INTRODUCTION

TOP ABSTRACT INTRODUCTION THE NA-K/H-K-ATPASE GENE FAMILY THE NONGASTRIC H-K-ATPASES:... THE NONGASTRIC H-K-ATPASES:... DISCREPANCIES BETWEEN THE... CONCLUSION REFERENCES

IN 1987, FIVE HUMAN GENES related to the Na-K-ATPase gene family were isolated using an Na-K-ATPase -subunit cDNA probe (53, 72, 75). Three of them encode Na-K-ATPase -subunit isoforms. A fourth one encodes an H-K-ATPase -subunit that has been isolated from rat colonic, amphibian toad bladder, or human skin cDNA libraries. Further cloning experiments allowed the identification of species variants in guinea pig and rabbit. The functional properties of these nongastric H-K-ATPases have been analyzed in expression systems such as the Xenopus laevis oocyte, the insect Sf9 cells, and the mammalian HEK 293 cells. Excellent reviews have been published recently on the subject (46, 81). The purpose of the present review is to describe our current knowledge of the molecular characteristics of these H-K-ATPase -subunits. Functional properties will be reported, on the basis of studies performed using exogenous expression in functional expression systems. These data indicate that the nongastric H-K-ATPase may be identified as a subgroup of the Na-K/H-K-ATPase gene family, apart from the Na-K-ATPase and the gastric H-K-ATPase subgroups. Comparison of the functional data with the physiological data reviewed in the accompanying paper by Silver and Soleimani (73a) reveals some discrepancies that will be discussed.

	THE NA-K/H-K-ATPASE GENE FAMILY

TOP ABSTRACT INTRODUCTION THE NA-K/H-K-ATPASE GENE FAMILY THE NONGASTRIC H-K-ATPASES:... THE NONGASTRIC H-K-ATPASES:... DISCREPANCIES BETWEEN THE... CONCLUSION REFERENCES

Classification

View larger version (22K): [in this window] [in a new window]   Fig. 1.   The Na-K/H-K-ATPase -subunit gene family is subdivided in three subgroups: the Na-K-ATPase, the gastric H-K-ATPase, and the nongastric H-K-ATPase. A: overall amino acid homologies between the -subunits of the three subgroups, indicating that the nongastric H-K-ATPase -subunits are equally related to (or divergent from) the Na-K-ATPase or the gastric H-K-ATPase -subunits. B: tree of sequences of various -subunits of the Na-K/H-K-ATPase gene family. For simplicity, only human, rat, Xenopus ("xen"), or Bufo marinus species variants are indicated. Length of branches indicates degree of divergence between two subgroups or two subunits. Three different subgroups appear early during evolution from a common ancestor gene. Nongastric H-K-ATPases probably cannot be considered as isoforms of the gastric H-K-ATPases, since these are as divergent from the gastric H-K-ATPases as they are from the Na-K-ATPases. The relationship between the various members of the nongastric H-K-ATPase subgroup is unclear. For example, the rat HK2 and human HK4 are as divergent as the rat NK1 and the rat HK2 and significantly more divergent than the rat and human NK1. This could suggest that rat HK2 and human HK4 are isoforms rather than species variants. The tree has been constructed using the PILEUP program of the Genetic Computer Group (GCG) software package (27).

Among each subgroup, several subunits have been characterized so far (Fig. 1B). For the Na-K-ATPase, these different -subunits can be identified as isoforms since 1) different genes have been characterized, 2) subunit-specific protein domains have been identified, and 3) subunit-specific functional properties have been observed. For the gastric H-K-ATPase subgroup, only one -subunit has been characterized in various species. It is referred to as HK1.

