Animal plasma membrane energization by proton-motive V-ATPases

Proton-translocating, vacuolar-type ATPases, well known energizers of eukaryotic, vacuolar membranes, now emerge as energizers of many plasma membranes. Just as Na⁺ gradients, imposed by Na⁺/K⁺ ATPases, energize basolateral plasma membranes of epithelia, so voltage gradients, imposed by H⁺ V-ATPases, energize apical plasma membranes. The energized membranes acidify or alkalinize compartments, absorb or secrete ions and fluids, and underwrite cellular homeostasis. V-ATPases acidify extracellular spaces of single cells such as phagocytes and osteoclasts and of polarized epithelia, such as vertebrate kidney and epididymis. They alkalinize extracellular spaces of lepidopteran midgut. V-ATPases energize fluid secretion by insect Malpighian tubules and fluid absorption by insect oocytes. They hyperpolarize external plasma membranes for Na⁺ uptake by amphibian skin and fish gills. Indeed, it is likely that ion uptake by osmotically active membranes of all fresh water organisms is energized by V-ATPases. Awareness of plasma membrane energization by V-ATPases provides new perspectives for basic science and presents new opportunities for medicine and agriculture. BioEssays 21:637–648, 1999. © 1999 John Wiley & Sons, Inc.     

Sodium,Potassium-ATPases in Algae and Oomycetes

We have investigated the presence of K⁺-transporting ATPases that belong to the phylogenetic group of animal Na⁺,K⁺-ATPases in the Pythium aphanidermatum Stramenopile oomycete, the Porphyra yezoensis red alga, and the Udotea petiolata green alga, by molecular cloning and expression in heterologous systems. PCR amplification and search in EST databases allowed one gene to be identified in each species that could encode ATPases of this type. Phylogenetic analysis of the sequences of these ATPases revealed that they cluster with ATPases of animal origin, and that the algal ATPases are closer to animal ATPases than the oomycete ATPase is. The P. yezoensis and P. aphanidermatum ATPases were functionally expressed in Saccharomyces cerevisiae and Escherichia coli alkali cation transport mutants. The aforementioned cloning and complementary searches in silicio for H⁺- and Na⁺,K⁺-ATPases revealed a great diversity of strategies for plasma membrane energization in eukaryotic cells different from typical animal, plant, and fungal cells.     

Differential expression of P-type ATPases in intestinal epithelial cells: Identification of putative new atp1a1 splice-variant

P-type ATPases are membrane proteins that couple ATP hydrolysis with cation transport across the membrane. Ten different subtypes have been described. In mammalia, 15 genes of P-type ATPases from subtypes II-A, II-B and II-C, that transport low-atomic-weight cations (Ca²⁺, Na⁺, K⁺ and H⁺), have been reported. They include reticulum and plasma-membrane Ca²⁺-ATPases, Na⁺/K⁺-ATPase and H⁺/K⁺-ATPases. Enterocytes and colonocytes show functional differences, which seem to be partially due to the differential expression of P-type ATPases. These enzymes have 9 structural motifs, being the phosphorylation (E) and the Mg²⁺ATP-binding (H) motifs the most preserved. These structural characteristics permitted developing a Multiplex-Nested-PCR (MN-PCR) for the simultaneous identification of different P-type ATPases. Thus, using MN-PCR, seven different cDNAs were cloned from enterocytes and colonocytes, including SERCA3, SERCA2, Na⁺/K⁺-ATPase α1-isoform, H⁺/K⁺-ATPase α2-isoform, PMCA1, PMCA4 and a cDNA-fragment that seems to be a new cassette-type splice-variant of the atp1a1 gen. PMCA4 in enterocytes and H⁺/K⁺-ATPase α2-isoform in colonocytes were differentially expressed. This cell-specific expression pattern is related with the distinctive enterocyte and colonocyte functions.     

