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Renal Vacuolar H⁺-ATPase

Institute of Physiology, University of Zurich, Zurich, Switzerland; Departments of Genetics, Cellular and Molecular Physiology, and Surgery, School of Medicine, Yale University, New Haven, Connecticut; and Program in Membrane Biology, Renal Unit, Massachusetts General Hospital, Harvard University, Boston, Massachusetts

ABSTRACT

I. INTRODUCTION

II. STRUCTURE AND GENERAL PROPERTIES OF VACUOLAR H+-ATPase

A. General Structure of the Vacuolar H+-ATPase

B. Role of Subunits of the Vacuolar H+-ATPase

1. The V1 domain

2. The V0 domain

3. The stalk

C. ''Kidney-Specific'' Subunits of the Vacuolar H+-ATPase

D. General Functional Properties of Vacuolar H+-ATPase

III. DISTRIBUTION OF THE VACUOLAR H+-ATPase IN THE KIDNEY

A. Proximal Tubule

B. Loop of Henle

C. Distal Tubule

D. Connecting Segment

E. Collecting Duct

F. Distribution of the Vacuolar H+-ATPase in Other Proton-Transporting Cells

1. Epididymis/vas deferens

2. Inner ear

3. Osteoclasts

G. Electron Microscopy of the Vacuolar H+-ATPase

1. Conventional electron microscopy

2. Rapid-freeze, deep-etch electron microscopy

3. Freeze-fracture electron microscopy and rod-shaped intramembraneous particles

IV. ROLE OF VACUOLAR H+-ATPases IN KIDNEY ACID-BASE TRANSPORT

A. Proximal Tubule

B. Loop of Henle and TAL

C. Late Distal Tubule, Connecting Segment, and Cortical Collecting Duct

D. Medullary Collecting Duct

V. CHLORIDE DEPENDENCE OF VACUOLAR H+-ATPase FUNCTION

A. ClC-5 (and ClC-4)

B. AQP-6

C. CFTR

VI. ENDOCYTOSIS AND ACIDIFICATION OF INTRACELLULAR VESICLES

A. Clathrin-Coated Pits/Vesicles

B. Endosomes, the Golgi/TGN, and Lysosomes

C. Vesicle Acidification and Recruitment of Endosomal Coat Components

D. Defective Vesicle Acidification and Proximal Tubule Pathophysiology

E. Dent's Disease

F. Acquired Fanconi Syndrome: Cadmium Nephrotoxicity

VII. INTERACTION OF THE VACUOLAR H+-ATPase WITH PROTEINS OTHER THAN ITS OWN SUBUNITS

A. Inhibitors and Activators of the Vacuolar H+-ATPase

B. Interaction of the Vacuolar H+-ATPase With SNARE Proteins

C. Subunit B1 of the Vacuolar H+-ATPase is a PDZ Binding Protein

D. The Vacuolar H+-ATPase Directly Interacts With the Actin Cytoskeleton

E. Interaction of the Vacuolar H+-ATPase With Enzymes of the Glycolytic Pathway

F. Interaction With Other Proteins

VIII. REGULATION OF H+-ATPase FUNCTION AND LOCALIZATION

A. General Mechanisms of Regulation of Vacuolar H+-ATPase Activity

1. Assembly and disassembly of vacuolar H+-ATPases

2. Altered abundance of vacuolar H+-ATPases

3. Disulfide bridges and changes in ATP-coupling efficiency

B. Polarized Expression of the Vacuolar H+-ATPase in Intercalated Cells

1. Acid-base regulation of vacuolar H+-ATPase polarity in intercalated cells

2. Plasticity of the intercalated cell phenotype

3. Plasticity of the principal and intercalated cell phenotypes

C. Adaptive Regulation of Vacuolar H+-ATPase Activity

1. Metabolic acidosis

2. Metabolic alkalosis

3. Respiratory acidosis and alkalosis

4. Electrolyte disturbances

D. Hormonal Regulation of Vacuolar H+-ATPase Activity

1. Regulation of bicarbonate reabsorption and vacuolar H+-ATPase activity by angiotensin II and aldosterone

A) THE RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM.

B) ANGIOTENSIN II.

C) ALDOSTERONE.

D) ENDOTHELIN.

E) KALLIKREIN.

F) THYROID HORMONES.

G) CALCITONIN.

H) VASOPRESSIN.

