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Renal Ion-Translocating ATPases
Ion motive ATPases involved in transcellular ion transport by renal epithelial cells include two molecular families: P-ATPases (Na,K-ATPase and H,K-ATPase) and V-ATPases. Na,K-ATPase is found in the plasma membrane of every vertebrate cell and exchanges three intracellular Na + for two intracellular K + for each hydrolyzed ATP molecule. Na,K-ATPase is highly expressed in the kidney tubule where it is located in the basolateral membrane and energizes Na + reabsorption. Na,K-ATPAse is highly regulated by hormones and local factors via modulation of its rate of synthesis, degradation and post translational events including regulatory phosphorylation and intracellular trafficking. Both gastric and non-gastric H,K-ATPases are expressed in the distal part of the kidney tubule where they reabsorb K + and secrete H + . Vacuaolar H-ATPases is found in intracellular compartments of every renal epithelial cell. In intercalated cells of connecting tubules and collecting ducts, it is localized in plasma membrane where it plays a key role in acid–base transport.
Keyword
ATPase; active ion transport; renal epithelial cell; sodium; potassium; proton
Active transport of solutes across membranes against their concentration or electrochemical gradients requires energy. For ion-motive ATPases (F-type, V-type, and P-type), this process is an exchange between energy contained in the electrochemical gradient and chemical energy provided by ATP hydrolysis. F-type ATPases or ATP synthases are responsible for the generation of ATP using energy of the proton gradient created by the respiratory chain in mitochondria or photosynthetic complexes in chloroplasts. V-type ATPases acidify vesicles by transporting protons from the cytoplasm to the lumen of intracellular organelles (endosomes, lysosomes, vacuoles, …). V-ATPases are also present in the plasma membrane of some epithelial cells. Despite sharing a common general architecture and a large number of subunits, F- and V-ATPases usually work in opposite directions. P-type ATPases (also called E1-, E2-ATPases) form a third group of ion-motive ATPases that perform unidirectional or exchange transport of monovalent (H + , Na + , K + ) or divalent (Ca 2+ , Cu 2+ , Mg 2+ , …) cations.
P-ATPases
While some members of the P-ATPase family are probably active as a single polypeptide, the functional unit of others consists of several subunits. The major subunit (α- or catalytic subunit for multimeric P-ATPases) consists of a series of hairpins formed by pairs of transmembrane segments linked by short extracellular loops. Two large intracellular loops make the connection between the first, second, and third hairpins. The largest cytoplasmic loop contains the ATP-binding domain and the phosphorylation site. Transient phosphorylation of this aspartate residue occurring during the transport cycle is a hallmark of P-ATPases ( Figure 3.1 ).
Structure and Function of Ca 2+ -ATPases (SERCA and PMCA)
SERCA is found in intracellular organelles related to the endoplasmic reticulum, such as the sarcoplasmic reticulum of cardiac and skeletal muscle cells. Three differently expressed genes have been identified: SERCA1 in fast-twitch skeletal muscle; SERCA2 in slow-twitch skeletal muscle, heart, and smooth muscle; and SERCA3 in blood, endothelial, and epithelial tissue. SERCA mediates uptake of Ca 2+ from the cytoplasm into the sarcoplasmic reticulum following calcium release from intracellular stores. It therefore acts as a terminator signal in excitation–contraction coupling processes in muscle, and plays a key role in excitation–secretion coupling in neurons and other secretory cells. Extensive structure–function studies have been performed with SERCA and a high resolution (2.6 Å) structure of this protein was obtained.
PMCA is expressed at the plasma membrane, where it extrudes Ca 2+ out of the cell. Four PMCA genes are known, with multiple splicing variants for each gene, resulting in the existence of about 20 isoforms. PMCA1 and PMCA4 are ubiquitous, while PMCA2 and PMCA3 are restricted to neurons, brain, muscle, and kidney. PMCA plays an important role in tubular reabsorption of calcium. The long C-terminal intracellular domain maintains PMCA in an inactive state by interacting with the catalytic site. A rise of cytosolic Ca 2+ concentration increases Ca 2+ –calmodulin-binding, allowing calmodulin to interact with the PMCA C-terminal domain. This releases PMCA autoinhibition, activating the pump. Conversely, calcium extrusion decreases cytosolic Ca 2+ concentration, and consequently its association with calmodulin. Calmodulin release from PMCA increases PMCA auto-inhibition. Regulatory inhibition of SERCA is mediated by the small associated protein phospholamban, which plays a role equivalent to that of the PMCA C-terminal domain. Phosphorylation of phospholamban by protein kinase C releases SERCA inhibition.