Evolution of the sodium pump

Eukaryotic plasma membranes (PMs) are energized by electrogenic P-type ATPases that generate either Na⁺ or H⁺ motive forces to drive Na⁺ and H⁺ dependent transport processes, respectively. For this purpose, animal rely on Na⁺/K⁺-ATPases whereas fungi and plants employ PM H⁺-ATPases. Prokaryotes, on the other hand, depend on H⁺ or Na⁺-motive electron transport complexes to energize their cell membranes. This raises the question as to why and when electrogenic Na⁺ and H⁺ pumps evolved? Here it is shown that prokaryotic Na⁺/K⁺-ATPases have near perfect conservation of binding sites involved in coordination of three Na⁺ and two K⁺ ions. Such pumps are rare in Eubacteria but are common in methanogenic Archaea where they often are found together with P-type putative PM H⁺-ATPases. With some exceptions, Na⁺/K⁺-ATPases and PM H⁺-ATPases are found everywhere in the eukaryotic tree of life, but never together in animals, fungi and land plants. It is hypothesized that Na⁺/K⁺-ATPases and PM H⁺-ATPases evolved in methanogenic Archaea to support the bioenergetics of these ancestral organisms, which can utilize both H⁺ and Na⁺ as energy currencies. Both pumps must have been simultaneously present in the first eukaryotic cell, but during diversification of the major eukaryotic kingdoms, and at the time animals diverged from fungi, animals kept Na⁺/K⁺-ATPases but lost PM H⁺-ATPases. At the same evolutionary branch point, fungi did loose Na⁺/K⁺-ATPases, and their role was taken over by PM H⁺-ATPases. An independent but similar scenery emerged during terrestrialization of plants: they lost Na⁺/K⁺-ATPases but kept PM H⁺-ATPases.     

Sodium or potassium efflux ATPase: A fungal, bryophyte, and protozoal ATPase

The K⁺ and Na⁺ concentrations in living cells are strictly regulated at almost constant concentrations, high for K⁺ and low for Na⁺. Because these concentrations correspond to influx-efflux steady states, K⁺ and Na⁺ effluxes and the transporters involved play a central role in the physiology of cells, especially in environments with high Na⁺ concentrations where a high Na⁺ influx may be the rule. In eukaryotic cells two P-type ATPases are crucial in these homeostatic processes, the Na,K-ATPase of animal cells and the H⁺-ATPase of fungi and plants. In fungi, a third P-type ATPase, the ENA ATPase, was discovered nineteen years ago. Although for many years it was considered to be exclusively a fungal enzyme, it is now known to be present in bryophytes and protozoa. Structurally, the ENA (from exitus natru: exit of sodium) ATPase is very similar to the sarco/endoplasmic reticulum Ca²⁺ (SERCA) ATPase, and it probably exchanges Na⁺ (or K⁺) for H⁺. The same exchange is mediated by Na⁺ (or K⁺)/H⁺ antiporters. However, in eukaryotic cells these antiporters are electroneutral and their function depends on a ΔpH across the plasma membrane. Therefore, the current notion is that the ENA ATPase is necessary at high external pH values, where the antiporters cannot mediate uphill Na⁺ efflux. This occurs in some fungal environments and at some points of protozoa parasitic cycles, which makes the ENA ATPase a possible target for controlling fungal and protozoan parasites. Another technological application of the ENA ATPase is the improvement of salt tolerance in flowering plants.     

Structural Basis of Substrate-Independent Phosphorylation in a P4-ATPase Lipid Flippase

