Regulation of respiration by Na+ and K+ in the halotolerant bacterium, Ba1

In the obligate aerobe, moderate halophile bacterium, Ba1, the ion composition of the medium was found to have a profound influence on the response of the respiratory system to changes in the external pH. In the pH range 6.5 to 8.5 the respiratory activity either increased or decreased progressively, depending whether K+ or Na+ ions were omitted from the medium. A nearly constant rate of respiration was observed in the entire pH range when both K+ and Na+ were present simultaneously. The stimulatory effect of Na+ was expressed especially in the alkaline pH range, where it induced acidification of the intracellular milieu. It was manifest in whole cells as well as in inverted membrane vesicles, and was not affected by either uncoupler or inhibitor of H+-ATPase. In contrast, the respiratory stimulation induced by K+ was most prominent in the acidic pH range and was accompanied by alkalinization of the internal pH. The effect of K+ was observed only in intact cells. Agents which interfered with energy transfer suppressed the effect of K+. With ethanol as the electron donor, Na+ was found to decrease the extent of reduction of the cellular NAD+ in the aerobic steady state, and to cause increased reduction of the cytochromes. K+ had no appreciable effect on the extent of reduction of any component in the respiratory chain. The implications of the above findings are discussed in relation to the mechanism(s) involved in the cation-mediated regulation of respiration and intracellular pH.     

Na+-ATPases of halotolerant microalgae

Na⁺ homeostasis in the cytoplasm is a common property of all organisms, irrespective of their taxonomic position. Low Na⁺ concentrations in the cytoplasm of living cells are maintained by specialized Na⁺-transporting molecular machines operating in the cell membranes. In eukaryotic cells, Na⁺-transporting ATPases of P-type play the important role in keeping the Na⁺ homeostasis. This review summarizes the authors’ investigations demonstrating the operation of the Na⁺-transporting P-type ATPases in the plasma membrane of green marine microalgae. Experiments described here provided the first evidence for the existence of the primary Na⁺-pump in plasma membranes of organisms attributed to the plant kingdom. The significance of the Na⁺-ATPases in halotolerant microalgae Dunaliella maritima and Tetraselmis viridis inhabiting saline environments is discussed.     

Na+-driven multidrug efflux pump VcmA from Vibrio cholerae non-O1, a non-halophilic bacterium

Corynebacterium diphtheriae strains express non-fimbrial surface proteins able to recognize and bind to specific host cells receptors. Protein extracts were obtained from bacterial cells by mechanical process and ammonium sulfate precipitation at 25 and 45% (w/v) saturation. SDS-PAGE analysis of the extracts detected two polypeptide bands of 67 and 72 kDa, named 67-72 p. The 67-72 p, rabbit anti-67-72 p IgG antibodies as well as human gastric mucin, N-acetylneuraminic acid and N-acetyl D-glucosamine molecules were able to inhibit bacterial hemagglutination. Hemagglutination assays using 67-72 p-coated latex beads and Western blot analysis of biotin-labeled 67-72 p and erythrocyte receptors demonstrated the binding of 67-72 p to human erythrocyte membranes. Immunolabeled colloidal gold-A protein transmission electron microscopy using anti-67-72 p revealed a diffuse distribution of non-fimbrial 67-72 p on the surface of C. diphtheriae strains of both sucrose-fermenting and non-fermenting biotypes. Non-fimbrial lectin-like surface 67-72 p may play a role as adhesins in bacterial attachment thereby facilitating the early steps in pathogenesis of both toxigenic and non-toxigenic C. diphtheriae.     

An NADH:quinone oxidoreductase of the halotolerant bacterium Ba1 is specifically dependent on sodium ions

