Institut National de la Santé et de la Recherche Médicale, Unité 478, Institut Fédératif de Recherche Cellules Épithéliales, Faculté de Médecine Xavier Bichat, Université Paris VII, F-75870 Paris Cedex 18, France
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ABSTRACT |
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The Na-K/H-K-ATPase gene family is divided in
three subgroups including the Na-K-ATPases, mainly involved in whole
body and cellular ion homeostasis, the gastric H-K-ATPase involved in
gastric fluid acidification, and the newly described nongastric
H-K-ATPases for which the identification of physiological roles is
still in its infancy. The first member of this last subfamily was first identified in 1992, rapidly followed by the molecular cloning of
several other members. The relationship between each member remains
unclear. The functional properties of these H-K-ATPases have been
studied after their ex vivo expression in various functional expression
systems, including the Xenopus laevis
oocyte, the insect Sf9 cell line, and the human HEK 293 cells. All
these H-K-ATPase -subunits appear to encode H-K-ATPases when
exogenously expressed in such expression systems. Recent data suggest
that these H-K-ATPases could also transport
Na+ in exchange for
K+, revealing a
complex cation transport selectivity. Moreover, they display a unique
pharmacological profile compared with the canonical Na-K-ATPases or the
gastric H-K-ATPase. In addition to their molecular and functional
characterizations, a major goal is to correlate the molecular
expression of these cloned H-K-ATPases with the native K-ATPases
activities described in vivo. This appears to be more complex than
anticipated. The discrepancies between the functional data obtained by
exogenous expression of the nongastric H-K-ATPases and the
physiological data obtained in native organs could have several
explanations as discussed in the present review. Extensive studies will
be required in the future to better understand the physiological role
of these H-K-ATPases, especially in disease processes including ionic
or acid-base disorders.
physiology; ion homeostasis; potassium-adenosinetriphosphatase; colon; kidney
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INTRODUCTION |
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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.
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THE NA-K/H-K-ATPASE GENE FAMILY |
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Classification
P-ATPases are membrane ATPases involved in ion transport (33, 37, 52). This large family includes among others the sarcoplasmic and endoplasmic reticulum Ca-ATPases (SERCA), the membrane Ca-ATPases (PMCA), and the various members of the Na-K/H-K-ATPase group (37). On the basis of the molecular and functional properties of each members, this last group could be subdivided into three different subgroups: the Na-K-ATPase, the gastric H-K-ATPase, and the so-called nongastric H-K-ATPase (Fig. 1A). Unique functional properties of the nongastric H-K-ATPases have been demonstrated compared with the Na-K-ATPase and the gastric H-K-ATPase groups, suggesting clearly that the nongastric H-K-ATPases do not functionally belong to the Na-K-ATPase and the gastric H-K-ATPase groups.
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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
Interestingly enough, as already observed by Kone (46), the amino acid homologies between the rat HK2 and the human HK4 are significantly lower (86%) than between the rat HK1 and the human HK1 (98%) or the rat NK1, NK2, or NK3 and the human NK1, NK2, or NK3 (97%, 99%, and 99%, respectively). This also appears clearly on Fig. 1B, representing the phylogenetic tree of theAt 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.
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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.
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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 HK
2a subunit. It is not known whether the colonic HK
2b
functionally differs from the full-length HK
2a, 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).
The H4 putative transmembrane domain has been involved in the cation specificity of the gastric H-K-ATPase compared with the Na-K-ATPase. The Asn351 residue in the H4 putative transmembrane domain plays an important role in determining the Na affinity of the Na-K pump (78). The gastric H-K-ATPase counterpart is a tyrosine residue, whereas the asparagine residue is conserved in the nongastric H-K-ATPase subgroup. The overall sequence homologies between the Na-K and nongastric H-K-ATPase subgroups may account for the functional similarities between these two pumps (see below). Two exceptions are Ile341 replaced by valine in the Na-K-ATPase and methionine in the gastric H-K-ATPase and the Thr363 replaced by a cysteine in both the Na-K and H-K-ATPases. These amino acids are therefore specific for the nongastric H-K-ATPase subgroup. Amino acid residues located in the H4 putative transmembrane domain that differ between the Na-K-ATPaseSorting 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.
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THE NONGASTRIC H-K-ATPASES: FUNCTIONAL CHARACTERIZATION |
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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 first H-K-ATPaseThe 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).
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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
(Nai) 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 Nai 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 Nai was also observed
when the rat colonic H-K-ATPase was expressed, regardless of the
coexpressed
-subunit (21). This effect on Nai 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 Nai 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).
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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
The rat colonic HKFunctional Expression in HEK 293 Cells
Functional expression of the human ATP1AL1 H-K-ATPase, the rat colonic H-K-ATPase, and the guinea pig H-K-ATPase has been reported in HEK 293 cells, a nonpolarized cell line derived from the embryonic kidney (4, 35, 47). The human ATP1AL1 H-K-ATPase requires coexpression ofGrishin 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.
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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 HK
2a or HK
2b 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 HK
2a and HK
2b 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
1
3 complexes have a
lower K affinity than Na-K-ATPase
1
1 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 effects of posttranslational modifications on the properties of the nongastric H-K-ATPases have not been analyzed so far. This contrasts with the extensive analysis of the Na-K-ATPaseThe 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
Finally, one explanation for the discrepancy between the functional expression ex vivo and the functional characteristics of the colonic and renal K-ATPase activities in vivo may be that other yet unrecognized isoforms of the nongastric H-K-ATPases have to be identified.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.
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CONCLUSION |
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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.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Manoocher Soleimani, Stefania Puttini, Nicolette Farman, and Jean-Daniel Horisberger for helpful comments on the manuscript. We are especially grateful to Jean-Daniel Horisberger for precious help in constructing the sequence comparisons. We also thank M. Cougnon and G. Planelles for stimulating collaborations as well as B. C. Rossier, J. D. Horisberger, and N. Farman for constant support.
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FOOTNOTES |
---|
This work was supported by Institut National de la Santé et de la Recherche Médicale. A. T. Beggah is supported by a fellowship of the Swiss National Fund for Scientific Research.
Address for reprint requests and other correspondence: F. Jaisser, Institut National de la Santé et de la Recherche Médicale, U. 478, Institut Fédératif de Recherche Cellules Épithéliales, and Faculté de Médecine Xavier Bichat, Université Paris VII, BP 416, F-75870 Paris Cedex 18, France (E-mail: jaisser{at}bichat.inserm.fr).
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