Structure of Na,K-ATPase and H,K-ATPase
Na,K- and H,K-ATPases are heteromeric proteins consisting of an α-subunit and a smaller glycosylated β-subunit. Na,K-ATPase hydrolytic activity, cation transport activity, and ouabain-binding properties were demonstrated by co-expression of α- and β-subunits in several expression systems (mammalian cells, Xenopus oocytes, baculovirus-infected insect cells, and yeast). Na- (or H-) and K-activated ATPase and cation transport activities (i.e., uphill cation transport driven by ATP hydrolysis) characteristic of Na,K- or H,K-ATPases have been demonstrated only in the presence of both α- and β-subunits. Expression of the α-subunit alone in insect cells resulted in Mg 2 -dependent ATPase activity that was not specifically activated by Na + and K + . The exact stoichiometry of the minimal functional unit is still a matter of debate. However, Na,K-ATPase activity is associated with solublized α–β protomers, and cross-linking experiments did not show evidence for a close interaction between α-subunits. A third subunit, the γ-subunit, can be associated with the α–β complex (see below).
Catalytic α-Subunit
Structure
Na,K- and H,K-ATPase α-subunit peptides range in length from about 1000 to 1040 amino acids. Their primary structure is characterized by a first group of four transmembrane segments, followed by a large cytoplasmic loop, and a second group of six transmembrane segments ( Figure 3.1 ). Crystal structures of pig and shark Na,K-ATPase at 3.5 and 2.4 Å resolutions, respectively, confirmed that the α-subunit has three cytoplasmic domains and 10 transmembrane helices, designated M1 to M10. Two-thirds of its mass is contained in the large cytoplasmic domain, while one-third spans the lipid bilayer. Of the total mass, only a small part is extracellular. Sequence homology between Na,K- and H,K-ATPases is high enough to safely predict H,K-ATPase structure, at least for the general architecture of this molecule, and for large domains where homology is highest.
Isoforms
Six different α-subunit genes have been identified in mammals: α1-4 isoforms of the Na,K-ATPase α-subunit; gastric H,K-ATPase α-subunit (αHKg); and colonic H,K-ATPase α-subunit (αHKc). Related isoforms have been identified in birds and amphibians. Na,K-ATPase sequences from Caenorhabditis elegans or Drosophila melanogaster do not show close similarity with any mammalian isoform. This suggests that the divergence between Na,K- and H,K-ATPase α-subunits precedes the divergence between mammals, amphibians and birds, and has occurred early in vertebrate evolution.
All Na,K-ATPase isoforms primarily maintain Na + and K + gradients across the cell membrane. The large inward electrochemical gradient for Na + is in turn used by numerous secondary active transport systems for various “housekeeping” functions: maintenance of intracellular pH via Na–H exchangers; extrusion of calcium via Na–Ca exchanger; control of cell volume via Na–K–2Cl symport and other coupled transport systems; and import of amino acids, nucleotides, and other nutrients or osmolytes through various Na + -coupled co-transport systems. The outward electrochemical gradient for K + is responsible for the intracellular negative membrane potential, because K + flows out of the cell through K + selective channels that are active in most cells. In addition to these general functions, Na + and K + gradients across cell membranes are essential for specialized functions, such as the generation and propagation of action potentials in excitable cells, neurotransmitter uptake, and transcellular transport of solutes and water by epithelial cells.
The α1 isoform is the most ubiquitous and abundant α isoform, and is responsible for the maintenance of whole-cell Na + and K + gradients necessary for housekeeping functions. Because of its abundance (it is the only α isoform present in many epithelial cells, including renal cells) it provides the driving force for solute and water transepithelial transport. The α2 isoform is found in skeletal and heart muscle, and in the nervous system (neurons and glial cells). The α3 isoform is essentially neuronal, but is also found in blood cells and macrophages. The α4 isoform is mostly expressed in testes, and plays a critical role in sperm motility.
αHKg is abundantly expressed in parietal cells of the gastric gland, where it plays a central role in proton secretion. Under resting conditions, it is mainly located in an intracellular tubulo–vesicular network that fuses with the apical membrane of parietal cells in response to stimuli, allowing the H,K-pump to secrete protons into the gastric gland lumen in exchange for potassium. αHKc is mainly expressed in the (rat) distal colon, but also in the kidney, uterus, and, to a lesser extent, in the heart.