2. Other hormones

IX. MODULATIONS IN VACUOLAR H+-ATPase FUNCTION IN DISEASE

A. Renal Tubular Acidosis

1. Primary dRTA

2. SCL4A1 (AE-1/band 3) mutation in autosomal dominant dRTA

3. Recessive dRTA

B. Mutations in the ATP6V1B1 (B1) Subunit of the Vacuolar H+-ATPase

1. Genome-wide linkage studies in rdRTA with kindreds

2. ATP6V1B1 mutation in rdRTA

3. Putative dysfunction of mutated B1 subunits

4. Hearing loss in dRTA patients harboring ATP6V1B1 mutations

5. ATP6V1B1 expression in other tissues

C. Vacuolar H+-ATPase B1 Subunit (Atp6v1b1)-Deficient Mice

D. ATP6V0A4 Mutations in Patients with Autosomal Recessive dRTA

1. Expression of a4 in the proximal tubule and collecting duct

2. Expression of the a4 isoform in male reproductive tract and ear

E. Evidence for Further Genes

F. Vacuolar H+-ATPase Loss or Dysfunction in Acquired Diseases

1. Sjogren’s disease

2. Acute ureteral obstruction

3. Drugs

X. SUMMARY AND OPEN QUESTIONS

ACKNOWLEDGMENTS

REFERENCES

	ABSTRACT

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	I. INTRODUCTION

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Vacuolar H⁺-ATPases are ubiquitous multisubunit complexes mediating ATP-driven vectorial transport of protons across membranes. They are expressed in virtually all eukaryotic cells in intracellular membranes or in certain specialized cells also in the plasma membrane. Whereas intracellular pH is generally regulated by Na⁺/H⁺ and Na⁺-dependent and independent Cl^–/HCO₃^– exchangers, the pH of many intracellular compartments such as the lysosome, the Golgi apparatus, secretory vesicles, and endosomes is regulated by vacuolar H⁺-ATPases (i.e., acidification of the compartment). The function of these organelles relies on an acidic intraorganellar pH to maintain optimal enzyme function. Disruption of this acidic intraorganellar pH leads to disturbance of organelle function and often to cell death. In neuronal cells, vacuolar H⁺-ATPases play an additional important role in synaptic transmission. Neurotransmitters taken up from the synaptic cleft are stored in vesicles acidified in an electrogenic manner by vacuolar H⁺-ATPases. The proton gradient (pH) or the potential gradient () generated by vacuolar H⁺-ATPases is then used by vesicular neurotransmitter transport proteins to accumulate neurotransmitters in storage vesicles (177). In contrast, vacuolar H⁺-ATPases localized in the plasma membrane mediate proton extrusion from the cell. Acidification of the cellular environment is intricately linked to specialized cell function as exemplified in osteoclasts where protons generated by the vacuolar H⁺-ATPases are used to dissolve bone matrix, or, macrophages where acidic pH is involved in killing and digesting neighboring cells or pathogens (513). In other cells vacuolar H⁺-ATPases mediate the regulation of extracellular pH of closed compartments such as in the inner ear and endolymph fluid (117, 167, 481), or the epididymis where seminal fluid is acidified (65, 68, 85). As reviewed here, in the kidney, in addition to the functions mentioned above, vacuolar H⁺-ATPases are involved in acid-base transport, thus contributing to overall body homeostasis.

Many of these functions have been reviewed elsewhere and are not the topic of this review (18, 174–177, 359, 384, 385, 392). This review focuses only on the function, regulation, and role of vacuolar H⁺-ATPases in renal physiology and pathophysiology. After a general introduction into the structure and function of the vacuolar H⁺-ATPase, we describe the localization of vacuolar H⁺-ATPases in the kidney and their role in acid-base transport as well as endo- and exocytosis. The regulation of these processes and the development of associated diseases caused by inherited or acquired states of vacuolar H⁺-ATPase dysfunction will also be addressed.

	II. STRUCTURE AND GENERAL PROPERTIES OF VACUOLAR H+-ATPase

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A. General Structure of the Vacuolar H⁺-ATPase

Vacuolar H⁺-ATPases belong to the large superfamily of ATPases that is subdivided into three subclasses: 1) P-type ATPases such as Na⁺-K⁺-ATPases, Ca²⁺-ATPases, and H⁺-K⁺-ATPases; 2) mitochondrial F₁F₀-ATPases; and 3) V-type (vacuolar) H⁺-ATPases (http://www.gene.ucl.ac.uk/nomenclature/) (370). Mitochondrial F₁F₀-ATPases and vacuolar H⁺-ATPases share many structural features such as subunit composition, high degrees of subunit similarities based on amino acid sequences, and subunit arrangement (212, 384). Functionally, however, they are distinguished by the fact that F₁F₀-ATPases use a proton gradient for ATP synthesis, whereas vacuolar H⁺-ATPases use ATP hydrolysis to generate a proton gradient. Some instances have been reported where vacuolar H⁺-ATPases may be functionally reversed and act as proton-driven ATP synthetases (132, 241). F₁F₀-ATPase structure and function have been investigated in detail over the past years, and recently, the three-dimensional structures of the F₁ sector and stalk have been resolved at high resolution (357). Furthermore, the resultant structural changes that occur in response to different states of enzyme activity have also been observed and mapped (583). Consequently, vacuolar H⁺-ATPase structure and function have been modelled based on results obtained from the F₁F₀-ATPases (384). In addition, much of the present knowledge on structure and function of vacuolar H⁺-ATPases comes from experiments conducted with the yeast vacuolar H⁺-ATPase, which also gave its name to this class of pump because of its role in acidifying the yeast food vacuole (384). Further extensive details on the structure of the vacuolar H⁺-ATPase and the roles of the respective subunits are found in some recent reviews (175, 176, 212, 384, 392).

In general, vacuolar H⁺-ATPases consist of two main domains, a peripheral catalytic V₁ domain (640 kDa) and a membrane-bound V₀ domain (240 kDa), together forming a protein complex of 900 kDa (Fig. 1). Both domains are connected through a stalklike structure that belongs to the V₁ domain (16–18, 177, 212, 384, 567). The stoichiometry of the vacuolar H⁺-ATPase subunits in yeast is thought to be A₃:B₃:C:D:E₂:F:G₂:H:a:d:c''(c':c)₆ (384, 420). The existence of the c' subunit in mammals is not resolved yet (see Table 1, Ref. 471).

View larger version (38K): [in this window] [in a new window]   FIG. 1. Structural model of the vacuolar H+-ATPase. The cytosolic V1 domain consists of the subunits A-H (denoted in capital letters), and the membrane-bound V0 domain comprises the subunits a-d with several isoforms of the c subunit (denoted in small letters). [Redrawn from Kawasaki-Nishi et al. (272).]

1. The V₁ domain

The cytosolic V₁ domain is composed of eight subunits denoted in capital letters (see Table 1). Homology to the - and -subunits of the F₁F₀-ATPase as well as mutational studies with yeast vacuolar H⁺-ATPases have established several functions for the A and B subunits. A and B subunits are arranged in an alternating manner in a pseudo-hexagonal head-piece (212). Whereas only one isoform of the A subunit has been identified, two highly homologous B subunits (ATP6V1B1 and ATP6V1B2) with a tissue- and cell-specific expression pattern exist in many species (53, 206, 389, 524, 556). Sequence comparisons demonstrate that the vacuolar H⁺-ATPase B subunits share 20–25% amino acid identity not only with the vacuolar H⁺-ATPase A subunit, but also with the - and -subunits of the mitochondrial F₁F₀-ATPase, suggesting that all four of these proteins evolved from a common ancestral nucleotide binding protein. As predicted by homology to - and -subunits of the F₁F₀-ATPase (1), both the A and B subunits of the vacuolar H⁺-ATPase have been demonstrated to participate in nucleotide binding (591); however, genetic and biochemical evidence suggests that it is the nucleotide binding site on the A subunit that is catalytic (165, 546). Nevertheless, inhibition of vacuolar H⁺-ATPase activity also occurred after modification of a single noncatalytic site of the B subunit by a photoactivated, nonhydrolyzable nucleotide analog, raising the possibility that the nucleotide binding site of the B subunit may play a role in the regulation of H⁺-ATPase activity (527). In addition, the yeast vacuolar H⁺-ATPase A subunit possesses a stretch of an additional 90 amino acids compared with -subunits. This additional segment may play a role in the coupling efficiency of ATP hydrolysis to proton transport and the dissociation of V₁ and V₀ complexes (461).

