Substrate-induced Changes in Domain Interaction of Vacuolar H+-Pyrophosphatase

Single molecule atomic force microscopy (smAFM) was employed to unfold transmembrane domain interactions of a unique vacuolar H⁺-pyrophosphatase (EC 3.6.1.1) from Vigna radiata. H⁺-Pyrophosphatase is a membrane-embedded homodimeric protein containing a single type of polypeptide and links PP_i hydrolysis to proton translocation. Each subunit consists of 16 transmembrane domains with both ends facing the lumen side. In this investigation, H⁺-pyrophosphatase was reconstituted into the lipid bilayer in the same orientation for efficient fishing out of the membrane by smAFM. The reconstituted H⁺-pyrophosphatase in the lipid bilayer showed an authentically dimeric structure, and the size of each monomer was ∼4 nm in length, ∼2 nm in width, and ∼1 nm in protrusion height. Upon extracting the H⁺-pyrophosphatase out of the membrane, force-distance curves containing 10 peaks were obtained and assigned to distinct domains. In the presence of pyrophosphate, phosphate, and imidodiphosphate, the numbers of interaction curves were altered to 7, 8, and 10, respectively, concomitantly with significant modification in force strength. The substrate-binding residues were further replaced to verify these domain changes upon substrate binding. A working model is accordingly proposed to show the interactions between transmembrane domains of H⁺-pyrophosphatase in the presence and absence of substrate and its analog.     

Membrane Na+-pyrophosphatases Can Transport Protons at Low Sodium Concentrations

Membrane-bound Na⁺-pyrophosphatase (Na⁺-PPase), working in parallel with the corresponding ATP-energized pumps, catalyzes active Na⁺ transport in bacteria and archaea. Each ∼75-kDa subunit of homodimeric Na⁺-PPase forms an unusual funnel-like structure with a catalytic site in the cytoplasmic part and a hydrophilic gated channel in the membrane. Here, we show that at subphysiological Na⁺ concentrations (<5 mm), the Na⁺-PPases of Chlorobium limicola, four other bacteria, and one archaeon additionally exhibit an H⁺-pumping activity in inverted membrane vesicles prepared from recombinant Escherichia coli strains. H⁺ accumulation in vesicles was measured with fluorescent pH indicators. At pH 6.2–8.2, H⁺ transport activity was high at 0.1 mm Na⁺ but decreased progressively with increasing Na⁺ concentrations until virtually disappearing at 5 mm Na⁺. In contrast, ²²Na⁺ transport activity changed little over a Na⁺ concentration range of 0.05–10 mm. Conservative substitutions of gate Glu²⁴² and nearby Ser²⁴³ and Asn⁶⁷⁷ residues reduced the catalytic and transport functions of the enzyme but did not affect the Na⁺ dependence of H⁺ transport, whereas a Lys⁶⁸¹ substitution abolished H⁺ (but not Na⁺) transport. All four substitutions markedly decreased PPase affinity for the activating Na⁺ ion. These results are interpreted in terms of a model that assumes the presence of two Na⁺-binding sites in the channel: one associated with the gate and controlling all enzyme activities and the other located at a distance and controlling only H⁺ transport activity. The inherent H⁺ transport activity of Na⁺-PPase provides a rationale for its easy evolution toward specific H⁺ transport.     

Unconventional Chemiosmotic Coupling of NHA2, a Mammalian Na+/H+ Antiporter,to a Plasma Membrane H+ Gradient

Human NHA2, a newly discovered cation proton antiporter, is implicated in essential hypertension by gene linkage analysis. We show that NHA2 mediates phloretin-sensitive Na⁺-Li⁺ counter-transport (SLC) activity, an established marker for hypertension. In contrast to bacteria and fungi where H⁺ gradients drive uptake of metabolites, secondary transport at the plasma membrane of mammalian cells is coupled to the Na⁺ electrochemical gradient. Our findings challenge this paradigm by showing coupling of NHA2 and V-type H⁺-ATPase at the plasma membrane of kidney-derived MDCK cells, resulting in a virtual Na⁺ efflux pump. Thus, NHA2 functionally recapitulates an ancient shared evolutionary origin with bacterial NhaA. Although plasma membrane H⁺ gradients have been observed in some specialized mammalian cells, the ubiquitous tissue distribution of NHA2 suggests that H⁺-coupled transport is more widespread. The coexistence of Na⁺ and H⁺-driven chemiosmotic circuits has implications for salt and pH regulation in the kidney.     