For the nongastric H-K-ATPases, which are the focus of the present review, several species variants have been cloned. In the currently accepted nomenclature (46, 81), the rat colonic H-K-ATPase is referred to as HK2, the toad bladder H-K-ATPase as HK3, and the human H-K-ATPase (ATP1AL1) as HK4. The recently characterized guinea pig colonic H-K-ATPase (GenBank accession no. D21854) would have to be referred to as HK5 and the one of the rabbit (31) as HK6. By analogy with the Na-K-ATPase -subunit classification, this nomenclature suggests that the rat, human, toad, and guinea pig -subunits are subunit isoforms. In the currently accepted nomenclature, the nongastric subunits are also considered to be isoforms of the gastric H-K-ATPase -subunit (HK1). However, it seems difficult to make such analogies between the H-K-ATPases, including gastric and nongastric H-K-ATPases, and the Na-K-ATPases for which true isoforms have been identified, both at the molecular and functional levels. The molecular and functional data reported in the literature and summarized in the present review indicate that it may be necessary to change the nomenclature in the future and more clearly distinguish the H-K-ATPase expressed in the stomach from those expressed in other organs.

Relationship Between Nongastric H-K-ATPases

At the functional level, characterizations of the rat, human, toad, and guinea pig -subunits do not reveal strong differences. The only functional difference observed so far deals with the ouabain sensitivity of the nongastric H-K-ATPases (see below). It should be noted, however, that the difference between rat HK2 and human HK4 is similar to that observed between rat NK1 and human NK1, two subunits that are not considered as isoforms. Therefore the current molecular and functional data indicate that the relationship between the various members of the nongastric H-K-ATPases remains unclear and needs to be directly addressed. As discussed below, molecular cloning of new members in the same species and/or comparative functional analysis will certainly help to clarify this point.

	THE NONGASTRIC H-K-ATPASES: MOLECULAR CHARACTERIZATION

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Nongastric H-K-ATPase -Subunits

The extensive description of the amino acid homologies between the subgroups is beyond the scope of the present review. Structure-function relationship analysis of the nongastric H-K-ATPase -subunit has not been performed so far. However, on the basis of previous studies performed on the Na-K-ATPase and gastric H-K-ATPase subgroups, several domains of the nongastric H-K-ATPase -subunits could be proposed to be important to support the function of these pumps. As a basis for future structure-function relationship studies, we have included in the present review part of the amino acid comparisons between the members of the Na-K/H-K-ATPase -subunit gene family (Fig. 2). It is interesting to note that several specific amino acid domains exist. They may support functional specificities between each subgroup members. Some of these domains that appear to be of particular interest will be described.

View larger version (194K): [in this window] [in a new window]   Fig. 2.   Amino acid comparison of several domains of the -subunits of the Na-K/H-K-ATPase gene family. Numbering is arbitrary and starts with the first amino acid of the B. marinus H-K-ATPase -subunit. NK refers to the Na-K-ATPases, gastric HK to the gastric H-K-ATPases, and nongastric HK to the nongastric H-K-ATPases, respectively. Xenopus refers to the X. laevis and B. to the B. marinus. The putative transmembrane domains H1-H8 are indicated by a bold line above the sequences. For convenience, amino acids or domains discussed in the text are indicated by a bar below the sequences. This amino acid sequence alignment has been obtained using the PILEUP program of the GCG software package (27).

The intracellular NH₂-terminal domain. Truncation of the NH₂-terminal domain of the Na-K-ATPase has been reported to induce modifications in the pump cycle, resulting in a lower apparent affinity for K (25, 80). This region may act as a cation-selective gate (37). Kone and Higham (47) recently reported the existence of two splice variants of the rat colonic H-K-ATPase that differ in the NH₂-terminal domain of the Na-K-ATPase. The HK2b subunit has a truncated NH₂-terminal domain, compared with the HK2a subunit. It is not known whether the colonic HK2b functionally differs from the full-length HK2a, since expression studies in the nonpolarized HEK 293 cells did not examine this point.