P4-ATPases define a eukaryotic subfamily of the P-type ATPases, and are responsible for the transverse flip of specific lipids from the extracellular or luminal leaflet to the cytosolic leaflet of cell membranes. The enzymatic cycle of P-type ATPases is divided into autophosphorylation and dephosphorylation half-reactions. Unlike most other P-type ATPases, P4-ATPases transport their substrate during dephosphorylation only, i.e. the phosphorylation half-reaction is not associated with transport. To study the structural basis of the distinct mechanisms of P4-ATPases, we have determined cryo-EM structures of Drs2p-Cdc50p from Saccharomyces cerevisiae covering multiple intermediates of the cycle. We identify several structural motifs specific to Drs2p and P4-ATPases in general that decrease movements and flexibility of domains as compared to other P-type ATPases such as Na⁺/K⁺-ATPase or Ca²⁺-ATPase. These motifs include the linkers that connect the transmembrane region to the actuator (A) domain, which is responsible for dephosphorylation. Additionally, mutation of Tyr380, which interacts with conserved Asp340 of the distinct DGET dephosphorylation loop of P4-ATPases, highlights a functional role of these P4-ATPase specific motifs in the A-domain. Finally, the transmembrane (TM) domain, responsible for transport, also undergoes less extensive conformational changes, which is ensured both by a longer segment connecting TM helix 4 with the phosphorylation site, and possible stabilization by the auxiliary subunit Cdc50p. Collectively these adaptions in P4-ATPases are responsible for phosphorylation becoming transport-independent.     

P-type ATPases of eukaryotes and bacteria: Sequence analyses and construction of phylogenetic trees

The amino acid sequences of 47 P-type ATPases from several eukaryotic and bacterial kingdoms were divided into three structural segments based on individual hydropathy profiles. Each homologous segment was (1) multiply aligned and functionally evaluated, (2) statistically analyzed to determine the degrees of sequence similarity, and (3) used for the construction of parsimonious phylogenetic trees. The results show that all of the P-type ATPases analyzed comprise a single family with four major clusters correlating with their cation specificities and biological sources as follows: cluster 1: Ca²⁺-transporting ATPases; cluster 2: Na⁺- and gastric H⁺-ATPases; cluster 3: plasma membrane H⁺-translocating ATPases of plants, fungi, and lower eukaryotes; and cluster 4: all but one of the bacterial P-type ATPases (specific for K⁺, Cd²⁺, Cu²⁺ and an unknown cation). The one bacterial exception to this general pattern was the Mg²⁺-ATPase of Salmonella typhimurium, which clustered with the eukaryotic sequences. Although exceptions were noted, the similarities of the phylogenetic trees derived from the three segments analyzed led to the probability that the N-terminal segments 1 and the centrally localized segments 2 evolved from a single primordial ATPase which existed prior to the divergence of eukaryotes from prokaryotes. By contrast, the C-terminal segments 3 appear to be eukaryotic specific, are not found in similar form in any of the prokaryotic enzymes, and are not all demonstrably homologous among the eukaryotic enzymes. These C-terminal domains may therefore have either arisen after the divergence of eukaryotes from prokaryotes or exhibited more rapid sequence divergence than either segment 1 or 2, thus masking their common origin. The relative rates of evolutionary divergence for the three segments were determined to be segment 2 < segment 1 < segment 3. Correlative functional analyses of the most conserved regions of these ATPases, based on published site-specific mutagenesis data, provided preliminary evidence for their functional roles in the transport mechanism. Our studies define the structural and evolutionary relationships among the P-type ATPases. They should provide a guide for the design of future studies of structure-function relationships employing molecular genetic, biochemical, and biophysical techniques. Correspondence to: M.H. Saier, Jr.     

Functional Identification of H<Superscript>+</Superscript>-ATPase and Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter in the Plasma Membrane Isolated from the Root Cells of Salt-Accumulating Halophyte <Emphasis Type="Italic">Suaeda altissima</Emphasis>

A membrane fraction enriched in plasma membrane (PM) vesicles was isolated from the root cells of a salt-accumulating halophyte Suaeda altissima (L.) Pall. by means of centrifugation in discontinuous sucrose density gradient. The PM vesicles were capable of generating ΔpH at their membrane and the transmembrane electric potential difference (Δψ). These quantities were measured with optical probes, acridine orange and oxonol VI, sensitive to ΔpH and Δψ, respectively. The ATP-dependent generation of ΔpH was sensitive to vanadate, an inhibitor of P-type ATPases. The results contain evidence for the functioning of H⁺-ATPase in the PM of the root cells of S. altissima. The addition of Na⁺ and Li⁺ ions to the outer medium resulted in dissipation of ΔpH preformed by the H⁺-ATPase, which indicates the presence in PM of the functionally active Na⁺/H⁺ antiporter. The results are discussed with regard to involvement of the Na⁺/H⁺ antiporter and the PM H⁺-ATPase in loading Na⁺ ions into the xylem of S. altissima roots.     

