K+ pump current measurements. After 3 days of incubation of noninjected Xenopus oocytes or 3 days after injection of oocytes with alpha 1 together with beta 1 or beta HK cRNAs, the maximal Na+-K+ pump current (Imax) was measured as described in MATERIALS AND METHODS in the presence of 10 mM K+. The current measured in noninjected oocytes was subtracted from that measured in cRNA-injected oocytes. Represented are means ± SE from 7-15 oocytes obtained from 2 different animals. * P < 0.05, lane 2 vs. lane 1. B: 86Rb uptake by AL1/beta complexes. Three days after injection of Xenopus oocytes with AL1 together with beta HK, Bufo bladder beta -subunit (beta  bl), or beta 1 cRNAs, 86Rb uptake was measured as described in MATERIALS AND METHODS in the presence of 1 µM ouabain, which inhibits endogenous, oocyte Na+-K+ pump activity. In lane 2, the inhibitory effect of 1 mM ouabain on AL1/beta HK complexes is shown. Represented are means ± SE from 7-11 oocytes obtained from 2 different animals. * P < 0.01, lane 2 vs. lane 1.

beta -Isoforms differentially influence the K+ activation of Na+-K+ pumps but not of nongastric H+-K+ pumps. To reveal a possible influence of beta -subunits on the transport properties of AL1, we compared the K+ activation of pump activities mediated by alpha 1 or AL1 coexpressed with different beta -subunits in Xenopus oocytes. As shown in Fig. 2 and in previous studies (18, 22), alpha 1 associated with beta HK produced Na+-K+ pumps with a significantly reduced K+ affinity compared with that of alpha 1 associated with beta 1 (Fig. 2, lanes 3 and 4). On the other hand, the K1/2 value for K+ was similar for AL1 associated with beta HK or beta 1 (Fig. 2, lanes 1 and 2), indicating that the K+ effect of beta -isoforms is less pronounced in nongastric H+-K+-ATPase than in Na+-K+-ATPase.


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Fig. 2.   Apparent K+ affinity of AL1 and alpha 1 coexpressed with different beta -subunits in Xenopus oocytes. Three days after injection of Xenopus oocytes with AL1 or alpha 1 cRNAs together with beta 1 or beta HK cRNAs, 86Rb uptake (solid bars) or Na+-K+ pump currents (hatched bars) were measured in the presence of different concentrations of K+, and the affinity constants for K+ (K1/2 K+) were calculated as described in MATERIALS AND METHODS. Represented are means ± SE from 13-20 oocytes obtained from 2 different animals. P < 0.01, lanes 1, 2, and 4 vs. lane 3.

Na+ transport function of AL1 and alpha 1/beta complexes. It has been reported that expression of colonic H+-K+-ATPase in Xenopus oocytes (10) or of AL1 in HEK cells (17) lowers [Na+]i, suggesting that nongastric H+-K+-ATPases may transport Na+ in exchange for K+. We compared the Na+ transport activity of AL1/beta HK, AL1/beta 1, and alpha 1/beta 1 complexes in oocytes in which [Na+]i was increased by the coexpression of the amiloride-sensitive epithelial Na+ channel. [Na+]i amounted to ~80 or 20 mM in oocytes expressing beta -subunits alone and treated (Fig. 3A, lane 1) or not (Fig. 3B, lane 1) with 1 µM ouabain to inhibit endogenous oocyte Na+-K+-ATPase. Similar to the colonic H+-K+-ATPase alpha -subunit coexpressed with beta HK (17), oocyte-expressed AL1/beta HK (Fig. 3A) or AL1/beta 1 (data not shown) complexes produced a significant, more than twofold decrease in the intracellular Na+ content (compare lane 3 to lane 1), which was comparable to that produced by Bufo alpha 1/beta 1 complexes (lane 2) or by human alpha 1/beta 1 complexes (Fig. 3B, compare lane 2 with lane 1). The specificity of the observed effect is supported by the fact that gastric alpha HK/beta HK complexes did not decrease significantly [Na+]i compared with control oocytes, despite an expression similar to that of AL1/beta complexes (data not shown).