Ouabain and Sch-28080 binding sites. Ouabain and Sch-28080 compounds are thought to be specific inhibitors of the Na-K/H-K-ATPases. The Na-K-ATPase is inhibited by ouabain and other digitalic glycosides but is insensitive to Sch-28080 (or omeprazole, an inhibitor of the gastric H-K-ATPase used for the treatment of peptidic ulcer), whereas the gastric H-K-ATPase is inhibited by both Sch-28080 and omeprazole, but not by ouabain. The nongastric H-K-ATPases have been reported to be sensitive to ouabain and, for some of them, to high concentrations of Sch-28080 (see below). Structure-function analyses have been extensively performed on the Na-K-ATPase and the gastric H-K-ATPase -subunits. Amino acid sequence homologies/differences between the nongastric H-K-ATPases and the other members of the Na-K/H-K-ATPase gene family may be useful to delineate the ouabain/Sch-28080 binding sites on the nongastric H-K-ATPase -subunits.

Putative cation binding sites. It is proposed that cation specificity resides in (or is highly dependent on) the structure of the transmembrane domains, which could form some kind of channel or pocket for the cations to be exchanged between the extracellular and the intracellular media (52, 79). Amino acid comparison indicates that there are more homologies between the nongastric H-K-ATPases and the Na-K-ATPases than homologies between the nongastric H-K-ATPases and the gastric H-K-ATPases. Specific amino acid domains of the gastric H-K-ATPase subgroup could be identified (Fig. 2). For example, the LQC domain of the gastric H-K-ATPase that is located in H1 is gastric H-K-ATPase specific, as well as Met¹²³ and Ala¹²⁶. Ala¹²⁶ (Glu in the Na-K-ATPases and nongastric H-K-ATPases) has been involved in the K affinity of the gastric H-K-ATPase (3). The tryptophan residue (Trp¹³¹) appears to be specific for the nongastric H-K group (Phe for Na-K-ATPase, Leu for gastric H-K-ATPase).

Sorting signals. The H4 putative transmembrane domain of the gastric H-K-ATPase has been recently reported to act as a dominant sorting signal. When the gastric H-K-ATPase H4 domain is transferred into an Na-K-ATPase chimera, it allows targeting of the mutant pump to the apical membrane rather than to the basolateral membrane of LLC-PK₁ transfected cells (28). The nongastric H-K-ATPases are believed to be targeted to the apical membrane (see below). However, the H4 transmembrane domain of the nongastric H-K-ATPases appears to be homologous to the Na-K-ATPase rather than to the gastric H-K-ATPase. Functional expression experiments in a polarized cell line are required to address this question. Indeed, recent data obtained in our laboratory suggest that, when it is expressed in a rat cortical collecting duct cell line, the rat colonic HK2a subunit is targeted to the basolateral domain (Jaisser and Beggah, unpublished results).

The putative / interaction domain. The extracellular segment between amino acid Asn⁹¹⁷ and Ala⁹⁴² has been reported to be involved in the interaction between the - and -subunits of the Na-K-ATPase (51). For the gastric H-K-ATPase, similar function was assigned to the extracellular segment between Arg⁹⁰⁸ and Arg⁹³² (59). Therefore the minimal / interaction domain may reside between amino acid residues 917 and 932. Recently, using the two-hybrid system and the alanine scanning mutagenesis approach, the results obtained in the laboratory of D. Fambrough indicate that the highly conserved SYGQ motif was required for / interactions (20). Whether the specific amino acids for each of the subgroups (like Glu⁹²⁰, Tyr⁹²⁹, Glu⁹³⁰) are important for specific / interactions remains to be analyzed. As discussed below, when expressed in heterologous expression systems, the nongastric H-K-ATPase -subunits are able to functionally associate with either the Na-K-ATPase 1-subunit, the Bufo marinus bladder -subunit, or the gastric H-K-ATPase -subunit. By contrast, the gastric H-K-ATPase -subunit appears to require specific interaction with its physiological counterparts for functional expression (34, 58), whereas the Na-K-ATPase is more permissive (41, 43). Functional expression of mutated or chimeric subunits would be interesting to determine whether in vivo interactions of the -subunit of each subgroup with a specific -subunit depend on the amino acid composition of this extracellular domain.

Putative phosphorylation sites. Protein kinase C (PKC) phosphorylation sites have been identified in the NH₂-terminal domain of the Na-K-ATPase 1-subunit (6, 8, 32). Phosphorylation may also occur (but at lower level) in the rat axolemma 3-subunit (32). Similarly, putative PKA and PKC phosphorylation motifs are found in the rat colonic H-K-ATPase -subunit (47).