Studies in yeast have suggested that the B subunit plays an essential role in vacuolar H⁺-ATPase function. In the yeast Saccharomyces cerevisiae, where the vacuolar H⁺-ATPase mediates acidification of the vacuole, disruption of the gene encoding the B subunit results in a conditional lethal phenotype, in which mutants can grow only in media with a narrow acidity range around pH 5.5 (383, 579). Along the same line, knockout of the vha55, encoding a B subunit homolog of Drosophila, produced a larval lethal phenotype (126).

Comparison of the B1 and B2 subunit isoform amino acid sequences from both human and cow reveals that the central 469 amino acids are highly conserved, while the 20–25 amino acids at both the amino and carboxy termini have diverged greatly, thus raising the possibility that these terminal regions provide specialized isoform-specific functions of the B subunit (386, 421). It has been speculated that the two B isoforms may confer differences in vacuolar H⁺-ATPases enzymatic activities or in vacuolar H⁺-ATPases sorting capacities, or alternatively, that these two isoforms may differ in their ability to regulate the membrane density of the vacuolar H⁺-ATPase (386). The carboxy terminus of the B1 subunit, but not the B2 subunit, terminates in a D-T-A-L sequence. This PDZ-binding motif is recognized by so-called PDZ proteins and mediates protein-protein interactions (see below). It has previously been suggested that interaction with PDZ domain-containing proteins may mediate tissue-specific interactions of the B1 subunit-containing vacuolar H⁺-ATPase with other proteins, which could perhaps mediate targeting or trafficking of the complex (70). However, none of the ATP6V1B1 missense mutations identified to date in dRTA kindreds is located in the carboxy-terminal region of the protein (268, 492). Thus genetic studies of dRTA kindreds unfortunately have not shed light into the requirement of the region of the B1 protein essential for normal vacuolar H⁺-ATPase-mediated proton secretion in intercalated cell (see below).

2. The V₀ domain

The proton translocating channel is formed by the V₀ domain involving the proteolipid c' and c'' subunits (420). Recent evidence from functional mutational analysis of the yeast Vph1 subunit (yeast analog of the mammalian a subunit) also suggests the involvement of the a subunit in the proton channel (304, 413). Targeted deletion of the proteolipid ATP6V0C (c) subunit in mouse leads to early embryonic lethality due to impaired nidation of the embryo (254). Proton translocation through the proton channel is potently inhibited in the nanomolar range by the macrolide antibiotics bafilomycin A₁ and concanamycin (59, 141). These selective vacuolar H⁺-ATPase inhibitors may act through interactions with the proteolipid c' and c'' subunits (58, 122, 248) as well as with the 100-kDa a subunit (590).

The a subunit (ATP6V0A1–4) of the V₀ domain has recently received special attention due to the fact that four isoforms of this subunit exist and that at least two human diseases are caused by mutations in two of these subunit isoforms (ATP6V0A3 and ATP6V0A4) (see below). In renal physiology, ATP6V0A4 (a4) is most interesting as this subunit isoform is only found in kidney and epididymis (472, 474). The ATP6V0A4 protein shares 61% amino acid identity with ATP6V0A1, which appears to be a widely transcribed a subunit isoform in mammals (412). The ATP6V0A4 protein also shares 47% identity with ATP6V0A3 (a3, also previously identified as OC116 or TCIRG1 on chromosome 11). Mutations in the ATP6V0A3 gene underlie one type of infantile malignant autosomal recessive osteopetrosis (181). ATP6V0A4 shares 40% amino acid identity with a subunit homologs as evolutionary distant as Neurospora and yeast, indicating the biological importance of this gene family (472).

Topological studies of yeast a subunit (Vph1p) of V-ATPase employing cysteine mutagenesis, labeling with membrane-permeant and -impermeant maleimides as well as using protease cleavage, led to an original topological model containing nine transmembrane domains in this subunit (305). Similarly, the alignment of mouse a1, a2, and a3 subunits with yeast Vph1p also predicted nine transmembrane domains (391). According to this model (272, 392) the amino-terminal domain is located on the cytoplasmic side of the membrane, while the carboxy terminus is located on the luminal side of the membrane. Hydropathy analysis of mouse a1, a2, a3, and a4 subunit isoforms performed in a different laboratory also predicted nine transmembrane domains in these subunits (397, 512). In contrast, another hydropathy analysis of the same mouse a1, a2, a3, and a4 isoform subunits as well as a human member (ATP6V0A4) of the a subunit family predicted six transmembrane domains (472, 494). According to this latter model, both amino-terminal and carboxy-terminal tails should be located on the cytoplasmic side of the membrane. Thus the transmembrane topology of the a subunits of the vacuolar H⁺-ATPase is still controversial, and additional structural/crystallography studies are needed to clarify this issue.

ATP6V0A4 contains several potential glycosylation sites, which were suggested to account for the difference between the 96-kDa predicted molecular mass of this subunit and the 116-kDa mass observed for previously reported a subunit homologs. However, analysis failed to detect either a signal sequence or a consensus sequence for polarized targeting to the lysosomal membrane or other organelles (472). In yeast and osteoclasts, pumps containing different a subunit isoforms localize to different intracellular organelles or reside in the cell membrane (271, 511). In addition, the coupling efficiency between ATP hydrolysis and proton pumping differed in pumps containing different a isoforms (271).

3. The stalk

The A and B subunits are connected to the V₀ domain through several subunits termed the "stalk," which also belongs to the V₁ domain. Homology modeling, using again the mitochondrial F₁F₀-ATPase, and cysteine cross-linking experiments in yeast, suggest that subunits G and E form part of the peripheral stalk connecting V₁ and V₀, whereas subunit D seems to localize to the central stalk (16). Further evidence from the crystal structure of the subunit C of Thermus thermophilus V-ATPase, homologous to eukaryotic subunit D of vacuolar H⁺-ATPases, suggests that this subunit belongs to the stalk and may help to attach the central stalk to the V₀ domain (257). Direct protein-protein interactions have been demonstrated for the E and H subunits in which deletion of the carboxy terminus of the E subunit decreases ATP hydrolysis and proton transport activity (323). In addition, at least the amino terminus of the a subunit (Vph1 in yeast) which otherwise belongs to the V₀ domain also interacts with the A and H subunits forming a "stator"-like structure. Mutations introduced into the amino terminus of the yeast a subunit abolished proton translocation but not ATP hydrolysis, suggesting that the statorlike structure may be important for coupling ATP hydrolysis to proton translocation (294, 295).