Pre‐steady‐state kinetics and solvent isotope effects support the “billiard‐type” transport mechanism in Na +‐translocating pyrophosphatase

Membrane‐bound pyrophosphatase (mPPase) found in microbes and plants is a membrane H⁺ pump that transports the H⁺ ion generated in coupled pyrophosphate hydrolysis out of the cytoplasm. Certain bacterial and archaeal mPPases can in parallel transport Na⁺ via a hypothetical “billiard‐type” mechanism, also involving the hydrolysis‐generated proton. Here, we present the functional evidence supporting this coupling mechanism. Rapid‐quench and pulse‐chase measurements with [³²P]pyrophosphate indicated that the chemical step (pyrophosphate hydrolysis) is rate‐limiting in mPPase catalysis and is preceded by a fast isomerization of the enzyme‐substrate complex. Na⁺, whose binding is a prerequisite for the hydrolysis step, is not required for substrate binding. Replacement of H₂O with D₂O decreased the rates of pyrophosphate hydrolysis by both Na⁺‐ and H⁺‐transporting bacterial mPPases, the effect being more significant than with a non‐transporting soluble pyrophosphatase. We also show that the Na⁺‐pumping mPPase of Thermotoga maritima resembles other dimeric mPPases in demonstrating negative kinetic cooperativity and the requirement for general acid catalysis. The findings point to a crucial role for the hydrolysis‐generated proton both in H⁺‐pumping and Na⁺‐pumping by mPPases.     

Essential amino acid residues in the central transmembrane domains and loops for energy coupling of Streptomyces coelicolor A3(2) H-pyrophosphatase

The H⁺-translocating inorganic pyrophosphatase is a proton pump that hydrolyzes inorganic pyrophosphate. It consists of a single polypeptide with 14−17 transmembrane domains, and is found in a range of organisms. We focused on the second quarter region of Streptomyces coelicolor A3(2) H⁺-pyrophosphatase, which contains long conserved cytoplasmic loops. We prepared a library of 1536 mutants that were assayed for pyrophosphate hydrolysis and proton translocation. Mutant enzymes with low substrate hydrolysis and proton-pump activities were selected and their DNAs sequenced. Of these, 34 were single-residue substitution mutants. We generated 29 site-directed mutant enzymes and assayed their activity. The mutation of 10 residues in the fifth transmembrane domain resulted in low coupling efficiencies, and a mutation of Gly¹⁹⁸ showed neither hydrolysis nor pumping activity. Four residues in cytoplasmic loop e were essential for substrate hydrolysis and efficient H⁺ translocation. Pro¹⁸⁹, Asp²⁸¹, and Val³⁵¹ in the periplasmic loops were critical for enzyme function. Mutation of Ala³⁵⁷ in periplasmic loop h caused a selective reduction of proton-pump activity. These low-efficiency mutants reflect dysfunction of the energy-conversion and/or proton-translocation activities of H⁺-pyrophosphatase. Four critical residues were also found in transmembrane domain 6, three in transmembrane domain 7, and five in transmembrane domains 8 and 9. These results suggest that transmembrane domain 5 is involved in enzyme function, and that energy coupling is affected by several residues in the transmembrane domains, as well as in the cytoplasmic and periplasmic loops. H⁺-pyrophosphatase activity might involve dynamic linkage between the hydrophilic and transmembrane domains.     

Induction of Na+ / K+ exchange in swollen heart mitochondria

Heart mitochondria swollen passively in nitrate salts contract in a respiration-dependent reaction which can be attributed to an endogenous cation/H⁺ exchange component (or components). The rate of contraction increases with increased extent of passive swelling in both Na⁺ and K⁺ salts. Since nearly constant internal cation concentrations are maintained during osmotic swelling, this result suggests that both Na⁺/H⁺ and K⁺/H⁺ exchange is enhanced by increased matrix volume. Endogenous Mg²⁺ is also lost with increased matrix volume, and this observation, in conjunction with other evidence available in the literature, suggests that monovalent cation/H⁺ exchanges may be regulated by divalent cations. Passive exchange of Na⁺/K⁺,⁴²K⁺/K⁺, and²⁴Na⁺/Na⁺ can be readily demonstrated in mitochondria swollen in nitrate. All these exchanges are low or not detectable in unswollen control mitochondria, and it appears that they are manifestations of the activated cation/H⁺ component (or components) functioning in the absence of pH.     