The rate of NADH oxidation by inverted membrane vesicles prepared from the halotolerant bacterium Ba1 of the Dead Sea is increased specifically by sodium ions, as observed earlier in whole cells. The site of this sodium effect is identified as the NADH: quinone oxidoreductase, similarly to the other such system known, Vibrio alginolyticus (H. Tokuda and T. Unemoto (1984) J. Biol. Chem. 259, 7785-7790). Sodium accelerates quinone reduction severalfold, but oxidation of the quinol, with oxygen as terminal electron acceptor, is unaffected. The sodium-dependent pathway of quinone reduction exhibits higher apparent affinity to extraneous quinone (Q-2) than the sodium-insensitive pathway, and is specifically inhibited by 2-heptyl-4-hydroxyquinoline N-oxide. ESR spectra of the membranes contain a feature at g = 1.98 which is tentatively identified as one originating from semiquinone. This feature is increased by NADH and decreased by addition of Na+, suggesting that, as proposed from different kinds of evidence for the V. alginolyticus system, sodium affects the semiquinone reduction step. As in the other system, the site of sodium stimulation in Ba1 probably corresponds to the site of sodium translocation, which was shown earlier (S. Ken-Dror, R. Shnaiderman, and Y. Avi-Dor (1984) Arch. Biochem. Biophys. 229, 640-649) to be linked directly to a redox reaction in the respiratory chain.     

Ba2+ unmasks K+ modulation of the Na+-K+ pump in the frog retinal pigment epithelium

This paper presents electrophysiological evidence that small changes in [K+]o modulate the activity of the Na+-K+ pump on the apical membrane of the frog retinal pigment epithelium (RPE). This membrane also has a large relative K+ conductance so that lowering [K+]o hyperpolarizes it and therefore increases the transepithelial potential (TEP). Ba2+, a K+ channel blocker, eliminated these normal K+-evoked responses; in their place, lowering [K+]o evoked an apical depolarization and TEP decrease that were blocked by apical ouabain or strophanthidin. These data indicate that Ba2+ blocked the major K+ conductance(s) of the RPE apical membrane and unmasked a slowing of the normally hyperpolarizing electrogenic Na+-K+ pump caused by lowering [K+]o. Evidence is also presented that [K+]o modulates the pump in the isolated RPE under physiological conditions (i.e., without Ba2+). In the intact retina, light decreases subretinal [K+]o and produces the vitreal-positive c-wave of the electroretinogram (ERG) that originates primarily in the RPE from a hyperpolarization of the apical membrane and TEP increase. When Ba2+ was present in the retinal perfusate, the apical membrane depolarized in response to light and the TEP decreased so that the ERG c-wave inverted. The retinal component of the c-wave, slow PIII, was abolished by Ba2+. The effects of Ba2+ were completely reversible. We conclude that Ba2+ unmasks a slowing of the RPE Na+-K+ pump by the light-evoked decrease in [K+]o. Such a response would reduce the amplitude of the normal ERG c-wave.     

Functional identification of electrogenic Na+-translocating ATPase in the plasma membrane of the halotolerant microalga Dunaliella maritima

The hypothesis that the primary Na+-pump, Na+-ATPase, functions in the plasma membrane (PM) of halotolerant microalga Dunaliella maritima was tested using membrane preparations from this organism enriched with the PM vesicles. The pH profile of ATP hydrolysis catalyzed by the PM fractions exhibited a broad optimum between pH 6 and 9. Hydrolysis in the alkaline range was specifically stimulated by Na+ ions. Maximal sodium dependent ATP hydrolysis was observed at pH 7.5-8.0. On the assumption that the ATP-hydrolysis at alkaline pH values is related to a Na+-ATPase activity, we investigated two ATP-dependent processes, sodium uptake by the PM vesicles and generation of electric potential difference (Deltapsi) across the vesicle membrane. PM vesicles from D. maritima were found to be able to accumulate 22Na+ upon ATP addition, with an optimum at pH 7.5-8.0. The ATP-dependent Na+ accumulation was stimulated by the permeant NO3- anion and the protonophore CCCP, and inhibited by orthovanadate. The sodium accumulation was accompanied by pronounced Deltapsi generation across the vesicle membrane. The data obtained indicate that a primary Na+ pump, an electrogenic Na+-ATPase of the P-type, functions in the PM of marine microalga D. maritima.     