Elucidation of antimicrobial activity and mechanism of action by N-substituted carbazole derivatives

Compounds belonging to a carbazole series have been identified as potent fungal plasma membrane proton adenosine triphophatase (H⁺-ATPase) inhibitors with a broad spectrum of antifungal activity. The carbazole compounds inhibit the adenosine triphosphate (ATP) hydrolysis activity of the essential fungal H⁺-ATPase, thereby functionally inhibiting the extrusion of protons and extracellular acidification, processes that are responsible for maintaining high plasma membrane potential. The compound class binds to and inhibits the H⁺-ATPase within minutes, leading to fungal death after 1–3 h of compound exposure in vitro. The tested compounds are not selective for the fungal H⁺-ATPase, exhibiting an overlap of inhibitory activity with the mammalian protein family of P-type ATPases; the sarco(endo)plasmic reticulum calcium ATPase (Ca²⁺-ATPase) and the sodium potassium ATPase (Na⁺,K⁺-ATPase). The ion transport in the P-type ATPases is energized by the conversion of ATP to adenosine diphosphate (ADP) and phosphate and a general inhibitory mechanism mediated by the carbazole derivative could therefore be blocking of the active site. However, biochemical studies show that increased concentrations of ATP do not change the inhibitory activity of the carbazoles suggesting they act as allosteric inhibitors. Furthermore decreased levels of intracellular ATP would suggest that the compounds inhibit the H⁺-ATPase indirectly, but Candida albicans cells exposed to potent H⁺-ATPase-inhibitory carbazoles result in increased levels of intracellular ATP, indicating direct inhibition of H⁺-ATPase.     

P-type ATPases as drug targets: Tools for medicine and science

P-type ATPases catalyze the selective active transport of ions like H⁺, Na⁺, K⁺, Ca²⁺, Zn²⁺, and Cu²⁺ across diverse biological membrane systems. Many members of the P-type ATPase protein family, such as the Na⁺,K⁺-, H⁺,K⁺-, Ca²⁺-, and H⁺-ATPases, are involved in the development of pathophysiological conditions or provide critical function to pathogens. Therefore, they seem to be promising targets for future drugs and novel antifungal agents and herbicides. Here, we review the current knowledge about P-type ATPase inhibitors and their present use as tools in science, medicine, and biotechnology. Recent structural information on a variety of P-type ATPase family members signifies that all P-type ATPases can be expected to share a similar basic structure and a similar basic machinery of ion transport. The ion transport pathway crossing the membrane lipid bilayer is constructed of two access channels leading from either side of the membrane to the ion binding sites at a central cavity. The selective opening and closure of the access channels allows vectorial access/release of ions from the binding sites. Recent structural information along with new homology modeling of diverse P-type ATPases in complex with known ligands demonstrate that the most proficient way for the development of efficient and selective drugs is to target their ion transport pathway.     

Mechanism and significance of P4 ATPase-catalyzed lipid transport: Lessons from a Na/K-pump

Members of the P₄ subfamily of P-type ATPases are believed to catalyze phospholipid transport across membrane bilayers, a process influencing a host of cellular functions. Atomic structures and functional analysis of P-type ATPases that pump small cations and metal ions revealed a transport mechanism that appears to be conserved throughout the family. A challenging problem is to understand how this mechanism is adapted in P₄ ATPases to flip phospholipids. P₄ ATPases form oligomeric complexes with members of the CDC50 protein family. While formation of these complexes is required for P₄ ATPase export from the endoplasmic reticulum, little is known about the functional role of the CDC50 subunits. The Na⁺/K⁺-ATPase and closely-related H⁺/K⁺-ATPase are the only other P-type pumps that are oligomeric, comprising mandatory β-subunits that are strikingly reminiscent of CDC50 proteins. Besides serving a role in the functional maturation of the catalytic α-subunit, the β-subunit also contributes specifically to intrinsic transport properties of the Na⁺/K⁺ pump. As β-subunits and CDC50 proteins likely adopted similar structures to accomplish analogous tasks, current knowledge of the Na⁺/K⁺-ATPase provides a useful guide for understanding the inner workings of the P₄ ATPase class of lipid pumps.     