On the basis of experiments using immobilized F₁ or V₁ sectors tagged with a bead on the stalk, rotations in both the mitochondrial F₁F₀-ATPase and a bacterial V₁-ATPase were observed (212, 252, 583). Similarly, tagging of the c subunit as part of the proton translocating pathway of the yeast vacuolar H⁺-ATPase showed counter-clockwise rotations of the c subunit relative to the stalk G subunit (240, 586). Thus it is thought that ATP binding to the B subunit and subsequent hydrolysis leads to a rotation of the central stalk structure relative to the A₃B₃ domain (212, 583). This conformational change may then induce the motion of the ring structure formed by the c and a subunits, thus inducing the transfer of protons across the membrane (212, 384).

In addition to true vacuolar H⁺-ATPase subunits, associated proteins have been identified. Some of these proteins are expressed in a highly tissue-specific manner, suggesting that these proteins may be involved in tissue-specific functions of the vacuolar H⁺-ATPase or may be involved in the targeting of the pump to specific intracellular structures (244, 500). Targeted disruption of the accessory Ac45 protein in mouse led to early developmental death of the blastocysts (450). In yeast, four homologous Vtc proteins (vacuolar transporter chaperones) have been identified associating with SNARE proteins and the V₀ domain of the vacuolar H⁺-ATPase. The function of these proteins is not fully understood yet; however, deletion of these proteins produces yeast strains with defects in vacuolar membrane fusion and H⁺-ATPase mediated acidification (369).

C. "Kidney-Specific" Subunits of the Vacuolar H⁺-ATPase

Of the identified subunits of the vacuolar H⁺-ATPase there are some isoforms that have been described in a limited number of tissues; these include the B1 isoform (ATP6V1B1), the a4 isoform (ATP6V0A4), the G3 isoform (ATP6V1G3), the C2 (ATP6V1C2), and the d2 isoform (ATP6V0D2) (80, 169, 386, 471, 472, 474, 499). All of these subunit isoforms appear to be expressed in the kidney, particularly in intercalated cells (498). The B1 isoform is part of the peripheral V₁ domain, and its yeast homolog is important for ATP binding before ATP hydrolysis by the A subunit (384). As mentioned above, two isoforms of the B subunit exist: the B2 isoform (ATP6V1B2) is ubiquitously expressed (49, 524), whereas in the kidney the B1 subunit is amplified in intercalated cells of the late distal tubule, connecting segment and cortical and medullary collecting duct (386), in narrow and clear cells of the epididymis (68, 80, 169), in the ciliary body of the eye (245, 558), and in the inner ear (interdental cell layer of the spiral limbus and some epithelial cells of the endolymphatic sac) (268). The a4 subunit (ATP6V0A4) is one of four isoforms of the a subunit which forms part of the membrane-bound V₀ sector. All four a subunits are expressed in the kidney as detected by Northern blot and RT-PCR analysis (397, 472, 512). The a4 isoform, however, is only expressed in narrow and clear cells of the epididymis (C. Pietremont, M. Futi, and S. Breton, unpublished observations; Ref. 472) the inner ear (492), and the kidney (proximal tubule, loop of Henle, and all subtypes of intercalated cells along the late distal tubule, connecting segment, and entire collecting duct) (472, 482). The distribution of the other a isoforms varies widely between tissues (347, 391, 512), but all isoforms are also expressed in the kidney with distribution patterns associated with various regions of the nephron in a differential manner (A. Hurtado-Lorenzo, D. Brown, M. Futai, and V. Marshansky, unpublished observations). Mutations in the a3 isoform (ATP6V0A3 or TCIRG1) have been identified in a severe form of infantile osteopetrosis, a disease of the osteoclasts (181).

Mutations in two "kidney-specific" subunit isoforms, B1 (ATP6V1B1) and a4 (ATP6V0A4), have been identified in patients with inherited forms of distal renal tubular acidosis with and without sensorineural deafness (see below).

D. General Functional Properties of Vacuolar H⁺-ATPase

The general functional properties, such as pH dependence and ratio of ATP-hydrolysis to H⁺ pumping, have been examined mainly in yeast, turtle urinary bladder preparations, or membrane vesicles obtained from brain or kidney. Some information was also derived from other organisms such as plant vacuolar H⁺-ATPases showing a high degree of functional similarities.

As discussed in more detail in section V, the movement of H⁺ across membranes results in a net charge translocation unless the movement of other ions electrically compensates, and thus facilitates further H⁺ pumping. In addition to this electrical gradient and similar to any other membrane transport system, the chemical gradient for H⁺ and the pH difference across the membrane also affect the pumping rate of vacuolar H⁺-ATPases. In turtle bladder, a sigmoidal relationship between H⁺-transporting rate and H⁺ gradient was observed between a normal intracellular pH (7.4) and extracellular pH from 4.5 to 7.0 (the rate being 0 below pH 4.5 and reaching saturation between pH 7.0 and 8.0) (14). Similarly, changes in the electrical gradient (i.e., an imposed voltage) resulted also in a sigmoidal curve for H⁺ transport in the range of a lumen-positive potential (relative to the serosa) of +120 mV (being zero) to –80 mV (being saturated between 0 and –30 mV) (14).