Regulation of pyrophosphate levels by H+-PPase is central for proper resumption of early plant development

The synthesis of DNA, RNA, and de novo proteins is fundamental for early development of the seedling after germination, but such processes release pyrophosphate (PPi) as a byproduct of ATP hydrolysis. The over-accumulation of the inhibitory metabolite PPi in the cytosol hinders these biosynthetic reactions. All living organisms possess ubiquitous enzymes collectively called inorganic pyrophosphatases (PPases), which catalyze the hydrolysis of PPi into two orthophosphate (Pi) molecules. Defects in PPase activity cause severe developmental defects and/or growth arrest in several organisms. In higher plants, a proton-translocating vacuolar PPase (H⁺­PPase) uses the energy of PPi hydrolysis to acidify the vacuole. However, the biological implications of PPi hydrolysis are vague due to the widespread belief that the major role of H⁺­PPase in plants is vacuolar acidification. We have shown that the Arabidopsis fugu5 mutant phenotype, caused by a defect in H⁺­PPase activity, is rescued by complementation with the yeast cytosolic PPase IPP1. In addition, our analyses have revealed that increased cytosolic PPi levels impair postgerminative development in fugu5 by inhibiting gluconeogenesis. This led us to the conclusion that the role of H⁺­PPase as a proton-pump is negligible. Here, we present further evidence of the growth-boosting effects of removing PPi in later stages of plant vegetative development, and briefly discuss the biological role of PPases and their potential applications in different disciplines and in various organisms.     

A stable Na+/H+ antiporter of thermophilic bacterium PS3

As a first step in the isolation of a stable Na⁺/H⁺ antiporter, its reaction in sonicated membrane vesicles of thermophilic bacterium PS3 has been characterized. The sonicated vesicles showed quenching of quinacrine fluorescence in either NADH oxidation or ATP hydrolysis. The quenching was reversed by the addition of Na⁺, Li⁺, Mn²⁺, Cd²⁺, and Co²⁺, but not of choline⁺ or Ca²⁺, regardless of their counter anions.²²Na⁺ was taken up into the vesicles by NADH oxidation, and the²²Na⁺ uptake was inhibited by the addition of an uncoupler. H⁺ release was observed on addition of Na⁺ to sonicated vesicles. The magnitude of the pH difference across the membrane induced by NADH oxidation was constant at pH 7.0 to 9.1, but the Na⁺/H⁺ antiport was affected by the pH of the medium (optimum pH=8.5). TheK _m 's of the antiporter for Na⁺ and Li⁺ were 2.5 and 0.1 mM, respectively, but theV _max values for the two ions were the same at pH 8.0. In the presence of Li⁺, no further decrease of fluorescence quenching was observed on addition of Na⁺ andvice versa. The Na⁺/H⁺ antiporter activity in PS3 was stable at 70°C, and the optimum temperature for activity was 55–60°C. In contrast to mesophilic cation/H⁺ antiporters, this antiporter was not inhibited by a thiol reagent.Abbreviations Tricine N-tris(hydroxymethyl)methylglycine - MOPS morpholinopropane sulfonic acid - TMAHO tetramethylammonium hydroxide - DCCD N,N-dicyclohexylcarbodiimide - FCCP carbonyl cyanidep-trifluoromethoxyphenylhydrazone - H⁺ — ATPase proton-translocating adenosine triphosphatase - electrochemical proton gradient across membrane - electrochemical Na⁺ gradient across membrane - pH pH difference across membrane     

Relation of ATPases in rat renal brush-border membranes to ATP-driven H+ secretion