A Novel Mechanism of P-type ATPase Autoinhibition Involving Both Termini of the Protein

The activity of many P-type ATPases is found to be regulated by interacting proteins or autoinhibitory elements located in N- or C-terminal extensions. An extended C terminus of fungal and plant P-type plasma membrane H⁺-ATPases has long been recognized to be part of a regulatory apparatus involving an autoinhibitory domain. Here we demonstrate that both the N and the C termini of the plant plasma membrane H⁺-ATPase are directly involved in controlling the pump activity state and that N-terminal displacements are coupled to secondary modifications taking place at the C-terminal end. This identifies the first group of P-type ATPases for which both ends of the polypeptide chain constitute regulatory domains, which together contribute to the autoinhibitory apparatus. This suggests an intricate mechanism of cis-regulation with both termini of the protein communicating to obtain the necessary control of the enzyme activity state.     

Mechanisms associated to impaired activity of cardiac P-type ATPases in endothelial nitric oxide synthase knockout mice

The effect of long-lasting in vivo restriction of nitric oxide (NO) bioavailability on cardiac and renal P-type ATPases critical for intracellular ion homeostasis is controversial. Previous work has shown in eNOS knockout (eNOS^?/?) mice hearts that Na⁺/K⁺- and Ca²⁺-ATPase activities were depressed but the underlying mechanisms are still unclear. The goal of this study was to characterize potential alterations responsible for impaired enzyme activity in eNOS^?/? mice. Na⁺/K⁺-ATPase activity from crude preparations of adult male eNOS^?/? mice hearts and kidneys was reduced compared with wild-type animals (32 %, p?<?0.05 and 16 %, p?<?0.0001, respectively). Immunoblot analysis showed that although the expression of the predominant (or exclusive, for the kidney) Na⁺/K⁺-ATPase α1 isoform was not significantly changed, there was an important downregulation of the less abundant α2 isoform in the heart (57 %, p?<?0.0001). In addition, although cardiac Ca²⁺-ATPase activity was unaltered, the expression of sarco/endoplasmic reticulum Ca²⁺-ATPase 2 protein in eNOS^?/? mice was very high (290 % compared with wild-type animals, p?<?0.0001) without any significant change in phospholamban expression. Consistent with these findings, the content of cardiac and renal free sulfhydryl groups, essential for the catalytic function of such ATPases, was decreased (23 %, p?<?0.01 and 35 %, p?<?0.05, respectively). Altogether, the present results suggest that the absence of eNOS promotes a compartmentalized altered redox balance that affects the activity and expression of ion transport ATPases.     

Ion transport and energy transduction of P-type ATPases: Implications from electrostatic calculations

This paper summarizes our present electrostatic calculations on P-type ATPases and their contribution to understand the molecular details of the reaction mechanisms. One focus was set on analyzing the proton countertransport of the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA1a). Protonation of acidic residues was calculated in dependence of pH for different enzyme states in the reaction cycle of the Ca²⁺-ATPase. We proposed that the acidic Ca²⁺ ligands Glu 771, Asp 800 and Glu 908 participate in the proton countertransport whereas Glu 309 is more likely to serve as a proton shuttle between binding site I and the cytoplasm. Complementary to infrared measurements, we assigned infrared bands to specific Ca²⁺ ligands that are hydrogen bonded. Ion pathways were proposed based on the calculations and structural data. Another focus was set on analyzing the energy transduction mechanism of P-type ATPases. In accordance to electrophysiological experiments, we simulated an electric field across the membrane. The impact of the electric field was studied by an accumulated number of residue conformational and ionization changes on specific transmembrane helices. Our calculations on the Ca²⁺-ATPase and the Na⁺/K⁺-ATPase indicated that the highly conserved transmembrane helix M5 is one structural element that is likely to act as energy transduction element in P-type ATPases. Perspectives and limitations of the electrostatic calculations for future computational studies are pointed out.     