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Fig. 3.   Na+ transport of AL1. A: AL1/beta complexes can reduce intracellular Na+ concentration ([Na+]i). Xenopus oocytes were injected with beta HK cRNA, with Bufo alpha 1 and beta 1 cRNAs, with AL1 and beta HK cRNAs, or with alpha HK and beta HK cRNAs together with cRNAs for the 3 subunits of the renal epithelial Na+ channel. Three days after cRNA injection, [Na+]i was determined as described in MATERIALS AND METHODS in the presence of 1 µM ouabain to inhibit the transport activity of endogenous, oocyte Na+-K+ pumps. Represented are means ± SE from 15-20 oocytes obtained from 2-4 different animals. * P < 0.01, lanes 2 and 3 vs. lane 1. B: reduction of [Na+]i by human alpha 1/beta 1 complexes. Xenopus oocytes were injected with beta 1 cRNA or with alpha 1 and beta 1 cRNAs together with cRNAs for the 3 subunits of the renal epithelial Na+ channel. [Na+]i was determined as described in MATERIALS AND METHODS in the absence of ouabain. Represented are means ± SE from 15-20 oocytes obtained from 2-4 different animals. * P < 0.01, lane 2 vs. lane 1. C: K+ transport of AL1/beta complexes depends on [Na+]i. Xenopus oocytes were injected with AL1 and beta HK cRNAs together with cRNAs for renal epithelial Na+ channel subunits. Three days after cRNA injection, [Na+]i was varied and determined as described in MATERIALS AND METHODS, and 86Rb uptake was measured in the presence of 1 µM ouabain. Represented are the results of 4 different experiments, and means ± SE were obtained from 8-10 oocytes. Inset: intracellular pH (pHi) measured in oocytes shown in C at 3 different ranges of [Na+]i. D: extracellular Na+ influences K+ transport of AL1/beta complexes. Three days after injection of Xenopus oocytes with AL1 and beta HK or beta 1 cRNAs, 86Rb uptake was measured in the presence of 1 µM ouabain and 0.5 mM K+ either in the absence (open bars) or presence (solid bars) of 90 mM extracellular Na+. Represented are means ± SE from 16-20 oocytes obtained from 2 different animals. * P < 0.05, plus extracellular Na+ vs. without extracellular Na+.

To analyze whether the Na+ efflux, mediated by AL1/beta HK complexes, is directly coupled to K+ influx, we measured the 86Rb uptake into oocytes that were loaded with different Na+ concentrations. As shown in Fig. 3C, the AL1/beta HK-mediated 86Rb flux is Na+ dependent and is half-maximally stimulated at ~9 mM Na+. pHi values, measured in oocytes with different Na+ loads, were not significantly different and ranged between 7.4 and 7.8 (Fig. 3C, inset), supporting the possibility that modulation of 86Rb flux is indeed mediated by changes in [Na+]i and not by changes in pHi.

Finally, to further characterize the Na+ transport function, we investigated the effect of extracellular Na+ on the K+ transport activity of AL1/beta HK and AL1/beta 1 complexes. Compared with 86Rb uptake measured in the presence of 90 mM extracellular Na+, 86Rb uptake (at 0.5 mM K+) by both complexes was slightly but significantly increased in the absence of extracellular Na+. This result may indicate that, similar to Na+-K+-ATPase (18), extracellular Na+ influences the apparent K+ affinity of AL1/beta complexes due to competition of Na+ and K+ for ion binding to extracellular sites.

Controlled proteolysis to probe the ligand dependence of AL1/beta , alpha 1/beta , and alpha 1-AL1/beta complexes. By using purified Na+-K+-ATPase preparations, it was previously shown that the proteolytic sensitivity and the production of specific proteolytic fragments of the alpha -subunit change in the presence of different ligands, as a reflection of its ligand-dependent conformational changes (25). We developed a controlled trypsinolysis assay to further characterize the substrate dependence of AL1 and to compare the influence of different beta -subunits on the functional properties of alpha 1 and AL1 proteins expressed in Xenopus oocytes. Because the antibodies used to detect alpha 1, and in particular AL1, did not efficiently recognize proteolytic alpha -fragments, the analysis was mainly based on the extent of the overall proteolytic sensitivity of the alpha -subunits in the presence of different ligands. With the exception of a small shift in the molecular mass of beta 1, beta -subunits were not significantly digested in any of the proteolysis conditions (Fig. 4, A and B, lanes 10-18). On the other hand, as expected (14), all alpha -subunits expressed without a beta -subunit were completely digested by trypsin under all conditions (data not shown).