H-K-ATPase -Subunits

Various P-ATPase -subunits have been characterized (37): the -subunit of the gastric H-K-ATPase; the -subunits of the Na-K-ATPase that include 1 and 2 (also called AMOG, for "adhesion molecule on glia"); a 3-subunit, first characterized in amphibian and recently isolated in human (56) and in the rat (69); and a -subunit isolated from the toad urinary bladder that may be the amphibian homolog of the mammalian Na-K-ATPase 2-subunit (40).

Immunoprecipitation using -subunit-specific antibodies (73) indicates that the Na-K-ATPase 1- and 2-subunits interact in vivo with the 1- and 3-subunits of the Na-K-ATPase, respectively, as does the gastric H-K-ATPase -subunit with the -subunit of the gastric H-K-ATPase (17). Using a rat colonic H-K-ATPase -subunit-specific antibody, it has been recently reported that the colonic H-K-ATPase -subunit physically associates with a so-called 1-subunit of the Na-K-ATPase in a renal medullary membrane preparation (18, 48) and in membrane preparation from the distal colon (18). This indicates that the 1-subunit of the Na-K-ATPase or a 1-related isoform may associate in vivo with the colonic H-K-ATPase -subunit in the kidney and in the distal colon.

The colonic H-K-ATPase -subunit has been reported to be expressed in the apical membrane of the distal colon and the principal cells of the collecting duct (50). This may be relevant to the finding of Marxer et al. (57) that described a 1-related protein in the apical membrane of the rat distal colon. Whether this 1-related protein is the known Na-K-ATPase 1-subunit or a -subunit isoform sharing a common epitope with Na-K-ATPase 1 remains to be established. Indeed, a so-called 3-subunit has been recently isolated in the rat distal colon (69). It is only 35-50% homologous to the previously characterized rat P-ATPase -subunits (69). Its colonic expression is upregulated by K depletion. This 3-subunit physically interacts with the colonic H-K-ATPase -subunit (70). Therefore, the rat colonic -subunit recently cloned by the group of H. Binder may be the proper -subunit that functionally associates in vivo in the distal colon with the colonic H-K-ATPase -subunit in case of K depletion. The respective roles and properties of the HK2/3 vs. HK2/1 heterodimers remain to be determined.

	THE NONGASTRIC H-K-ATPASES: FUNCTIONAL CHARACTERIZATION

TOP ABSTRACT INTRODUCTION THE NA-K/H-K-ATPASE GENE FAMILY THE NONGASTRIC H-K-ATPASES:... THE NONGASTRIC H-K-ATPASES:... DISCREPANCIES BETWEEN THE... CONCLUSION REFERENCES

To analyze the functional properties of the nongastric H-K-ATPases that have been characterized so far and, subsequently, to understand their physiological roles in ion homeostasis, functional expression of the -subunits of the nongastric H-K-ATPases has been done in various expression systems, including the X. laevis oocyte, the insect Sf9 cell line, and the nonpolarized human cell line HEK 293.

Functional Expression in X. laevis Oocytes

The second nongastric H-K-ATPase that was functionally expressed in Xenopus oocyte was the human skin P-ATPase. This P-ATPase -subunit is encoded by the human ATP1AL1 gene (62). Its functional properties are roughly identical to the amphibian one, i.e., it encodes a moderately ouabain-sensitive, poorly Sch-28080-sensitive H-K-ATPase. This emphasizes the high conservation of the properties of the nongastric H-K-ATPases during evolution, a feature usually considered to reflect an important and specialized role in organism homeostasis.

Functional expression of the rat colonic P-ATPase in Xenopus oocytes has been reported by two independent groups (Fig. 3). This pump is a moderately ouabain-sensitive, Sch-28080-insensitive K-ATPase (19, 23). Proton transport capacity was evaluated by Cougnon et al. (23). The authors showed that this pump could be considered as an H-K-ATPase, allowing K-dependent, ouabain-sensitive internal pH alkalinization.