Summary In the presence of inhibitors for mitochondrial H⁺-ATPase, (Na⁺+K⁺)- and Ca²⁺-ATPases, and alkaline phosphatase, sealed brush-border membrane vesicles hydrolyse externally added ATP demonstrating the existence of ATPases at the outside of the membrane (ecto-ATPases). These ATPases accept several nucleotides, are stimulated by Ca²⁺ and Mg²⁺, and are inhibited by N,N-dicyclohexylcarbodiimide (DCCD), but not by N-ethylmaleimide (NEM). They occur in both brushborder and basolateral membranes. Opening of brush-border membrane vesicles with Triton X-100 exposes ATPases located at the inside (cytosolic side) of the membrane. These detergent-exposed ATPases prefer ATP, are activated by Mg²⁺ and Mn²⁺, but not by Ca²⁺, and are inhibited by DCCD as well as by NEM. They are present in brush-border, but not in basolateral membranes. As measured by an intravesicularly trapped pH indicator, ATP-loaded brush-border membrane vesicles extrude protons by a DCCD- and NEM-sensitive pump. ATP-driven H⁺ secretion is electrogenic and requires either exit of a permeant anion (Cl^–) or entry of a cation, e.g., Na⁺ via electrogenic Na⁺/d-glucose and Na⁺/l-phenylalanine uptake. In the presence of Na⁺, ATP-driven H⁺ efflux is stimulated by blocking the Na⁺/H⁺ exchanger with amiloride. These data prove the coexistence of Na⁺-coupled substrate transporters, Na⁺/H⁺ exchanger, and an ATP-driven H⁺ pump in brush-border membrane vesicles. Similar location and inhibitor sensitivity reveal the identity of ATP-driven H⁺ pumps with (a part of) the DCCD- and NEM-sensitive ATPases at the cytosolic side of the brush-border membrane.     

Role of Tonoplast Proton Pumps and Na+/H+ Antiport System in Salt Tolerance of Populus euphratica Oliv.

Populus euphratica has been used as a plant model to study resistance against salt and osmotic stresses, with recent studies having characterized the tonoplast and the plasma membrane ATPases, and two Na⁺/H⁺ antiporters, homologs of the Arabidopsis tonoplast AtNHX1, were published in databases. In the present work we show that P. euphratica suspension-cultured cells are highly tolerant to high salinity, being able to grow with up to 150 mM NaCl in the culture medium without substantial modification of the final population size when compared to the control cells in the absence of salt. At a salt concentration of 300 mM, cells were unable to grow but remained highly viable up to 17 days after subculture. The addition of a 1-M-NaCl pulse to unadapted cells did not promote a significant loss in cell viability within 48 h. In tonoplast vesicles purified from cells cultivated in the absence of salt and from salt-stressed cells, vacuolar H⁺-pyrophosphatase (V-H⁺-PPase) seemed to be the primary tonoplast proton pump; however, there appears to be a decrease in V-H⁺-PPase activity with exposure to NaCl, in contrast to the sodium-induced increase in the activity of vacuolar H⁺-ATPase (V-H⁺-ATPase). Despite reports that in P. euphratica there is no significant difference in the concentration of Na⁺ in the different cell compartments under NaCl stress, in the present study, confocal and epifluorescence microscopic observations using a Na⁺-sensitive probe showed that suspension-cultured cells subject to a salt pulse accumulated Na⁺ in the vacuole when compared with control cells. Concordantly, a tonoplast Na⁺/H⁺ exchange system is described whose activity is upregulated by salt and, indirectly, by a salt-mediated increase of V-H⁺-ATPase activity.     

Identification of a new 130 bp <Emphasis Type="Italic">cis</Emphasis>-acting element in the <Emphasis Type="Italic">TsVP1</Emphasis> promoter involved in the salt stress response from <Emphasis Type="Italic">Thellungiella halophila</Emphasis>

Background  

Salt stress is one of the major abiotic stresses affecting plant growth and productivity. Vacuolar H⁺-pyrophosphatase (H⁺-PPase) genes play an important role in salt stress tolerance in multiple species.     

Lack of immunological cross reactivity between the transport enzymes (Na+ + K+)-ATPase and (K+ + H+)-ATPase

Goat antisera against (Na⁺ + K⁺)-ATPase and its isolated subunits and against (K⁺ + H⁺)-ATPase have been prepared in order to test for immune cross-reactivity between the two enzymes, whose catalytic subunits show great chemical similarity. None of the (Na⁺ + K⁺)-ATPase antisera cross-reacted with (K⁺ + H⁺)-ATPase or inhibited its enzyme activity. The same was true for the (K⁺ + H⁺)-ATPase antiserum with regard to (Na⁺ + K⁺)-ATPase and its subunits and its enzyme activity. So not withstanding the chemical similarity of their subunits, there is no immunological cross-reactivity between these two plasma membrane ATPases.Number LIII in the series Studies on (Na⁺ + K⁺)-Activated ATPase.     