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Fig. 4.   Ligand-dependent conformational changes of alpha 1/beta complexes probed by controlled trypsinolysis. The alpha 1 and beta 1 (A) or alpha 1 and beta HK (B) cRNAs were injected into Xenopus oocytes, and, after metabolic labeling for 24 h in the presence of brefeldin A, microsomes were prepared and aliquots were subjected to controlled trypsinolysis as described in MATERIALS AND METHODS in the presence of the indicated ligands. After trypsinolysis, samples were either first immunoprecipitated with alpha -antibodies before gel electrophoresis (lanes 1-9) or directly subjected to SDS-PAGE after Endo H treatment (lanes 10-18). Indicated are the positions of alpha 1 and of deglycosylated beta 1 and beta HK. The tryptic pattern of alpha -subunits were similar in oocytes that were not treated with brefeldin A (data not shown). Dots indicate the positions of ligand-dependent tryptic fragments of the alpha -subunit observed in immunoprecipitated (lanes 1-9) or nonimmunoprecipitated (lanes 10-18) samples. Shown is a representative example out of 2-4 similar experiments. C: quantification of data represented in A and B. Represented is the percentage of the trypsin-resistant alpha -subunit remaining after trypsinolysis in the presence of different ligands. Solid bars, alpha 1/beta 1 complexes; open bars, alpha 1/beta HK complexes; ouab, ouabain.

As a basis for comparison, we first applied controlled trypsinolysis to alpha 1 expressed in Xenopus oocytes with beta 1 or beta HK. Figure 4, A and C, shows that alpha 1, coexpressed with beta 1, produced ~20% trypsin-resistant alpha -subunits at a trypsin-to-protein ratio of 0.05 and in the presence of Mg2+. Addition of K+ and/or ATP increased its trypsin resistance by about twofold. The highest stability of alpha 1 was achieved when Na+ was combined with Mg2+/ATP. Ouabain influenced the overall stability of alpha 1 neither in the presence of Na+/Mg2+/ATP nor in the presence Mg2+/Tris-Pi. However, the ligand-dependent proteolytic pattern of immunoprecipitated (Fig. 4A, compare lanes 3 and 7) or nonimmunoprecipitated (compare lanes 12 and 16, lanes 11, 17, and 18) alpha 1 changed in the presence of ouabain, indicating that it delicately influences the conformational state of alpha 1/beta 1 complexes.

Association of alpha 1 with beta HK, instead of beta 1, resulted in a significant reduction in the overall trypsin resistance of alpha 1 in all ionic conditions (Fig. 4, B and C), which may be responsible for the pronounced change of ligand-dependent proteolytic patterns (compare Fig. 4, A and B). Moreover, in contrast to alpha 1 associated with beta 1, alpha 1 associated with beta HK failed to increase its trypsin resistance in response to K+ in the presence of Mg2+ (Fig. 4B, compare lanes 2 and 4, and Fig. 4C). Altogether, these results are consistent with the observed difference in K+ affinity between alpha 1/beta HK and alpha 1/beta 1 complexes (Fig. 2).

The results of these experiments validate the use of controlled trypsinolysis as a tool to probe the ligand-dependence of alpha -subunits of P-type ATPases synthesized in intact cells and confirm the significant effect of beta -isoforms on the ligand-dependent conformational changes of the Na+-K+-ATPase alpha -subunit. Based on these results, we used controlled proteolysis assays to assess the ligand spectrum of AL1 and its dependence on the type of the associated beta -subunit.

K+-dependent conformational changes of AL1 in the presence of different beta -subunits. To analyze whether the well-established ATP-dependent K+ transport of nongastric H+-K+-ATPases is reflected in ligand-dependent conformational changes of AL1, we performed controlled proteolysis assays on AL1 associated with different beta -subunits in the presence of K+, Mg2+, and/or ATP. In general, AL1 associated with beta HK was more trypsin sensitive than AL1 associated with beta  bl or beta 1 (Fig. 5, A-E). This result compares with the higher trypsin sensitivity of alpha 1/beta HK than of alpha 1/beta 1 complexes (compare Fig. 4C and Fig. 5, D and E). However, in contrast to alpha 1 in alpha 1/beta HK complexes (Fig. 4, B and C), AL1 in AL1/beta HK complexes significantly increased its trypsin resistance in the presence of K+/Mg2+ compared with that in the presence of Mg2+ alone (Fig. 5, A and D). This result may reflect the differences in the apparent K+ affinity of alpha 1 and AL1 associated with beta HK (Fig. 2). Similar to AL1 associated with beta HK, AL1 associated with beta  bl or beta 1 increased its trypsin resistance in the presence of K+ or K+/Mg2+ compared with that in the presence of Mg2+ alone (Fig. 5E). Because beta 1 but not beta HK can support a stabilizing effect of K+ on alpha 1 in the presence of Mg2+, but all beta -subunits produce a stabilizing effect on AL1 under these conditions, it may be suggested that beta -isoforms have a less pronounced effect on the cation affinity of AL1 than on that of alpha 1. This conclusion is also supported by the similar apparent K+ affinity of AL1/beta HK and AL1/beta 1 complexes (Fig. 2).