The two groups also evaluated the influence of various P-type ATPase -subunits on the functional properties of the rat colonic H-K-ATPase. Codina et al. (19) showed that this -subunit could indifferently associate with the rat Na-K-ATPase 1- and the rat gastric H-K -subunit. Their functional properties are not affected by the Na-K-ATPase 1- or the gastric H-K-ATPase -subunits (19) nor by the amphibian Na-K-ATPase 3- or bladder P-ATPase -subunits (Cougnon et al., unpublished results). These results differ with what has been previously reported for the Na-K-ATPase. The Na-K-ATPase -subunit has been shown to be involved in the pump cycle through its interaction with the -subunit (12, 29, 30, 41). However, one should keep in mind that the oocyte system may be inappropriate to detect subtle differences related to pharmacological properties (see below). These data also indicate that in such an expression system, heterologous / association are possible, a feature that may reflect a permissive / interaction. It would be interesting to compare the functional properties of the colonic HK2/rat 1-subunits and the colonic HK2/rat 3-subunits. Putative distinct properties may explain the functional differences that have been reported about K-ATPase activities in the colon and in the kidney (see Table 1).

View this table: [in this window] [in a new window]   Table 1.   Comparison of the functional characteristics of the rat renal colonic H-K-ATPase -subunit

More recently, Cougnon and coworkers (21) reported evidences in support of a yet unrecognized functional property that appears to be common to all nongastric H-K-ATPases. They measured the intracellular Na (Na_i) activity in steady-state conditions in Xenopus oocytes expressing various subunits of the Na-K/H-K-ATPase gene family (21). As anticipated, steady-state Na_i was markedly decreased in oocytes expressing the B. marinus Na-K-ATPase compared with oocytes expressing the gastric H-K-ATPase or a -subunit alone (Fig. 3). Interestingly enough, a similar decrease in Na_i was also observed when the rat colonic H-K-ATPase was expressed, regardless of the coexpressed -subunit (21). This effect on Na_i depends on the presence of extracellular K. Due to electrochemical concerns, it was shown that this effect relies to an active transport process. Ouabain, 2 mM, increased Na_i value in Xenopus oocytes expressing the rat colonic H-K-ATPase or the B. marinus Na-K-ATPase, indicating that Na transport was ouabain sensitive (21). These data support the hypothesis that the nongastric H-K-ATPase may have a direct role in Na⁺ extrusion and strongly suggest that the rat colonic H-K-ATPase is indeed a (Na/H)-K-ATPase, when expressed in Xenopus oocytes. It should be emphasized that this "Na transport" capacity occurs in "physiological" experimental conditions and that this finding is specific for the nongastric H-K-ATPase and not for the canonical gastric H-K-ATPase functionally tested in the same experimental conditions. This clearly differs with the "Na transport" capacity of the gastric H-K-ATPase or the "proton transport" capacity of the Na-K-ATPase reported to occur in very special experimental conditions (65, 66).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Functional characteristics of the rat colonic H-K-ATPase -subunit, when expressed in the X. laevis oocyte. The rat colonic H-K-ATPase 2a-subunit has been expressed in Xenopus oocytes, with or without the Na-K-ATPase 1-subunit. When indicated, the B. marinus Na-K-ATPase 1-subunit has been coexpressed with the B. marinus Na-K-ATPase 1-subunit. Results were obtained from Codina et al. (19) for A or from Cougnon et al. (21) for B and C. These data indicate that 1) the rat colonic H-K-ATPase 2-subunit functionally associates with the rat Na-K-ATPase 1-subunit; 2) the 2a/1 heterodimer mediates K transport (as estimated from 86RbCl uptake); 3) the 2a/1 heterodimer mediates H transport, resulting in a strong steady-state intracellular alkalinization of the oocyte, at variance with the Na-K-ATPase 1/1 heterodimer; and 4) the 2a/1 heterodimer mediates Na transport, resulting in a strong decrease of steady-state intracellular Na activity, as does the Na-K-ATPase 1/1 heterodimer, but at variance with the gastric H-K-ATPase heterodimer. These transport properties are inhibited by ouabain (data not shown). Similar results have been obtained with the B. marinus H-K-ATPase -subunit (unpublished observations and Refs. 21 and 39). pHi, intracellular pH; Na+i, intracellular sodium. ***P < 0.001.