β-Subunit
Isoforms
As stated above, the β-subunit is an essential constituent of functional Na,K-ATPase and H,K-ATPase. Five different genes encoding similar proteins are known in mammalian genomes: β1; β2; β3; βHK; and βm (“m” emphasizes its predominant expression in skeletal muscle). β1, β2, and β3 are clearly Na,K-ATPase β-subunit isoforms, while βHK is co-expressed with gastric H,K-ATPase. Although usually described as ubiquitous, β1 appears to be absent, or at best is only a minor component, in several tissues such as liver and red blood cells. The β2 isoform was initially identified in glial cells, but is also present in other cell types, including neurons, blood cells, and epithelial cells. The β3 isoform, initially identified in nervous systems, is also widely distributed, being most abundant in testes, liver, and lungs and less so in skeletal muscle and kidney. Despite sharing sufficient sequence similarity to be classified in the same family, βm does not associate with any known mammalian α-subunit.
Structure and α–β Interaction
β-subunit peptides range in length from 288 to 315 amino acids, and show a lower degree of homology (about 30%–40% identity between isoforms) than α isoforms. Crystal structures of Na,K-ATPase obtained at resolutions of 3.5 Å and 2.4 Å have lent detailed insight into β-subunit structure. These studies, together with experimental evidence, show that the β-subunit is a type II membrane protein consisting of a single transmembrane segment, a ~35-amino acid N-terminal domain, and a large extracellular domain containing two to seven glycosylation sites, depending on the isoform, and six cysteine residues that form three disulfide bridges ( Figure 3.1 ).
Experimental modeling and analysis of Na,K-ATPase crystal structures has revealed complex interactions between α- and β-subunits. The transmembrane helix of the β-subunit forms several hydrogen bonds and numerous contacts with M7 and M10 transmembrane helices of the α-subunit, primarily via clusters of aromatic residues. At the extracellular side of the β-subunit, a stretch of amino acids adjacent to its transmembrane domain interacts with the α M7 / M8 extracellular loop that contains a consensus sequence SYGQ. Further downstream, Lys250 of the β-subunit forms a salt bridge with Glu899, located in the α M7 / M8 extracellular loop.
Except for gastric αHK and βHK, which are most abundantly expressed in a single cell type (parietal cells of gastric glands), there is no obvious common pattern of distribution between α isoforms and β isoforms that would define preferential physiological associations. Indeed, some cells even express as many as three α isoforms and at least two β isoforms. Unless formation of specific complexes is favored or repressed by unknown mechanisms, numerous combinations are possible, as suggested by studies using artificial expression systems. β1 is abundantly expressed in tissues in which α1 predominates, such as the kidney, strongly suggesting that α1β1 represents the predominant isozyme in these tissues. The nature of the β-subunit associated with αHKc is also a matter of debate, since all β isoforms are able to associate with αHKc, depending on the expression system used.
Functional Role
The functional interaction between α- and β-subunits has been studied and reviewed in detail. By acting as a molecular chaperone, the β-subunit plays a critical role in the maturation of the α-subunit. Indeed, the α-subunit reaches a mature and functional conformation, ready to be translocated from the endoplasmic reticulum to the plasma membrane, only when associated with a β-subunit.
The β-subunit contributes to intrinsic transport properties of the whole enzyme in several expression systems by influencing its apparent K + and Na + affinities. Biochemical analysis and crystallization of Na,K-ATPase has lent some mechanistic insight as to how this is achieved. By interacting with the M7 transmembrane domain of the α-subunit, Tyr40 and Tyr44 of the β-subunit transmembrane helix help confer intrinsic transport properties of the Na,K-ATPase enzyme, as suggested by a mutagenic study. Unwinding of M7 via hydrogen bonding of Tyr44 with Gly855 appears to be of central importance to K + binding. The role of the β-subunit ectodomain in modulating cation transport is further illustrated by the complex interactions between this domain and the α M7 / M8 extracellular loop.
The role of the β-subunit in cell–cell adhesion will be discussed in the section “New Physiological Functions of Na,K-ATPase.”