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Fig. 5.   K+-dependent conformational changes of AL1/beta HK, AL1/beta bl, and AL1/beta 1 complexes. AL1 and beta HK (A), AL1 and beta  bl (B), or AL1 and beta 1 (C) cRNAs were injected into Xenopus oocytes and subjected to controlled trypsinolysis as described in Fig. 4 and MATERIALS AND METHODS in the presence of the indicated ligands. Shown is a representative example out of 2-6 similar experiments. D: quantifications of data obtained with AL1/beta HK complexes shown in A. Represented are means ± SE (n = 3-6) with the exception of the ligandless and Mg2+/ATP conditions (n = 2). * P < 0.01. E: quantifications of data obtained with AL1/beta bl complexes (shaded bars) shown in B and of data obtained with AL1/beta 1 complexes (open bars) shown in C. Represented are means ± SE for AL1/beta bl under Mg2+ and K+/Mg2+ conditions (n = 3-4). * P < 0.05; no SE given for AL1/beta 1 complexes (n = 2).

In the sole presence of ATP, AL1 was highly trypsin sensitive in all AL1/beta complexes (Fig. 5, A-E). Addition of K+ but not Mg2+ increased the trypsin resistance of AL1 associated with beta HK (Fig. 5A, lanes 6-8, Fig. 5D), whereas AL1 associated with beta  bl and beta 1 became more trypsin resistant with additional K+ as well as Mg2+ (Fig. 5, B and C, lanes 6-8, and E). In conclusion, the results obtained on ligand-induced conformational changes of AL1 are consistent with the ability of AL1-beta complexes to perform Mg2+/ATP-dependent K+ transport. Furthermore, our results suggest that, in nongastric H+-K+-ATPases, beta -isoforms have a less pronounced K+ effect than in Na+-K+-ATPase but nevertheless influence differentially the conformational state under certain ligand conditions.

Na+-dependent conformational changes of AL1. To provide further evidence for the Na+ transport capacity of AL1-beta complexes, we next examined whether AL1/beta complexes could undergo Na+-dependent conformational changes. The trypsin resistance of AL1 in AL1/beta bl and AL1/beta 1 complexes, which are intrinsically more stable than AL1/beta HK complexes, tended to decrease in the presence of Na+ or Na+/Mg2+ compared with that in the presence of Mg2+ (Fig. 6B, lanes 2-7, and Fig. 6D). On the other hand, the trypsin resistance of AL1/beta HK complexes rather increased when Na+ was present alone or together with Mg2+/ATP (Fig. 6A, lanes 3 and 7, and Fig. 6C). However, this Na+ effect was statistically not significant, probably due to the high intrinsic trypsin sensitivity of AL1 in beta HK complexes, which renders quantifications difficult. Overall, these results support that AL1/beta complexes interact with Na+ and respond with a conformational change that is, however, more discreet than that induced by K+.


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Fig. 6.   Na+-dependent conformational changes of AL1/beta HK, AL1/beta bl, and AL1/beta 1 complexes. AL1 and beta HK (A), AL1 and beta  bl (B), or AL1 and beta 1 cRNAs were injected into Xenopus oocytes and subjected to controlled trypsinolysis as described in Fig. 4 and MATERIALS AND METHODS in the presence of the indicated ligands. Shown is a representative example of 2-6 similar experiments. C: quantifications of data with AL1/beta HK complexes represented in A. Represented are means ± SE (n = 3-6) with the exception of the ligandless and Mg2+/ATP conditions (n = 2). D: quantifications of data obtained with AL1/beta bl complexes (shaded bars) shown in B and of data obtained with AL1/beta 1 complexes (open bars; data not shown). Represented are means ± SE (n = 3-4) with the exception of the ligand-less and Mg2+/ATP conditions (n = 2). * P = 0.02; SE given for AL1/beta 1 complexes (n = 2).