A primary respiratory Na+ pump of an anaerobic bacterium: the Na+-dependent NADH:quinone oxidoreductase of Klebsiella pneumoniae

Membranes of Klebsiella pneumoniae, grown anaerobically on citrate, contain a NADH oxidase activity that is activated specifically by Na⁺ or Li⁺ ions and effectively inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Cytochromes b and d were present in the membranes, and the steady state reduction level of cytochrome b increased on NaCl addition. Inverted bacterial membrane vesicles accumulated Na⁺ ions upon NADH oxidation. Na⁺ uptake was completely inhibited by monensin and by HQNO and slightly stimulated by carbonylcyanide-p-trifluoromethoxy phenylhydrazone (FCCP), thus indicating the operation of a primary Na⁺ pump. A Triton extract of the bacterial membranes did not catalyze NADH oxidation by O₂, but by ferricyanide or menadione in a Na⁺-independent manner. The Na⁺-dependent NADH oxidation by O₂ was restored by adding ubiquinone-1 in micromolar concentrations. After inhibition of the terminal oxidase with KCN, ubiquinol was formed from ubiquinone-1 and NADH. The reaction was stimulated about 6-fold by 10 mM NaCl and was severely inhibited by low amounts of HQNO. Superoxide radicals were formed during electron transfer from NADH to ubiquinone-1. These radicals disappeared by adding NaCl, but not with NaCl and HQNO. It is suggested that the superoxide radicals arise from semiquinone radicals which are formed by one electron reduction of quinone in a Na⁺-independent reaction sequence and then dismutate in a Na⁺ and HQNO sensitive reaction to quinone and quinol. The mechanism of the respiratory Na⁺ pump of K. pneumoniae appears to be quite similar to that of Vibrio alginolyticus.     

Biochemical and molecular characterization of a Na+-translocating F1Fo-ATPase from the thermoalkaliphilic bacterium Clostridium paradoxum

Clostridium paradoxum is an anaerobic thermoalkaliphilic bacterium that grows rapidly at pH 9.8 and 56 degrees C. Under these conditions, growth is sensitive to the F-type ATP synthase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), suggesting an important role for this enzyme in the physiology of C. paradoxum. The ATP synthase was characterized at the biochemical and molecular levels. The purified enzyme (30-fold purification) displayed the typical subunit pattern for an F1Fo-ATP synthase but also included the presence of a stable oligomeric c-ring that could be dissociated by trichloroacetic acid treatment into its monomeric c subunits. The purified ATPase was stimulated by sodium ions, and sodium provided protection against inhibition by DCCD that was pH dependent. ATP synthesis in inverted membrane vesicles was driven by an artificially imposed chemical gradient of sodium ions in the presence of a transmembrane electrical potential that was sensitive to monensin. Cloning and sequencing of the atp operon revealed the presence of a sodium-binding motif in the membrane-bound c subunit (viz., Q28, E61, and S62). On the basis of these properties, the F1Fo-ATP synthase of C. paradoxum is a sodium-translocating ATPase that is used to generate an electrochemical gradient of + that could be used to drive other membrane-bound bioenergetic processes (e.g., solute transport or flagellar rotation). In support of this proposal are the low rates of ATP synthesis catalyzed by the enzyme and the lack of the C-terminal region of the epsilon subunit that has been shown to be essential for coupled ATP synthesis.     

Genome sequence of the halotolerant bacterium Imtechella halotolerans K1T

We report the 3.087-Mb genome sequence of Imtechella halotolerans K1(T), isolated from an estuarine water sample collected from Kochi, Kerala, India. Strain K1 was recently reported as a novel genus of the family Flavobacteriaceae.     

Gene cloning and characterization of VcrM,a Na+-coupled multidrug efflux pump,from Vibrio cholerae non-O1

We cloned a DNA fragment responsible for drug resistance from chromosome of Vibrio cholerae non-O1. Nucleotide sequence analysis of this fragment revealed the presence of a single open reading frame encoding a protein consisting of 445 amino acid residues. We designated the gene as vcrM. Hydropathy analysis of the deduced amino acid sequence of VcrM suggests the presence of 12 trans-membrane segments. A dendrogram showed that VcrM is a member of the DinF-subfamily within the MATE family of multidrug efflux pumps. Expression of the cloned vcrM gene in drug-hypersensitive Escherichia coli KAM32 cells made them resistant to acriflavine, 4', 6-diamidino-2-phenylindole, Hoechst 33342, rhodamine 6G, tetraphenylphosphonium chloride (TPPCl) and ethidium bromide. Efflux of acriflavine due to VcrM was dependent on Na+ or Li+. Moreover, Na+ efflux was observed with VcrM when TPPCl was added to Na+-loaded cells. Therefore, we conclude that VcrM is a Na+/drug antiporter-type multidrug efflux pump.     