FXYD Proteins
Isoforms
FXYD proteins are a third component of Na,K-ATPase. There are seven isoforms in mammals, ranging from 61 to 95 amino acids in length, except for FXYD5, which consists of 178 amino acids due to an N-terminal extension. Most FXYD proteins are small type 1 membrane proteins containing an extracellular N-terminus. This family of proteins is so named since all members contain a FXYD (Phe-X-Tyr-Asp) sequence located immediately downstream of the transmembrane segment. All members also contain two conserved glycine residues in the transmembrane domain, as well as a serine residue located further downstream. As with α and β isoforms, the tissue distribution of FXYD proteins is isoform-specific. FXYD1 (phospholemman) is predominately expressed in heart and skeletal muscle and, to a lesser extent, in brain, FXYD2 (γ-subunit) in kidney, FXYD3 (MAT-8) in stomach and colon, FXYD4 (CHIF) in kidney and distal colon, FXYD5 (RIC) in kidney, intestine and lung, and FXYD6 and FXYD7 in brain. In the kidney FXYD2 is mostly expressed in proximal tubule and thick ascending limb of Henle, while FXYD4 is exclusively found in the collecting duct where its expression increases from cortical to medullary portions.
Function and Interaction with α- and β-Subunits
FXYD proteins were first thought to be regulators of ion channels or even to act as ion channels themselves. It has now been demonstrated that FXYD proteins interact with Na,K-ATPase. Contrary to the β-subunit, most FXYD proteins do not associate with H,K-ATPase and do not appear to act as a molecular chaperone. Rather, they regulate Na,K-ATPase functional properties. As recently reviewed, analysis of FXYD-deficient mice and in vitro modulation of Na,K-ATPase activity by different FXYD proteins has shown that FXYD1–4, 6, and 7 all decrease Na,K-ATPase apparent affinity for Na + and/or K + , with the exception of FXYD4 which has been shown to decrease apparent affinity for K + but increase that for Na + . FXYD5 does not appear to influence Na,K-ATPase affinity for either Na + or K + , but enhances maximal transport activity. By modulating Na,K-ATPase activity, FXYD proteins play important physiological roles, each isoform playing a different role depending on its tissue-specific distribution. For instance, FXYD1 influences myocardial contractility by modulating Na,K-ATPase activity and calcium handling, as demonstrated in FXYD1-deficient mice that display depressed cardiac contractile function and increased cardiac mass. FXYD6, the only FXYD isoform expressed in the inner ear, may play an auditory and vestibular role by contributing to endolymph ionic compostion, whose production depends on Na,K-ATPase activity. Reflecting altered expression levels in various types of cancer, FXYD3 may be implicated in the control of cell differentiation, proliferation, and/or apoptosis through regulation of Na,K-ATPase activity. Finally, the renal tubule segment-specific distribution of FXYD2 and FXYD4 may explain, at least in part, the higher apparent Na + affinity of collecting duct Na,K-ATPase as compared to that of more proximal nephron segments.
Crystallization of the Na,K-ATPase holoenzyme has deciphered FXYD structure, and revealed how it interacts with α- and β-subunits, providing some insight into how it modulates Na,K-ATPase activity. This notably involves interaction between the transmembrane domain of FXYD proteins, particularly Gly34, with the α M9 transmembrane domain. Hydrogen bonds between Cys31 and αGlu960 may additionally play a structural role important for FXYD functional regulation. The FXYD motif helps confer β conformational structure. This is partly achieved via Phe12, which anchors this segment to the β-subunit, and Tyr14, which forms a cluster of aromatic residues with Tyr69 of the β-subunit and Trp987 of the α M9 / M10 extracellular loop.
Properties of Na,K-ATPase and H,K-ATPase
Ion Transport
Under physiological conditions, Na,K-ATPase exchanges three intracellular Na + for two extracellular K + at the expense of one ATP during each cycle, generating an outward current of one net charge per cycle. The outward current generated by the Na,K-pump tends to hyperpolarize the cell membrane to a few millivolts under steady-state conditions. The majority of the 50 to 80 mV resting-membrane potential is due to flow of K through K channels, which relies on Na,K-pump activity, since high intracellular K + concentration is maintained by the Na,K-pump. The apparent affinities for Na + and K + are dependent on experimental conditions, but under physiological conditions intracellular Na + activates the Na,K-pump with a K½ of 10 to 20 mM and a Hill coefficient between 2 and 3. Extracellular K + has a K½ of about 1 mM, with a Hill coefficient between 1 and 2.