Ouabain-dependent conformational changes of AL1. AL1-beta complexes are moderately sensitive to ouabain (34). To verify whether this property is reflected in a conformational change of AL1, we performed controlled proteolysis assays on AL1/beta HK, AL1/beta bl, and AL1/beta 1 complexes in ouabain/Na+/ATP or ouabain/Mg2+/Tris-Pi conditions. Compared with the control conditions (Na+/ATP, Mg2+/Tris-Pi), ouabain had only slight or no effect on the trypsin sensitivity of AL1/beta complexes (data not shown). A conformational effect of ouabain on AL1 was most apparent in AL1/beta bl complexes, in which ouabain decreased the trypsin resistance of AL1 in the presence of Mg2+/Pi compared with that in the presence of Mg2+/Tris-Pi alone (Fig. 7, lanes 1-5). Under these conditions, ouabain also modulated the production of distinct proteolytic fragments produced under control conditions and visible either in immunoprecipitated (Fig. 7, lanes 2 and 3) or in nonimmunoprecipitated (lanes 4 and 5) AL1 preparations. Thus, at least in AL1/beta bl complexes, the moderate ouabain sensitivity is reflected in a conformational change of AL1.


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Fig. 7.   Ouabain-dependent conformational changes of AL1/beta bl complexes. AL1 and beta  bl cRNAs were injected into Xenopus oocytes and subjected to controlled trypsinolysis as described in Fig. 4 and MATERIALS AND METHODS in the presence of the indicated ligands. Samples were either first immunoprecipitated with AL1 antibodies before gel electrophoresis (lanes 1-3) or directly subjected to SDS-PAGE (lanes 4 and 5). Shown is a representative example of 2-3 similar experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study provides new information on two pending questions concerning the structure-function relationship in nongastric H+-K+-ATPases, namely, on their ability to transport Na+ and on the influence of different beta -isoforms on their functional properties.

Na+ transport function of AL1. It has recently been reported that rat colonic H+-K+-ATPase (10) or human AL1/beta HK complexes (17) expressed in Xenopus oocytes or in HEK-293 cells, respectively, decrease [Na+]i, suggesting that nongastric X+-K+-ATPases may not only transport H+ but also Na+ in exchange for K+. On the other hand, measurements of the Na+ dependence of ATPase activity of nongastric H+-K+-ATPases have produced conflicting results. Whereas AL1-beta HK complexes expressed in Sf21 insect cells exhibit an ATPase activity that is inhibited rather than stimulated by Na+ (1), a Na+-dependent K+-ATPase activity was demonstrated in apical membranes of distal colon (8). In this latter study, it can, however, not entirely be excluded that a contaminating fraction of Na+-K+-ATPase in the apical membrane preparation participates in the measured Na+-dependent ATPase activity. In any case, both results, activation or inhibition of ATPase activity, are consistent with Na+ binding to nongastric H+-K+-ATPases.

Our results presented in this study lend further support for the interaction of Na+ with nongastric X+-K+-ATPases and in particular for the transport of Na+ by these pumps. We indeed show that, as for the colonic H+-K+-ATPase (10), AL1/beta complexes expressed in Xenopus oocytes can decrease [Na+]i to an extent similar to Na+-K+-ATPase. More importantly, we document that Na+ efflux is indeed mediated by AL1, since it is coupled to K+ uptake, with a half-maximal stimulation of K+ uptake at ~9 mM [Na+]i, in the absence of significant changes in pHi. The ability of AL1 to transport Na+ is also supported by the observation that Na+ can induce conformational changes in AL1, as probed by a controlled trypsinolysis assay, and that it may compete for K+ binding at external sites similar to those for Na+-K+-ATPase.