Genome sequence of the halotolerant marine bacterium Myxococcus fulvus HW-1

Myxococcus fulvus HW-1 (ATCC BAA-855) is a halotolerant marine myxobacterium. This strain exhibits complex social behaviors in the presence of low concentrations of seawater but adopts an asocial living pattern under oceanic conditions. The whole genome of M. fulvus HW-1 will enable us to further investigate the details of its evolution.     

Angiotensin regulates the selectivity of the Na+-K+ pump for intracellular Na+

Treatment of rabbits with angiotensin-converting enzyme (ACE)inhibitors increases the apparent affinity of theNa⁺-K⁺pump for Na⁺. To explore themechanism, we voltage clamped myocytes from control rabbits and rabbitstreated with captopril with patch pipettes containing 10 mMNa⁺. When pipette solutions wereK⁺ free, pump current(I_p) formyocytes from captopril-treated rabbits was nearly identical to thatfor myocytes from controls. However, treatment caused a significantincrease in I_pmeasured with pipettes containingK⁺. A similar difference wasobserved when myocytes from rabbits treated with the ANG II receptorantagonist losartan and myocytes from controls were compared.Treatment-induced differences in I_p wereeliminated by in vitro exposure to ANG II or phorbol 12-myristate 13-acetate or inclusion of the protein kinase C fragment composed ofamino acids 530-558 in pipette solutions. Treatmentwith captopril had no effect on the voltage dependence ofI_p. We concludethat ANG II regulates the pump's selectivity for intracellularNa⁺ at sites near the cytoplasmicsurface. Protein kinase C is implicated in the messenger cascade.

     

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.     

Respiratory Na+ pump and Na+-dependent energetics inVibrio alginolyticus

The marine bacteriumVibrio alginolyticus was found to possess the respiratory Na⁺ pump that generates an electrochemical potential of Na⁺, which plays a central role in bioenergetics ofV. alginolyticus, as a direct result of respiration. Mutants defective in the Na⁺ pump revealed that one of the two kinds of NADH: quinone oxidoreductase requires Na⁺ for activity and functions as the Na⁺ pump. The Na⁺ pump composed of three subunits was purified and reconstituted into liposomes. Generation of membrane potential by the reconstituted proteoliposomes required Na⁺. The respiratory Na⁺ pump coupled to the NADH: quinone oxidoreductase was found in wide varieties of Gramnegative marine bacteria belonging to the generaAlcaligenes, Alteromonas, andVibrio, and showed a striking similarity in the mode of electron transfer and enzymic properties. Na⁺ extrusion seemed to be coupled to a dismutation reaction, which leads to the formation of quinol and quinone from semi-quinone radical.     

Functional characterization of the transmembrane segment VII of the NHE1 isoform of the Na+/H+ exchanger

The Na+/H+ exchanger isoform 1 is an integral membrane protein that regulates intracellular pH. It extrudes 1 intracellular H+ in exchange for 1 extracellular Na+. It has 2 large domains, an N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the cysteine accessibility of amino acids of the critical transmembrane segment TM VII. Residues Leu 255, Leu 258, Glu 262, Leu 265, Asn 266, Asp 267, Val 269, Val 272, and Leu 273 were all mutated to cysteine residues in the cysteineless NHE1 isoform. Mutation of amino acids E262, N266, and D267 caused severe defects in activity and targeting of the intact full length protein. The balance of the active mutants were examined for sensitivity to the sulfhydryl reactive reagents, positively charged MTSET ((2- (trimethylammonium)ethyl)methanethiosulfonate) and negatively charged MTSES ((2-sulfonatoethyl)methanethiosulfonate). Leu 255 and Leu 258 were sensitive to MTSET but not to MTSES. The results suggest that these amino acids are pore-lining residues. We present a model of TM VII that shows that residues Leu 255, Leu 258, Glu 262, Asn 266, and Asp 267 lie near the same face of TM VII, lining the ion transduction pore.     