In addition to this intrinsic dependency on the transmembrane electrochemical gradient, the transport rate can also be modulated by alterations in the rate of ATP hydrolysis and its coupling to H⁺ translocation. A recent detailed analysis of current-voltage relationships in the absence and presence of several ions, ATP or ADP and imposing different pH gradients, described different coupling ratios for vacuolar H⁺-ATPases from yeast. In the presence of large pH gradients (4 pH units), the approximate ratio was 2 H⁺/ATP and increased to more than 4 H⁺/ATP for small or no pH gradients (273). Similar findings have also been reported from plant vacuolar H⁺-ATPases (125). Experiments on brain vesicular H⁺-ATPase suggested that the coupling efficiency can be modulated by the presence of Ca²⁺ or Mg²⁺ but with differential mechanisms. The effect of Ca²⁺ was dependent on the membrane potential, whereas Mg²⁺ supported vacuolar H⁺-ATPase activity independent from the voltage (121). Another factor influencing the coupling ratio of yeast vacuolar H⁺-ATPase is the availability of glucose (530), a fact that may be related to recent findings of interaction between vacuolar H⁺-ATPase subunits and glucolytic enzymes (see sect. VII). Even though the underlying molecular mechanism for these variable stoichiometries is unkown at present, some observations point to the involvement of at least two subunits. Changes in the coupling of ATP hydrolysis and H⁺ transport have been observed in some mutants of the A subunit of the V₁ sector as well as in different isoforms of the a subunit in the V₀ sector. As described above, ATP hydrolysis is mainly mediated by sites on the A subunit, and mutants have been identified in yeast vacuolar H⁺-ATPase that alter the efficiency of coupling between ATP hydrolysis and H⁺ translocation (461). Similarly, different coupling stoichiometries have been observed in yeast vacuolar H⁺-ATPase expressing different isoforms of the a subunit yeast homolog (271).

A complete uncoupling of ATP hydrolysis and H⁺ transport has been observed after application of azide, which inhibits proton transport without affecting ATP hydrolysis (528).

	III. DISTRIBUTION OF THE VACUOLAR H+-ATPase IN THE KIDNEY

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As discussed below, one of the major functions of the kidney is to maintain a constant plasma bicarbonate concentration by reabsorbing filtered bicarbonate and by regenerating bicarbonate that is consumed in buffering acids generated by metabolism. The vacuolar H⁺-ATPase is one of several acid-base regulating proteins that are involved in this process (222). While the vacuolar H⁺-ATPase is generally considered to be an "intracellular" protein, studies over the past several years have clearly shown that it is inserted into the plasma membrane of many cell types in the kidney and other tissues, where it plays a key role in different physiological processes including bicarbonate reabsorption, sperm maturation and storage, bone reabsorption, and hearing.

A. Proximal Tubule

Immunocytochemical observations using specific antibodies raised against various subunits of the vacuolar H⁺-ATPase, as well as numerous functional studies on intact tubules and isolated brush-border membrane vesicles, have shown that a bafilomycin- and N-ethylmaleimide (NEM)-sensitive vacuolar H⁺-ATPase is located in the apical membrane and in intracellular organelles of proximal tubule epithelial cells (146, 173, 191, 238, 260, 439, 440, 442, 543, 544). The 31-kDa E2 subunit of the vacuolar H⁺-ATPase is present in rat proximal tubule segments along with the "brain" or B2 (ATP6V1B2) isoform of the 56-kDa subunit. The "kidney" or B1 (ATP6V1B1) isoform is amplified in collecting duct intercalated cells (386) (see below). The B2 subunit isoform of the vacuolar H⁺-ATPase has a truncated carboxy-terminal domain that lacks the PDZ-binding domain found in the B1 (intercalated cell) isoform (386, 421). This subunit does not interact with the PDZ protein, NHERF1 in proximal tubules, and the localization of these proteins in the apical region of proximal tubule epithelial cells is distinct (70). While NHERF1 is expressed mainly in the brush-border microvilli (where it colocalizes with NHE-3), the vacuolar H⁺-ATPase is found in the submicrovillar region (Fig. 2, A and B). The functional relevance of expressing the B2 56-kDa isoform in proximal tubules is unknown, but it may allow the vacuolar H⁺-ATPase to function and recycle without interference from the complex PDZ and PDZ-binding protein interactions that would otherwise occur in this region of the cell.

View larger version (122K): [in this window] [in a new window]   FIG. 2. A: proximal tubule stained for the H+-ATPase (green) and NHERF-1 (red). NHERF-1 is located in the brush-border microvilli, and the H+-ATPase is present in submicrovillar vesicles and invaginations. The two proteins are not colocalized. Bar = 10 µm. B: a differential interference contrast image is shown for orientation purposes. C: localization of the E subunit of the H+-ATPase in a proximal tubule cell by immunogold electron microscopy. H+-ATPase-associated gold particles are concentrated at the base of the brush-border microvilli (small arrows) and are excluded from the clathrin-coated pit domain. The clathrin coat appears as an electron-lucent band on the cytoplasmic side of the membrane invaginations (arrowheads). Bar = 0.5 µm. [Modified from Breton et al. (70) and Brown et al. (79).]

View larger version (17K): [in this window] [in a new window]   FIG. 3. Acridine orange fluorescence quenching can be used to measure the functional acidification capacity of endosomes isolated from rat kidney cortex. In the presence of ATP alone (ATP, –Baf), the rapid fluorescence quenching reflects V-ATPase-dependent proton translocation into the endosomal lumen. No detectable intraluminal acidification occurs when ATP is added in the presence the specific V-ATPase inhibitor bafilomycin (ATP, +Baf). After the generation of an acidic lumen, the endosomal pH can be alkalinized by the subsequent addition of bafilomycin (Baf), which inhibits further proton translocation into the endosomes. Protons exit the endosomes into the bulk bath solution by passive proton leakage across the endosomal membrane (this experiment was performed in a sodium-free solution, thus excluding the contribution of a Na+/H+ exchanger to the observed alkalinization). This alkalinization can be further enhanced by the V-ATPase inhibitor CCCP and by the proton ionophore nigericin (Nig), which allows complete pH equilibration with the bath solution to occur. (From V. Marshansky, unpublished results.)