Interestingly, this property appears to be common to the nongastric H-K-ATPase subfamily, i.e., the rat colonic H-K-ATPase (21), the B. marinus bladder H-K-ATPase (21), and the human ATP1AL1 H-K-ATPase (35, 36). Whether Na may be transported in vivo in exchange for K by the corresponding K-ATPases remains to be examined.

Recent data collected by Cougnon and collaborators (22) indicate that understanding the physiological properties of the rat colonic H-K-ATPase (and probably those of the other nongastric H-K-ATPases) is more difficult than anticipated. Using the well-characterized oocytes as an expression system, these authors report that ammonium ions can also be transported by the rat colonic H-K-ATPase in "standard" experimental conditions (22). Ammonium competes with potassium for transport by the colonic H-K-ATPase. Similar to K⁺, NH⁺₄ appears to be exchanged for proton or Na⁺, since, in the absence of K⁺ but the presence of ammonium in the extracellular medium, proton or Na⁺ could be extruded from the oocyte intracellular compartment. Ammonium transport is also sensitive to ouabain, similar to that of K⁺, proton, or Na⁺ transports.

Ammonium transport by the colonic H-K-ATPase may be of physiological relevance in the kidney medulla in certain physiopathological conditions. Interestingly, these results contrast with the data reported recently indicating that, in K⁺ depletion, the colonic H-K-ATPase can function in NH⁺₄/K⁺ exchange mode (1). Indeed, Silver and Soleimani (73a) propose in the accompanying review that the colonic H-K-ATPase could mediate the transport of intracellular NH⁺₄ in exchange for luminal K⁺ in the case of potassium depletion, mediating secretion of NH⁺₄ in the lumen. One possible explanation for this apparent discrepancy may be that, in the inner medullary collecting duct, the rat colonic H-K-ATPase is indeed targeted to the basolateral membrane, instead of the apical membrane. If ammonium is transported in place of K⁺, then this would result in a net secretion of ammonium from the interstitium to the lumen.

Functional Expression in Sf9 Insect Cells

Functional Expression in HEK 293 Cells

Grishin and coworkers (35) showed that the proton fluxes mediated by the human ATP1AL1 H-K-ATPase was 10-fold lower than those of K⁺, suggesting that the stoichiometry of H/K transport differs from 1/1 or that this observation results from the exchange of another cation against K⁺. Interestingly, the full inhibition of the endogenous ouabain-sensitive Na-K-ATPase that leads to cell death in wild-type HEK 293 cells was compensated by the expression of the skin ATP1AL1 K-ATPase (35, 36) or the rat colonic H-K-ATPase (47). This result supports the fact that the nongastric H-K-ATPases may also transport Na⁺, resulting in a (Na/H)-K-ATPase, as proposed by Cougnon et al. (21). Grishin and Caplan (36) recently reported that the human ATP1AL1 H-K-ATPase mediates ouabain-sensitive Na⁺ efflux, when the ATP1AL1 -subunit is coexpressed with the gastric H-K-ATPase -subunit in HEK 293. Correlation with ⁸⁶Rb influx (as K⁺ surrogate) indicates that the pump formed by the human ATP1AL1 -subunit and the gastric H-K-ATPase -subunit mediates primarily Na/K rather than H/K exchange, when expressed in HEK 293 cells. Analysis of Na⁺ and proton transports by the various nongastric H-K-ATPases remains to be done by direct measurement of Na⁺ dependence and proton dependence of the K-ATPase activities mediated by the nongastric H-K-ATPases, a difficult task in expression systems such as the Xenopus oocyte or transfected cell lines.