Immunocytochemical localization of Na+, K+-ATPase in primary cultures of rat retina

Conditions have been established which allow growth of embryonic rat retinal cells in dissociated cell culture for up to one month. Na⁺,K⁺-ATPase localization was stuied in both neuronal and mixed neuronal-glial (flat cell) cultures. High Na⁺,K⁺-ATPase-like-immunoreactivity was associated with plasma membranes of neuronal cell bodies and their processes. Markedly lower immunoreactivity was found in the underlying flat cells in mixed cultures. Staining was generally uniform over perikaryal plasma membranes and showed a bead-like appearance in neuronal processes, supporting previous studies in brain tissue which used histocytochemical procedures specific for the Na⁺,K⁺-ATPase. This system should be useful for examining distribution of the enzyme in developing nerve and glial cells and may help to resolve questions regarding Na⁺–K⁺ homeostasis by neurons and glia.Dedicated to Henry McIlwain.     

Variable ATPase composition of human tumor plasma membranes

Purified plasma membranes from several transplantable human tumors exhibit very high Mg²⁺-dependent ATPase activities. Three types of Mg²⁺-dependent ATPases can be demonstrated: (1) an ouabain sensitive Na⁺, K⁺-ATPase, which is a minor component of the tumor plasma membrane ATPase, (2) a Mg²⁺-activated ATPase, which is a non-specific nucleoside triphosphatase, and (3) an ATPase activity stimulated by Na⁺ (or K⁺) alone. In three human melanomas, only the first two activities are found. In an astrocytoma and an oat cell carcinoma, all three activities are found. In the same two tumors, the plasma membrane Mg²⁺-ATPase is also stimulated by Con A. The relationship of these ATPases are discussed.     

Na+-transporting ATPase in the plasma membrane of halotolerant microalga Dunaliella maritima operates as a Na+ uniporter

A fraction of inside-out membrane vesicles enriched in plasma membranes (PM) was isolated from Dunaliella maritima cells. Attempts were made to reveal ATP-driven Na⁺-dependent H⁺ efflux from the PM vesicles to external medium, as detected by alkalization of the vesicle lumen. In parallel experiments, ATP-dependent Na⁺ uptake and electric potential generation in PM vesicles were investigated. The alkalization of the vesicle lumen was monitored with an impermeant pH-sensitive optical probe pyranine (8-hydroxy-1,3,6-pyrenetrisulfonic acid), which was loaded into vesicles during the isolation procedure. Sodium uptake was measured with ²²Na⁺ radioactive label. The generation of electric potential in PM vesicles (positive inside) was recorded with a voltage-sensitive probe oxonol VI. Appreciable Na⁺-and ATP-dependent alkalization of vesicle lumen was only observed in the presence of a protonophore CCCP (carbonyl cyanide-chlorophenylhydrazone). In parallel experiments, CCCP accelerated the ATP-dependent ²²Na⁺ uptake and abolished the electric potential generated by the Na⁺-ATPase at the vesicle membrane. A permeant anion NO^? ₃ accelerated ATP-dependent ²²Na⁺ uptake and promoted dissipation of the electric potential like CCCP did. At the same time, NO^? ₃ inhibited the ATP-and Na⁺-dependent alkalization of the vesicle lumen. The results clearly show that the ATP-and Na⁺-dependent H⁺ efflux from PM vesicles of D. maritima is driven by the electric potential generated at the vesicle membrane by the Na⁺-ATPase. Hence, the Na⁺-transporting ATPase of D. maritima carries only one ion species, i.e., Na⁺. Proton is not involved as a counter-ion in the catalytic cycle of this enzyme.