As for the proximal tubule, proton and bicarbonate transport in the thick ascending limb of Henle's loop are mediated by the vacuolar H⁺-ATPase, along with other transporters including the Na⁺/H⁺ exchanger NHE-3 (96, 554). Both the electroneutral NBC(N)1 (538) and electrogenic NBC4 (577) are expressed in the thick ascending limb (TAL). The Cl^–/HCO₃^– exchanger AE2 has also been localized in the basolateral membrane of the TAL in rat and mouse kidney (12, 493) and might provide a potential route for bicarbonate reabsorption in this membrane domain. The vacuolar H⁺-ATPase in this segment is located in numerous cytoplasmic vesicles, which are concentrated at the apical pole of the cell (Fig. 4A). Immunogold electron microscopy clearly shows that some vacuolar H⁺-ATPase is in fact in the apical plasma membrane, and some is associated with subapical vesicles (79). Furthermore, freeze-fracture studies have shown that the apical membrane of thick ascending limb cells contains clusters of rod-shaped intramembrane particles (Fig. 4, B and C), which are associated with the presence of the vacuolar H⁺-ATPase in some other cell types, such as intercalated cells (73). Subunit-specific antibodies show that the B2 isoform of the vacuolar H⁺-ATPase is expressed in the TAL (unpublished data). So far, the physiological regulation of vacuolar H⁺-ATPase recycling in the TAL has not been demonstrated.

View larger version (108K): [in this window] [in a new window]   FIG. 4. A: immunofluorescence localization of the E1 subunit of the H+-ATPase in thick ascending limbs of Henle. A bright staining is seen in the apical membrane and subapical vesicles. Bar = 10 µm. B: freeze-fracture electron micrograph of the basolateral (BL) and apical (Ap) membrane of cells from the thick ascending limb of Henle. The localization of mitochondria within the basolateral membrane invaginations is characteristic of this kidney segment. The lipid bilayer of the apical membrane contains typical rod-shaped intramembranous particles (IMPs). Bar = 0.5 µm. C: higher magnification image showing the apical membrane subdomains containing clusters of rod-shaped IMPs. These rod-shaped IMPs are thought to represent part of the V0 sector of the H+-ATPase. Bar = 80 nm. (From D. Brown and S. Breton, unpublished data.)

C. Distal Tubule

In normal animals, bicarbonate reabsorption in this segment is negligible, but it can be increased by as much as fivefold in acidotic animals (307). By immunostaining, the vacuolar H⁺-ATPase (containing the B1 56-kDa subunit) is present at relatively high levels on the apical plasma membrane of distal convoluted tubule (DCT) cells, where it forms a very sharp line at the level of the membrane (79). Relatively little staining of intracellular vesicles is seen in the DCT, and the capacity of the vacuolar H⁺-ATPase to recycle in this segment is unknown. The late DCT also expresses apical H⁺-ATPase in "DCT" cells, but in addition, intercalated cells with very high H⁺-ATPase expression make their first appearance in this tubule segment.

D. Connecting Segment

The connecting segment joins the DCT with the cortical collecting duct. In rabbits, this segment forms long arcades that are easily distinguished, whereas in other species such as rodents it is shorter and less distinct. Specific connecting tubule cells can be distinguished by their high content of calbindin 28 in the rat (316, 435), which is expressed at lower levels in the DCT and the collecting duct. Intercalated cells are present in the connecting segment, where they show the range of phenotypes described in more detail below for the cortical collecting duct. However, connecting segments tend to have a greater percentage of B-cells and more of the subclass of intercalated cells that have apical vacuolar H⁺-ATPase but no basolateral AE1 (278). In addition, connecting tubule cells have a distinct apical band of vacuolar H⁺-ATPase staining similar to the staining of DCT cells (79).

E. Collecting Duct

Intercalated cells of the collecting duct express the highest levels of vacuolar H⁺-ATPases among all acid-base transporting cells in the kidney (Fig. 5). The 56-kDa B1 subunit of the vacuolar H⁺-ATPase, which contains a carboxy-terminal PDZ-binding motif (DTAL) that interacts with NHERF1, is expressed in intercalated cells (70). Other subunits including subunits A, B2, E2, G1, G3, a4, and d1 are also expressed in kidney intercalated cells (78, 79, 411, 498, 499). The classification of intercalated cells grows more complex as more is learned about their functional properties. In the initial study showing the cellular distribution of the vacuolar H⁺-ATPase in different intercalated cells, it was recognized that (in addition to the staining of numerous cytoplasmic vesicles in most intercalated cells), A-cells have apical vacuolar H⁺-ATPase, B-cells have basolateral vacuolar H⁺-ATPase, but many cells have a diffuse or even a bipolar vacuolar H⁺-ATPase distribution (34, 78). In the cortical collecting duct, all subtypes of intercalated cells (IC) are detectable (Fig. 5A). In the outer stripe of the outer medulla, A-cells predominate, but a few residual B-cells can be found. In the inner stripe of the outer medulla, only A-IC are present, and they represent 40% of the epithelial cell population of the collecting duct. In the inner medulla, the epithelium initially contains between 5 and 10% A-IC, and these cells disappear from the epithelium in the middle and terminal portions of the inner medullary collecting duct (79, 114).

View larger version (13K): [in this window] [in a new window]   FIG. 5. A: cortical collecting duct double-stained for the E1 subunit of the H+-ATPase (green) and for the chloride/bicarbonate exchanger AE-1 (yellow). In this figure, three patterns of intercalated cell staining are seen. Typical type A intercalated cells have apical H+-ATPase and basolateral AE-1 (small arrows). Typical type B intercalated cells have basolateral H+-ATPase and no basolateral AE-1 (arrowhead). Some type B intercalated cells, negative for AE-1, show bipolar H+-ATPase (large arrows). Some AE-1 negative cells can also have apical H+-ATPase (not shown). B: in the inner stripe of the outer medulla, only type A intercalated cells are seen, with basolateral chloride/bicarbonate exchanger AE-1 (yellow) and apical H+-ATPase (green) (arrows). (From D. Brown and S. Breton, unpublished data.)

The "kidney" 56-kDa B1 (ATP6V1B1) subunit is also present at lower levels in principal cells, where it is associated with endosomes that contain aquaporin (AQP)-2 water channels (447). Since these endosomes do not acidify their lumen, it was proposed that this vacuolar H⁺-ATPase subunit might be involved in the recycling of AQP-2 water channels in a way that is independent of its proton pumping activity (218).

F. Distribution of the Vacuolar H⁺-ATPase in Other Proton-Transporting Cells

The vacuolar H⁺-ATPase plays a pivotal role in various transporting epithelia that are not directly involved in systemic acid-base balance. This pump can also act as an energizer of plasma membranes, particularly the apical membranes of epithelial cells, by imposing proton electrochemical gradients across the membrane, which provides the driving force for a variety of ion transport processes (566). Thus the vacuolar H⁺-ATPase is found on the plasma membranes of a variety of cell types in addition to those described above in the kidney.