Conversion of monensin from an ionophore to an inhibitor of Na+ uptake by SV3t3 membrane vesicles as a function of Na+ concentration

Monensin is a Na⁺ ionophore in membrane vesicles from SV3T3 cells; but its ability to stimulate Na⁺ flux is inhibited by increasing concentrations of Na⁺. At greater than 20-mM Na⁺, monensin inhibits Na⁺ uptake by the vesicles. Cs⁺ and NH₄⁺ also cause monensin to inhibit Na⁺ uptake, but general alterations in ionic strength do not convert the ionophore to an inhibitor. Monensin does not cause Na⁺ loss during collection of the vesicles on filters; nor is inhibition the result of the vesicle lumen being made alkaline by H⁺ loss in exchange for Na⁺. The specificity for cation and ionophore indicates that a precise interaction between the cation, ionophore, and membrane is required for inhibition.     

Identification of amino acid residues participating in the energy coupling and proton transport of Streptomyces coelicolor A3(2) H-pyrophosphatase

The H⁺-translocating inorganic pyrophosphatase is a proton pump that hydrolyzes inorganic pyrophosphate. It consists of a single polypeptide with 14-17 transmembrane domains (TMs). We focused on the third quarter region of Streptomyces coelicolor A3(2) H⁺-pyrophosphatase, which contains a long conserved cytoplasmic loop. We assayed 1520 mutants for pyrophosphate hydrolysis and proton translocation, and selected 34 single-residue substitution mutants with low substrate hydrolysis and proton-pump activities. We also generated 39 site-directed mutant enzymes and assayed their activity. The mutation of 5 residues in TM10 resulted in low energy-coupling efficiencies, and mutation of conserved residues Thr⁴⁰⁹, Val⁴¹¹, and Gly⁴¹⁴ showed neither hydrolysis nor pumping activity. The mutation of six, five, and four residues in TM11, 12, and 13, respectively, gave a negative effect. Phe³⁸⁸, Thr³⁸⁹, and Val³⁹⁶ in cytoplasmic loop i were essential for efficient H⁺ translocation. Ala⁴³⁶ and Pro⁵⁶⁰ in the periplasmic loops were critical for coupling efficiency. These low-efficiency mutants showed dysfunction of the energy-conversion and/or proton-translocation activity. The energy efficiency was increased markedly by the mutation of two and six residues in TM9 and 12, respectively. These results suggest that TM10 is involved in enzyme function, and that TM12 regulate the energy-conversion efficiency. H⁺-pyrophosphatase might involve dynamic linkage between the hydrophilic loops and TMs through the central half region of the enzyme.     

The Na+/H+ Exchanger Present in Trout Erythrocyte Membranes is Not Responsible for the Amiloride-insensitive Na+/Li+ Exchange Activity

The protein responsible for the Na⁺/Li⁺ exchange activity across the erythrocyte membrane has not been cloned or isolated. It has been suggested that a Na⁺/H⁺ exchanger could be responsible for the Na⁺/Li⁺ exchange activity across the erythrocyte membrane. Previously, we reported that in the trout erythrocyte, the Li⁺/H⁺ exchange activity (mediated by the Na⁺/H⁺ exchanger βNHE) and the Na⁺/Li⁺ exchange activity respond differently to cAMP, DMA (dimethyl-amiloride) and O₂. We concluded that the DMA insensitive Na⁺/Li⁺ exchange activity originates from a different protein. To further examine these findings, we measured Li⁺ efflux in fibroblasts expressing the βNHE as the only Na⁺/H⁺ exchanger. Moreover, the internal pH of these cells was monitored with a fluorescent probe. Our findings indicate that acidification of fibroblasts expressing the Na⁺/H⁺ exchanger βNHE, induces a Na⁺ stimulated Li⁺ efflux activity in trout erythrocytes. This exchange activity, however, is DMA sensitive and therefore differs from the DMA insensitive Na⁺/Li⁺ exchange activity. In these fibroblasts no significant DMA insensitive Na⁺/Li⁺ exchange activity was found. These results support the hypothesis that the trout erythrocyte Na⁺/Li⁺ exchange activity is not mediated by the Na⁺/H⁺ exchanger (βNHE) present in these membranes. Received: 6 December 1996/Revised: 11 August 1997     