Na+ transport adds to the previously established H+ and NH<UP><SUB>4</SUB><SUP>+</SUP></UP> transport of nongastric H+-K+-ATPases. Whereas Na+ and H+ are exchanged with K+, the question whether nongastric H+-K+-ATPases exchange (36) and/or cotransport (8, 9) NH<UP><SUB>4</SUB><SUP>+</SUP></UP> with K+ is not resolved. Also, little is known about the stoichiometry of the exchange of the different ions with K+. It has been observed that K+ influx mediated by AL1 expressed in HEK-293 cells is much greater than the H+ efflux (16) but, on the other hand, the transport of nongastric Bufo H+-K+-ATPase expressed in Xenopus oocytes is electroneutral (4). Theoretically, it is possible that all ions are transported simultaneously, but it may be expected that Na+, H+, and NH<UP><SUB>4</SUB><SUP>+</SUP></UP> compete for common binding sites and that the proportion of each ion transported depends on changes in intra- and extracellular ion concentrations. It has been suggested that, in contrast to gastric H+-K+-ATPase, colonic H+-K+-ATPase in the kidney does not play a role in H+ secretion or K+ reabsorption under normal physiological conditions (for review and references see Refs. 44 and 47). However, colonic H+-K+-ATPase shows adaptive regulation in pathophysiological states such as K+ and Na+ depletion or proximal renal tubular acidosis, suggesting an important role for this exchanger in K+, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and Na+ reabsorption. A recent report proposes that, under conditions of K+ depeletion, the NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-transport-competent colonic H+-K+-ATPase in the renal collecting duct replaces the NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-transport-incompetent gastric H+-K+-ATPase and enhances NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (and H+) secretion into the urinary space facilitated by K+, which is recycled through ROMK-1 channels (36). On the other hand, it has been suggested that, in the colon, nongastric H+-K+-ATPase is responsible for NH<UP><SUB>4</SUB><SUP>+</SUP></UP> absorption (9). Similar to NH<UP><SUB>4</SUB><SUP>+</SUP></UP> transport, the potential physiological or pathophysiological relevance of the Na+ transport capacity of nongastric X+-K+-ATPases has not yet been elucidated. Nongastric H+-K+- ATPases may be indirectly involved in increased Na+ reabsorption in Na+-depleted conditions (44, 45), but it is unlikely that Na+ transport of nongastric H+-K+-ATPases plays a role, since urinary Na+ is decreased and not increased in this condition (44). As observed in our study, AL1 transport activity is strongly stimulated by changes in intracellular Na+. Therefore, future studies on the putative physiological role of Na+ transport should concentrate on examination of the exchange mode of nongastric H+-K+-ATPases under salt-loaded conditions in which nongastric H+-K+- ATPases may favor Na+ secretion in certain nephron segments and the colon. In this context, the interesting hypothesis has been put forward that, in macula densa cells, which apparently lack functionally and immunologically detectable Na+-K+-ATPase, apical colonic H+-K+-ATPase may regulate [Na+]i (38).

The functional role of beta -isoforms in AL1. beta -Subunits of X+-K+-ATPases are necessary for maturation of the enzymes and were shown to influence the intrinsic transport properties of gastric H+-K+- and Na+-K+-ATPases (13). Authentic beta -subunits in gastric H+-K+-ATPase and in the three Na+-K+-ATPase isozymes have been identified and are represented by one gastric H+-K+-ATPase and three Na+-K+-ATPase beta -isoforms. Significantly, for nongastric H+-K+-ATPase, no specific beta -subunit has been identified and the real subunit composition in situ is still unknown. Indeed, both Na+-K+-ATPase beta 1 (6, 29) and beta 3 (40) isoforms have been suggested as the real partner subunit of nongastric H+-K+-ATPase in native tissue based on immunological evidence. These predictions are, however, not supported by the observation that only beta HK and Na+-K+-ATPase beta 2-like isoforms, but neither beta 1 nor beta 3, can act as efficient chaperones for the maturation of nongastric H+-K+-ATPase alpha -subunits after expression in different cells (2, 15, 39). On the other hand, in agreement with Codina et al. (7), we observed in this study that expression of a nongastric H+-K+-ATPase with beta 1-subunits produces a number of functional pumps at the cell surface of Xenopus oocytes similar to that produced after expression of AL1 with gastric beta HK, despite the poor chaperone activity of beta 1. This result indicates that, under conditions of marked overexpression as can be achieved in Xenopus oocytes, a certain number of AL1/beta 1 complexes, which based on their poor stability must be partially misfolded, can escape the endoplasmic reticulum (ER) quality control that normally retains these proteins in the ER. Because Xenopus oocytes have a limited capacity for cell surface expression of endogenous and exogenous proteins (3, 42), it may result that a similar number of inefficiently assembled AL1/beta 1 and efficiently assembled AL1/beta HK become expressed at the cell surface. Thus the functional expression of AL1/beta 1 complexes in oocytes cannot be used as an argument to support beta 1 as the authentic subunit of nongastric H+-K+-ATPases in situ.