Despite extensive studies, the difference in transport properties between Na,K-ATPase α isoforms has not been entirely resolved. Initial studies performed in native tissues indicated that α2/α3 isoforms have a higher affinity for Na + . More recent studies comparing isoforms in the same artificial expression system revealed that α2 has a slightly lower (about 20 mM), and α3 a much lower (30–70 mM), affinity for Na. However, other factors, such as the type of associated β-subunit and FXYD protein, also modulate Na,K-ATPase transport properties (see above).
The mechanism of cation translocation by Na,K-ATPase can be summarized as follows ( Figure 3.2 ). Na,K-ATPase exists under two main conformations, E1 and E2, and transport activity is performed via a cycle in which the protein is transiently phosphorylated, and alternately adopts E1 or E2 conformations. These two conformations differ in their apparent affinities for Na + and K + . The E1 conformation has high affinity sites for Na + exposed at the intracellular side of the membrane, while the E2 conformation has high affinity sites for K + exposed at the extracellular side of the membrane. Three Na + and two K + ions are alternately bound to the enzyme and then “occluded,” that is, tightly bound inside the protein. This model is compatible with the reported structure of the major conformations of SERCA, and the E2-P conformation of Na,K-ATPase.
Non-electrogenic transport is achieved by gastric H,K-ATPase, indicating that a symmetrical number of H + and K + ions are translocated across the membrane during each cycle. Transport stoichiometry depends on pH. Under conditions of high or near neutral pH, the stoichiometry is 2H,2K + -1ATP, and shifts to 1H,1K + -1ATP under physiological conditions, i.e., conditions of very low extracellular pH. The transport properties of colonic H,K-ATPase and its close relatives (human ATP1AL1 and toad Bufo marinus bladder H,K-pump) are not yet completely defined. Artificial systems have demonstrated an inward transport of K + , and an outward transport of H + . However, data obtained in heterologous expression systems have suggested that these enzymes may exchange Na + for K + .
Pharmacology
A group of natural compounds known as “cardiac steroids,” so named because they contain a steroid nucleus attached to a lactone ring and are used for treatment of congestive heart failure, are potent Na,K-ATPase inhibitors. In addition, one or several endogenously related compounds may also act as hormonal agents that participate in regulating Na,K-ATPase activity.
Cardiac steroid interaction with Na,K-ATPase, particularly ouabain, has been extensively studied. Differences of ouabain affinity between Na,K-ATPase isoforms, together with mutagenesis studies and recently obtained crystal structures of ouabain bound to Na,K-ATPase, all show that ouabain binds to a deep cavity formed by the transmembrane helices M1 , M2 , M4 , M5 , and M6 at the proximity of the K + -binding site. The slow kinetics of ouabain binding may be associated with partial unwinding of the M4 helix.
Large differences in ouabain sensitivity occur among animal species. The α1 isoform is ouabain-resistant in rat, mouse, and toad Bufo marinus , but is sensitive in human, rabbit, sheep, and Xenopus . The α2 and α3 isoforms are more sensitive than the α1 isoform in “resistant” species. However, in humans, little difference of equilibrium binding is found among α1, α2, and α3 isoforms, except for a slightly higher K I for α2. However, the α2 isoform also exhibits a faster ouabain association and dissociation rate constant than α1 and α3. The “resistant” phenotype of some species is linked to charged amino acids in the first extracellular loop between M1 and M2 . The presence of endogenous circulating inhibitors of Na,K-ATPase (“endo-ouabain”) is well-demonstrated. However, their precise chemical nature needs to be clarified, their controlled synthesis and release better understood, and their specific effects more precisely described, before the hypothesis of controlled Na,K-ATPase activity by such circulating hormones can be considered as fully established.
Gastric H,K-ATPase is insensitive to ouabain, as demonstrated by an absence of detectable effects at millimolar concentrations of ouabain. On the other hand, non-gastric H,K-ATPases, expressed in Xenopus oocytes, show some sensitivity to ouabain and exhibit inhibitory constants (K I ) between 10 and 100 mM. Two types of gastric H,K-pump inhibitors are known. SCH-28080 is a reversible inhibitor that competes with extracellular K + , while substituted benzimidazole compounds irreversibly inhibit gastric H,K-ATPase by forming a covalent (disulfide) bond between the sulfonamide form of the compound (produced in very acid pH) and the thiol group of a cysteine residue exposed at the cell surface.
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