Identification and Characterization of the Na+/H+ Antiporter Nhas3 from the Thylakoid Membrane of Synechocystis sp. PCC 6803

Na⁺/H⁺ antiporters influence proton or sodium motive force across the membrane. Synechocystis sp. PCC 6803 has six genes encoding Na⁺/H⁺ antiporters, nhaS1–5 and sll0556. In this study, the function of NhaS3 was examined. NhaS3 was essential for growth of Synechocystis, and loss of nhaS3 was not complemented by expression of the Escherichia coli Na⁺/H⁺ antiporter NhaA. Membrane fractionation followed by immunoblotting as well as immunogold labeling revealed that NhaS3 was localized in the thylakoid membrane of Synechocystis. NhaS3 was shown to be functional over a pH range from pH 6.5 to 9.0 when expressed in E. coli. A reduction in the copy number of nhaS3 in the Synechocystis genome rendered the cells more sensitive to high Na⁺ concentrations. NhaS3 had no K⁺/H⁺ exchange activity itself but enhanced K⁺ uptake from the medium when expressed in an E. coli potassium uptake mutant. Expression of nhaS3 increased after shifting from low CO₂ to high CO₂ conditions. Expression of nhaS3 was also found to be controlled by the circadian rhythm. Gene expression peaked at the beginning of subjective night. This coincided with the time of the lowest rate of CO₂ consumption caused by the ceasing of O₂-evolving photosynthesis. This is the first report of a Na⁺/H⁺ antiporter localized in thylakoid membrane. Our results suggested a role of NhaS3 in the maintenance of ion homeostasis of H⁺, Na⁺, and K⁺ in supporting the conversion of photosynthetic products and in the supply of energy in the dark.Na⁺/H⁺ antiporters are integral membrane proteins that transport Na⁺ and H⁺ in opposite directions across the membrane and that occur in virtually all cell types. These transporters play an important role in the regulation of cytosolic pH and Na⁺ concentrations and influence proton or sodium motive force across the membrane (1, 2). In Escherichia coli, three Na⁺/H⁺ antiporters (NhaA, NhaB, and ChaA) have been described in detail. Of these, NhaA is the functionally best characterized transporter. The crystal structure of NhaA has been resolved (3). In addition, mutants of nhaA, nhaB, and chaA as well as the triple mutant have been generated (4). The triple mutant was shown to be hypersensitive to extracellular Na⁺. The genome of the cyanobacterium Synechocystis sp. PCC 6803 contains six genes encoding Na⁺/H⁺ antiporters (NhaS1–5 and sll0556). NhaS1 (slr1727) has also been designated SynNhaP (5, 6). Null mutants of nhaS1, nhaS2, nhaS4, and nhaS5 have been generated; however, a null mutant of nhaS3 could not be obtained, indicating that it is an essential gene (6–8). By heterologous expression in E. coli, Na⁺/H⁺ exchange activities could be shown for NhaS1–5 (5, 6). Inactivation of nhaS1 and nhaS2 results in retardation of growth of Synechocystis (5, 6). It has been reported that in these mutants the concentration of Na⁺ in cytosol and intrathylakoid space (lumen) increases and impairs the photosynthetic and/or respiratory activity of the cell (9, 10). Therefore the Na⁺ extrusion by Synechocystis Na⁺/H⁺ antiporters similar to E. coli NhaA, NhaB, and ChaA is essential for the adaptation to salinity stress.In contrast to the case in E. coli, Na⁺ is an essential element for the growth of some cyanobacteria (11, 12). Interestingly, the Na⁺/H⁺ antiporter homolog NhaS4 was identified as an uptake system for Na⁺ from the medium during a screen for mutations in Synechocystis that result in lack of growth at low Na⁺ concentrations (7). The requirement of a Na⁺ uptake antiporter for cell growth is consistent with the physiology of Synechocystis. Specifically, photoautotrophic bacteria like cyanobacteria share some components (plastoquinone, cytochrome b₆f, and c₆) of the thylakoid membrane for electron transport for both photophosphorylation and respiratory oxidative phosphorylation. Na⁺/H⁺ antiporters therefore may coordinate both H⁺ and Na⁺ gradients across the plasma and thylakoid membranes to adapt to daily environmental changes (11). It remains to be determined whether the six Na⁺/H⁺ antiporters are localized to the plasma membrane or to the thylakoid membrane in Synechocystis. Information on the membrane localization will also provide information on the physiological role in Synechocystis. In this study, we explored the membrane localization of NhaS3, the role of specific amino acid residues for its function, and the effect of CO₂ concentration and circadian rhythms on the expression pattern of nhaS3 to gain insight into the physiological role of NhaS3 in Synechocystis.     