In view of the uncertainty about the real subunit composition, it is important to know whether different beta -isoforms differentially influence the intrinsic functional properties of nongastric H+-K+-ATPases to be able to attribute a physiological relevance to kinetic and transport properties determined on different nongastric H+-K+-ATPase alpha /beta complexes artificially expressed in cells. Interestingly, we found that, in contrast to gastric H+-K+-ATPase or Na+-K+-ATPase (for review see Ref. 13), the transport properties of AL1 are not significantly affected by the association of different beta -isozymes. In agreement with previous observations that showed that colonic H+-K+-ATPase associated with beta HK or beta 1 have indistinguishable affinities for K+ or ouabain (7), we observed that the K1/2 value for K+ and the ability to transport Na+ are similar in AL1/beta HK and AL1/beta 1 complexes expressed in Xenopus oocytes. The less important cation effect of beta -isoforms on AL1 than on Na+-K+-ATPase alpha -subunits is also reflected by controlled proteolysis assays, which show that, for example, the K+-dependent conformational changes of AL1 associated with beta HK or beta 1 are similar, whereas those of alpha 1 associated with these beta -isoforms differ. The reasons for the apparent differences between the influence of beta -isoforms on gastric H+-K+-ATPase and Na+-K+-ATPase, on the one hand, and on nongastric H+-K+-ATPases, on the other hand, are so far not known. However, it is possible that this is a reflection of the intermediate position of nongastric H+-K+-ATPases between Na+-K+- and gastric H+-K+-ATPases with respect to their structural, functional, and pharmacological characteristics. A consequence of the observed lack of a significant functional effect of beta -isoforms on nongastric H+-K+-ATPases is that, despite the uncertainty of the authentic beta -subunit, the functional properties of nongastric H+-K+-ATPases determined in various expression systems with different beta -subunits are likely to be physiologically relevant.

According to our results, controlled proteolysis assays represent a valuable, additional experimental tool not only to further refine the analysis of beta -dependent and beta -independent ligand interactions in X+-K+- ATPases but also to study the mechanistic basis of the beta -subunit effects on their intrinsic structural and functional properties. Indeed, our proteolysis assays show that, compared with beta 1, beta HK has a pronounced negative effect on overall trypsin resistance, and thus conformational stability, of both Na+-K+-ATPase and nongastric H+-K+-ATPase alpha -subunits. Potentially, this characteristic of beta HK assembly represents an argument against a promiscuous association of beta HK with nongastric H+-K+-ATPases or Na+-K+-ATPase alpha -subunits in situ.

In conclusion, based on the results of transport studies and of controlled proteolysis assays, which reflect ligand-dependent conformational changes, the present study provides new evidence that nongastric H+-K+-ATPases can transport Na+ in exchange for K+. Moreover, our results reveal that the type of beta -subunit associated has a global structural influence on nongastric H+-K+-ATPase alpha -subunits similar to that on Na+-K+-ATPase alpha -subunits but that beta -subunits influence to a lesser extent the transport properties of nongastric H+-K+-ATPases than those of Na+-K+-ATPase.


    ACKNOWLEDGEMENTS

We thank Sophie Roy and Daniele Schaer for excellent technical assistance. We thank G. Sachs for the cDNAs coding for rabbit gastric H+-K+-ATPase alpha - and beta -subunits, K. Kawakami for the cDNA coding for human Na+-K+-ATPase beta 1-subunit, and F. Jaisser for the cDNA coding for the beta -subunit of Bufo bladder.


    FOOTNOTES

This work was supported by Swiss National Fund for Scientific Research Grants 31-53721.98 and 31-64793.01 (to K. Geering) and National Heart, Lung, and Blood Institute Grant HL-36573 (to N. N. Modyanov).

Address for reprint requests and other correspondence: K. Geering, Institute Of Pharmacology and Toxicology, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland (E-mail: kaethi.geering{at}ipharm.unil.ch).

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.

First published March 20, 2002;10.1152/ajpcell.00590.2001

Received 11 December 2001; accepted in final form 17 March 2002.


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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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