	DISCREPANCIES BETWEEN THE FUNCTIONAL PROPERTIES OF THE CLONED NONGASTRIC H-K-ATPASES AND THOSE OF THE NATIVE COLONIC AND RENAL K-ATPASES

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The molecular and the functional characterizations of the various nongastric H-K-ATPases should help to achieve a difficult goal: to identify the molecular entities encoding the physiologically relevant K-ATPases. So far, several major discrepancies exist between the physiological studies analyzing the cell-specific expression and the functional properties of the endogenous colonic and renal K-ATPases on one hand and the functional characteristics of the cloned nongastric H-K-ATPases -subunits on the other hand.

This is outlined by Silver and Soleimani in the accompanying review (73a) and clearly shown in the data summarized in Table 1. All the data reported in Table 1 deal with the rat colonic H-K-ATPases, excluding possible species differences.

It is tempting to deduce from physiological studies that the type III K-ATPase from the kidney is encoded by the rat colonic H-K-ATPase. However, the pharmacological properties of the rat colonic H-K-ATPase when exogenously expressed in oocytes, Sf9 cells, or HEK 293 cells differ drastically from the pharmacological properties of the native K-ATPases (Table 1).

A major task for the next few years would be to explain these discrepancies. The following reasons may be involved: 1) the functional expression systems are not appropriate to analyze the functional properties of the cloned nongastric H-K-ATPase -subunits; 2) posttranslational modifications or splicing variants exist in vivo in the "normal" cell context. This may explain why the full-length or the unprocessed subunits display different functional properties than do the endogenous complexes; 3) other subunits may exist and need to be characterized. We would like to discuss these points, and we hope that the bulk of results that will be accumulated in the future will help to discriminate between all these nonmutually exclusive possibilities.

Functional Expression Systems May Not Be Appropriate To Analyze the Functional Properties of the Cloned Nongastric H-K-ATPase -Subunits

Putative -subunit. Recent experiments indicate that the -subunit of the Na-K-ATPase is required for proper function of the Na-K-ATPase at early stages of embryonic development (45). The -subunit also affects the functional properties of the heterodimer when expressed in the Xenopus oocyte (7). The -subunit modifies the K affinity of the complexes, without effects on the other functions tested (7). Minor et al. (61) recently reported that the human Na-K-ATPase -subunit induces cation channel activity when expressed in Xenopus oocytes. Moreover, these authors observed an increase in the Na and K uptake mediated by the exogenously expressed Na-K-ATPase, when the human Na-K-ATPase -subunit was coexpressed with the rat 1- and 1-subunits in Sf9 cells (61).

Oligomerization of the -subunits. The P-ATPases have been reported to act as a heterodimer consisting of at least two - and two -subunits. The dimerization of the gastric -subunit is required for proper function (63, 71). If this finding is extended to the other K-ATPases, then one could imagine that the natural nongastric H-K-ATPase -subunit dimer could be present in the target cells as an / homodimer between two nongastric HK2a or HK2b subunits, or as an / oligodimer between the two 2a and 2b splice variants, or even between two different -subunits such as the Na-K-ATPase -subunit/nongastric H-K-ATPase -subunit. Indeed, Kone and Higham (47) reported that the NH₂-terminal truncated colonic 2b-subunit is expressed at a higher levels (up to 5-fold) than the 2a-subunit in both the colon and the kidney medulla. The ratio between the two HK2a and HK2b splice variants may influence the functional properties of the native H-K-ATPase. It would be easy to test this hypothesis using functional expression systems.

Functional importance of the -subunit. It has been reported that the nature of the -subunit involved in the Na-K-ATPase heterodimer influences the functional properties of the complexes. For example, the K affinity, as well as Na affinity, depends of the -subunit present in the Na-K-ATPase heterodimer (12, 29, 30, 41). Na-K-ATPase 13 complexes have a lower K affinity than Na-K-ATPase 11 complexes. The -subunit also influences Na interactions in the Na-K-ATPase pump cycle (30). However, neither ouabain nor Sch-28080 affinities of the nongastric H-K-ATPase appear to be influenced by the -subunit (19, 23). One cannot exclude that an unrecognized -subunit could behave differently.