1. Epididymis/vas deferens

The epididymis and vas deferens of the male reproductive tract contain a subpopulation of epithelial cells that express very high levels of the vacuolar H⁺-ATPase on their apical membrane and intracellular vesicles (68, 80). Numerous subunits of the H⁺-ATPase, including the B1, E1, A (70 kD), and a4 subunits, are expressed in these epithelial cells (69, 80, 169; Breton et al., unpublished observations). These cells resemble kidney intercalated cells and help maintain a low luminal pH and low bicarbonate concentration that are critical for spermatozoa maturation and storage in the epididymis. Sperm motility is triggered, during ejaculation, by neutralization of the epididymal fluid by the prostatic and seminal vesicle fluid.

2. Inner ear

In the inner ear, immunocytochemical data showed that epithelial cells lining the endolymphatic sac and interdental cells of the cochlea express subunits B1 and E1 of the proton pump on their apical membrane (138, 481). Mutations of the B1 subunit have been related to sensorineural deafness (268) probably due to alkalinization of the endolymph and subsequent impairment of the contractile response of hair cells to mechanostimuli. These results support the notion that active acidification of the endolymph by the vacuolar H⁺-ATPase is essential for adequate auditory function.

3. Osteoclasts

Osteoclasts are specialized macrophages that are involved in bone remodeling. Osteoclasts attach themselves to bone matrix and reabsorb mineralized bone by creating an acidic environment between their bone-facing apical plasma membrane and the bone surface. Their apical membrane "ruffled border" contains high levels of the vacuolar H⁺-ATPase, which works in parallel with a "basolateral" Cl^–/HCO₃^– exchanger AE1 (35). The vacuolar H⁺-ATPase 56-kDa subunit expressed in osteoclasts is the "brain B2" isoform (298), whereas the 116-kDa (a) subunit isoform is the a3 (ATP6V0A3). Mutations in the gene encoding for the a3 subunit have been identified in patients suffering from infantile malignant osteopetrosis, a disease where the bone marrow cavity is not formed due to impaired osteoclast activity (181). The vacuolar H⁺-ATPase is, therefore, a major player in modulating bone resorption and might be a suitable target for therapeutic intervention for osteoporosis and other skeletal diseases.

G. Electron Microscopy of the Vacuolar H⁺-ATPase

As described above, the vacuolar H⁺-ATPase is a complex, multisubunit protein that comprises a transmembrane V₀ sector and a much larger cytoplasmic V₁ sector. Because of its large size, the vacuolar H⁺-ATPase can be visualized by electron microscopy using several techniques.

1. Conventional electron microscopy

Electron microscopic studies on ion-transporting epithelia in insects revealed a dense array of 10-nm studlike projections associated with some plasma membrane domains (217). These structures, named "portasomes" (227), closely resembled the mitochondrial F₁F₀-ATPase (102). Similar structures were identified on the plasma membranes of proton transporting epithelial cells of the turtle urinary bladder (484, 485) and of kidney intercalated cells (83, 331, 333, 378, 486), where they coated not only the plasma membrane (Fig. 6, A and C) but also the cytoplasmic surface of many intracellular vesicles. These 10-nm projections were identified as the V₁ sector of the vacuolar H⁺-ATPase by direct immunogold labeling of intercalated cell plasma membranes with specific antivacuolar H⁺-ATPase subunit antibodies (Fig. 6B) (77).

View larger version (142K): [in this window] [in a new window]   FIG. 6. A: electron micrograph of the apical region of a kidney collecting duct intercalated cell. The underside of the plasma membrane is decorated with an array of electron-dense projections, or studs, which measure 20 nm in length (arrows). These studs represent the V1 sectors of the H+-ATPase. Bar = 60 nm. B: localization of the H+-ATPase by immunogold electron microscopy in a single intercalated cell apical microvillus. The submembrane electron-dense studs are less discernable under these conditions, but the gold particles are closely associated with this dense band on the cytoplasmic surface of the membrane (arrows). Bar = 40 nm. C: high magnification image showing the H+-ATPase-associated studs on the cytoplasmic side of the plasma membrane (arrows). Bar = 60 nm. D: apical plasma membrane of a proton-secreting mitochondria-rich cell from a toad urinary bladder visualized after rapid-freeze, deep-etch treatment. Each individual stud represents the V1 sector of a single H+-ATPase molecule. The 10-nm-diameter structures form a tightly packed, paracrystalline array. This illustrates the dense clustering of H+-ATPase molecules in these specialized membrane domains. Bar = 30 nm. [Modified from Brown et al. (77).]

This membrane-coating material was identified as the V₁ sector of the vacuolar H⁺-ATPase complex by rapid-freeze, deep-etch electron microscopy. The underside of fragments of apical plasma membrane from toad urinary bladder epithelium was examined following rapid-freezing and rotary shadowing with platinum and carbon (77). Figure 6D shows the underside of the plasma membrane from a proton-secreting mitochondria-rich (MR) cell. The paracrystalline arrays of densely packed, 10-nm-diameter projections were also found on the cytoplasmic surface of vesicles inside these cells. Identical structures were seen when the purified enzyme was incorporated into liposomes and examined by the rapid-freeze, deep-etch procedure, confirming their identity as the vacuolar H⁺-ATPase (77). The vacuolar H⁺-ATPase complex on the contractile vacuole of Paramecium was also examined by the rapid-freeze, deep-etch technique, and similar studs (referred to as "pegs") were detected (115, 237). Coupled with immunocytochemical labeling at the light and electron microscopic level with specific antivacuolar H⁺-ATPase subunit antibodies, this work provided direct evidence that the portasomes originally observed in insect cells were composed of the cytoplasmic V₁ sectors of the vacuolar H⁺-ATPase.