ATP Dependence of Na+/H+ Exchange : Nucleotide Specificity and Assessment of the Role of Phospholipids

We studied the ATP dependence of NHE-1, the ubiquitous isoform of the Na⁺/H⁺ antiporter, using the whole-cell configuration of the patch-clamp technique to apply nucleotides intracellularly while measuring cytosolic pH (pH_i) by microfluorimetry. Na⁺/H⁺ exchange activity was measured as the Na⁺-driven pH_i recovery from an acid load, which was imposed via the patch pipette. In Chinese hamster ovary (CHO) fibroblasts stably transfected with NHE-1, omission of ATP from the pipette solution inhibited Na⁺/H⁺ exchange. Conversely, ATP perfusion restored exchange activity in cells that had been metabolically depleted by 2-deoxy-d-glucose and oligomycin. In cells dialyzed in the presence of ATP, no “run-down” was observed even after extended periods, suggesting that the nucleotide is the only diffusible factor required for optimal NHE-1 activity. Half-maximal activation of the antiporter was obtained at ∼5 mM Mg-ATP. Submillimolar concentrations failed to sustain Na⁺/H⁺ exchange even when an ATP regenerating system was included in the pipette solution. High ATP concentrations are also known to be required for the optimal function of other cation exchangers. In the case of the Na/Ca²⁺ exchanger, this requirement has been attributed to an aminophospholipid translocase, or “flippase.” The involvement of this enzyme in Na⁺/H⁺ exchange was examined using fluorescent phosphatidylserine, which is actively translocated by the flippase. ATP depletion decreased the transmembrane uptake of NBD-labeled phosphatidylserine (NBD-PS), indicating that the flippase was inhibited. Diamide, an agent reported to block the flippase, was as potent as ATP depletion in reducing NBD-PS uptake. However, diamide had no effect on Na⁺/H⁺ exchange, implying that the effect of ATP is not mediated by changes in lipid distribution across the plasma membrane. K-ATP and ATPγS were as efficient as Mg-ATP in sustaining NHE-1 activity, while AMP-PNP and AMP-PCP only partially substituted for ATP. In contrast, GTPγS was ineffective. We conclude that ATP is the only soluble factor necessary for optimal activity of the NHE-1 isoform of the antiporter. Mg²⁺ does not appear to be essential for the stimulatory effect of ATP. We propose that two mechanisms mediate the activation of the antiporter by ATP: one requires hydrolysis and is likely an energy-dependent event. The second process does not involve hydrolysis of the γ-phosphate, excluding mediation by protein or lipid kinases. We suggest that this effect is due to binding of ATP to an as yet unidentified, nondiffusible effector that activates the antiporter.     

Na+-K+ transport in roots under salt stress

Salinity causes billion dollar losses in annual crop production. So far, the main avenue in breeding crops for salt tolerance has been to reduce Na⁺ uptake and transport from roots to shoots. Recently we have demonstrated that retention of cytosolic K⁺ could be considered as another key factor in conferring salt tolerance in plants. A subsequent study has shown that Na⁺-induced K⁺ efflux in barley root epidermis occurs primarily via outward rectifying K⁺ channels (KORC). Surprisingly, expression of KORC was similar in salt- tolerant and sensitive genotypes. However, the former were able to better oppose Na⁺-induced depolarization via enhanced activity of plasma membrane H⁺-ATPase (thus minimizing K⁺ leak from the cytosol). In addition to highly K⁺-selective KORC channels, activities of several types of non-selective cation channels were detected at depolarizing potentials. Here we show that the expression of one of them, NORC, was significantly lower in salt-tolerant genotypes. As NORC is capable of mediating K⁺ efflux coupled to Na⁺ influx, we suggest that the restriction of its activity could be beneficial for plants under salt stress.Key words: salinity tolerance, barley, ion flux, K⁺ homeostasis, KOR, non-selective channels, patch-clamp