Influence of the functional expression system. Finally, the nature of the functional expression system may also directly affect the functional properties of the exogenously expressed K-ATPases. This could explain the discrepancies reported in studies using either the insect Sf9 cell or the X. laevis oocyte for the functional expression of the rat colonic HK2a subunit. In this case, the ouabain sensitivity was opposite in the two functional expression systems (Table 1).

Posttranslational Modifications of the Nongastric H-K-ATPases

The recent cloning of two splice variants of the rat colonic H-K-ATPases -subunit may be the first step in recognizing such a phenomenon. The two splice variants have a different NH₂ terminus, with the 2b having a shorter NH₂-terminal domain than the 2a and missing two putative PKA and PKC phosphorylation sites (47). In the Na-K-ATPase 1-subunit, one of these sites has been shown to be phosphorylated in vivo by the PKC (6, 32, 54). Such a phosphorylation event has not been analyzed either ex vivo or in vivo for the nongastric H-K-ATPases.

Other Nongastric H-K-ATPase Subunits Have To Be Characterized

Against this is the fact that only one gene has been identified in human (called ATP1AL1, encoding the skin H-K-ATPase -subunit) (75) and in the mouse (P. Meneton, personal communication). To clone such unrecognized isoforms of the nongastric H-K-ATPase subgroup, we have previously used degenerated oligonucleotides directed against highly conserved domains of the -subunits of the Na-K/H-K-ATPase gene family (38). This approach was successful for the cloning of the colonic H-K-ATPase -subunit cDNA both in the distal colon (38) and in the rat collecting tubule (Jaisser, unpublished data). We have not been able to isolate another H-K-ATPase isoform from these tissues. A negative result is of course a weak argument.

In favor of the "missing -subunit" hypothesis, it should be noted that two different K-ATPase activities with different pharmacological profiles have been consistently reported to be present in the distal colon. Recently, Rajendran et al. (67) described a highly ouabain-sensitive K-ATPase in the crypt cells of the distal colon. The colonic H-K-ATPase 2-subunit is not expressed in the crypt cells. Its expression is strictly restricted to the surface cells (38, 42, 50). As shown in Table 1, the ouabain- and Sch-28080-sensitive type III K-ATPase described by Doucet and collaborators (13, 82) may be encoded by a new isoform, which could be identical to the colonic "crypt" isoform. Another explanation proposed by some authors is that the cloned colonic H-K-ATPase acquires Sch-28080 sensitivity (and an increased ouabain sensitivity) in vivo during K depletion (13). On the basis of the functional studies reported in the literature for the Na-K-ATPase, the gastric H-K-ATPase, and the nongastric ATPases, the "missing subunit" hypothesis is more attractive than a drastic change of the pharmacological profile dependent on the physiopathological status. Further cloning efforts are required to argue in favor or against the "missing H-K-ATPase -subunit isoform" hypothesis.

	CONCLUSION

TOP ABSTRACT INTRODUCTION THE NA-K/H-K-ATPASE GENE FAMILY THE NONGASTRIC H-K-ATPASES:... THE NONGASTRIC H-K-ATPASES:... DISCREPANCIES BETWEEN THE... CONCLUSION REFERENCES

The purpose of the present review was to describe the molecular and functional properties of the cloned nongastric H-K-ATPases. The molecular data indicate that these nongastric H-K-ATPases could be identified as a subgroup of the Na-K/H-K-ATPase gene family, apart from the canonical Na-K-ATPases and gastric H-K-ATPases. Functional data obtained from ex vivo expression in various functional expression systems support this classification. The unique cation transport capacity of the nongastric H-K-ATPase as well as its unique pharmacological profile indicate that these H-K-ATPases may have a specialized physiological role. The physiological relevance of the cell-specific expression of these H-K-ATPases and their regulations are discussed in the accompanying review (73a). It should be noted that several questions remain to be answered, particularly the existence of yet unrecognized isoforms or the existence of functional variants due to gene variants or to posttranslational modifications.