3. Freeze-fracture electron microscopy and rod-shaped intramembraneous particles

By freeze-fracture electron microscopy, the lipid bilayer of many cells that express high levels of the vacuolar H⁺-ATPase contains characteristic, elongated, or rod-shaped intramembraneous particles (IMPs) (73). These particles are usually assumed to represent the transmembrane V₀ sector of the vacuolar H⁺-ATPase (Fig. 4C). In some instances, an increase in the proton secretory activity of membranes has been associated with an increase in the number and density of rod-shaped IMPs in the plasma membrane (286, 486) or with the apparent activation of intercalated cells (486). However, while all membrane domains that contain rod-shaped IMPs also contain the vacuolar H⁺-ATPase as detected by immunocytochemistry, some cells that have abundant membrane-associated vacuolar H⁺-ATPase do not show rod-shaped IMPs when examined by the freeze-fracture technique. Renal proximal tubules, for example, have apical plasma membrane vacuolar H⁺-ATPase but no rod-shaped IMPs (401). Furthermore, H⁺-ATPase-rich narrow cells in the proximal regions of the epididymis have many rod-shaped IMPs (81), whereas those in the distal epididymis, which are known as clear cells, have no detectable rod-shaped IMPs (82). The precise relationship between the characteristic rod-shaped IMPs and the vacuolar H⁺-ATPase remains to be established but by analogy with the F₀F₁ ATP synthase (92), it is possible that they represent dimeric structures comprised of two V₀ domains (which form the rod-shaped IMP) and two V₁ domains, which are visible on the cytoplasmic surface of the membrane as studs or portasomes.

	IV. ROLE OF VACUOLAR H+-ATPases IN KIDNEY ACID-BASE TRANSPORT

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Overall body pH (acid-base) homeostasis is controlled mainly by the exhalation of CO₂ and by the reabsorption, generation, or secretion of bicarbonate as well as the secretion of acid and acid equivalents by the kidneys (for review, see Ref. 222). About 180–200 liters of blood are filtered daily in the glomeruli of the kidneys; 99% of the filtered load has to be reabsorbed along the nephron to avoid excessive loss of solutes and fluid. This is achieved by the reabsorption of solutes (i.e., amino acids, phosphate, glucose), NaCl, bicarbonate, and water mediated by specialized transport systems.

A. Proximal Tubule

Bicarbonate reabsorption in the proximal tubule accounts for 70–80% of the filtered load and occurs mainly in the initial segments (222). It is mediated by the concerted action of several transport proteins and enzymes located on the apical and basolateral membranes (Fig. 7A). On the apical side two main transport systems mediate H⁺ secretion, the first step in bicarbonate reabsorption. The bulk of proton secretion involves Na⁺/H⁺ exchange, and several isoforms of Na⁺/H⁺ exchangers have been localized in the brush-border membrane of the proximal tubule, including NHE-2, NHE-3, and NHE-8 (209). About 50% of overall apical NHE activity may be mediated by NHE-3, the remainder by another isoform(s) (111). Genetic knock-out of NHE-2 has no effect on renal function, whereas complete or kidney-specific knock-out of NHE-3 results in a reduction of proximal tubular bicarbonate reabsorption (555) as well as Na⁺ and water loss (451, 573). Up to 40% of proximal tubule bicarbonate reabsorption is Na⁺ independent and is sensitive to the vacuolar H⁺-ATPase inhibitor bafilomycin (103, 555), and it has thus been postulated to be mediated by vacuolar H⁺-ATPases expressed in the brush-border membrane. The extent of Na⁺-independent, bafolimycin-sensitive bicarbonate reabsorption varies among different species, being more extensive in carnivores than in herbivores (146). Indeed, vacuolar H⁺-ATPases are strongly expressed in the proximal tubule (see below), and the activity has been measured as NEM-, bafilomycin-, or concanamycin-sensitive H⁺ extrusion in several proximal tubular preparations such as in brush-border membrane vesicles (101, 234, 260, 329, 446, 475), isolated proximal tubule fragments (146, 173, 276, 543, 544, 596), and isolated perfused proximal tubules (191, 334) and using in vivo microperfusion experiments (103, 555).

View larger version (29K): [in this window] [in a new window]   FIG. 7. Schematic model of transport processes involved in renal bicarbonate absorption and proton secretion. A: in the proximal tubule, protons are secreted via apical Na+/H+ exchangers and vacuolar H+-ATPases. The secreted H+ combine with filtered HCO3– under the influence of a membrane-bound carbonic anhydrase (CA IV) to form H2O and CO2. After diffusion into the cell, CO2 is rehydrated by the cytosolic carbonic anhydrase II (CA II), the H+ secreted again, and the HCO3– released into the interstitium via the basolateral Na+/HCO3– cotransporter NBC1. B: type A intercalated cells are characterized by the expression of the basolateral Cl–/HCO3– exchanger AE-1 and the presence of an apical vacuolar H+-ATPase. H+ and HCO3– are formed by a cytosolic CA II and secreted into the lumen and the interstitium, respectively. Type A intercalated cells may also express an apical H+-K+-ATPase. C: type B intercalated cells are characterized by the absence of the AE-1 Cl–/HCO3– exchanger on the basolateral side and the presence of the vacuolar H+-ATPase which can be found on both sides of the cell. In addition, type B intercalated cells express an apical Cl–/HCO3– exchanger that may be represented by pendrin. As suggested by some authors, a third subtype of intercalated cells, non-A/non-B, is characterized by the apical expression of vacuolar H+-ATPases and the absence of AE-1.

B. Loop of Henle and TAL

About 15–20% of the filtered bicarbonate is reabsorbed in the loop of Henle, mainly in the TAL involving Na⁺/H⁺ exchange and NEM-sensitive vacuolar H⁺-ATPase (56, 97, 98, 557). The main Na⁺/H⁺ exchanger isoform is NHE-3, but NHE-2 may also play a role (98, 554). The reabsorbed HCO₃^– is excreted into blood possibly through a K⁺-dependent HCO₃^– pathway (56), the molecular identity of which is not resolved. The expression of the Cl^–/HCO₃^– exchanger isoform AE2 in the TAL has been described and functionally analyzed (12, 150). In addition, both the electroneutral NBC(n)1 (538) and electrogenic NBC4 (577) are expressed in the TAL and might be involved in bicarbonate reabsorption in this segment. The TAL cells contain extensive apical and subapical vesicular H⁺-ATPase (see below), but a definitive role in bicarbonate reabsorption by this nephron segment has not been shown. However, bafilomycin does inhibit intracellular pH recovery from an acid load by TAL cells in suspension (184). Several hormones and conditions such as o