Further biochemical characterization of an Na+ pump inhibitor purified from human urine

An increase in endogenous Na+,K+-ATPase inhibitor(s) with digitalis-like properties has been reported in chronic renal insufficiency, in Na+-dependent experimental hypertension and in some essential hypertensive patients. The present study specifies some properties and some biochemical characteristics of a semipurified compound from human urine having digitalis-like properties. The urine-derived inhibitor (endalin) inhibits Na+,K+-ATPase activity and [3H]-ouabain binding, and cross-reacts with anti-digoxin antibodies. The inhibitory effect on ATPases of endalin is higher on Na+,K+-ATPase than on Mg2+-ATPase and Ca2+-ATPase. The mechanism of endalin action on highly purified Na+,K+-ATPase was compared to that of ouabain and was similar in that it reversibly inhibited Na+,K+-ATPase activity; it inhibited Na+,K+-ATPase non-competitively with ATP; its inhibitory effect was facilitated by Na+; K+ decreased its inhibitory effect on Na+,K+-ATPase; it competitively inhibited ouabain binding to the enzyme; its binding was maximal in the presence of Mg2+ and Pi; it decreased the Na+ pump activity in human erythrocytes; it reduced serotonin uptake by human platelets; and it was diuretic and natriuretic in rat bioassay. The endalin differed from ouabain in only three aspects: its inhibitory effect was not really specific for Na+,K+-ATPase; its binding to the enzyme was undetectable in the presence of Mg2+ and ATP; it was not kaliuretic in rat bioassay. Endalin is a reversible and partial specific inhibitor of Na+,K+-ATPase, its Na+,K+-ATPase inhibition closely resembles that of ouabain and it could be considered as one of the natriuretic hormones.     

Functional characterization of a Na+-dependent dicarboxylate transporter from Vibrio cholerae

The SLC13 transporter family, whose members play key physiological roles in the regulation of fatty acid synthesis, adiposity, insulin resistance, and other processes, catalyzes the transport of Krebs cycle intermediates and sulfate across the plasma membrane of mammalian cells. SLC13 transporters are part of the divalent anion:Na⁺ symporter (DASS) family that includes several well-characterized bacterial members. Despite sharing significant sequence similarity, the functional characteristics of DASS family members differ with regard to their substrate and coupling ion dependence. The publication of a high resolution structure of dimer VcINDY, a bacterial DASS family member, provides crucial structural insight into this transporter family. However, marrying this structural insight to the current functional understanding of this family also demands a comprehensive analysis of the transporter’s functional properties. To this end, we purified VcINDY, reconstituted it into liposomes, and determined its basic functional characteristics. Our data demonstrate that VcINDY is a high affinity, Na⁺-dependent transporter with a preference for C₄- and C₅-dicarboxylates. Transport of the model substrate, succinate, is highly pH dependent, consistent with VcINDY strongly preferring the substrate’s dianionic form. VcINDY transport is electrogenic with succinate coupled to the transport of three or more Na⁺ ions. In contrast to succinate, citrate, bound in the VcINDY crystal structure (in an inward-facing conformation), seems to interact only weakly with the transporter in vitro. These transport properties together provide a functional framework for future experimental and computational examinations of the VcINDY transport mechanism.     

Functional characterization of a Na+-coupled dicarboxylate transporter from Bacillus licheniformis

The Na⁺-coupled dicarboxylate transporter, SdcL, from Bacillus licheniformis is a member of the divalent anion/Na⁺ symporter (DASS) family that includes the bacterial Na⁺/dicarboxylate cotransporter SdcS (from Staphyloccocus aureus) and the mammalian Na⁺/dicarboxylate cotransporters, NaDC1 and NaDC3. The transport properties of SdcL produced in Escherichia coli are similar to those of its prokaryotic and eukaryotic counterparts, involving the Na⁺-dependent transport of dicarboxylates such as succinate or malate across the cytoplasmic membrane with a K_m of ～ 6 μM. SdcL may also transport aspartate, α-ketoglutarate and oxaloacetate with low affinity. The cotransport of Na⁺ and dicarboxylate by SdcL has an apparent stoichiometry of 2:1, and a K_0.5 for Na⁺ of 0.9 mM. Our findings represent the characterization of another prokaryotic protein of the DASS family with transport properties similar to its eukaryotic counterparts, but with a broader substrate specificity than other prokaryotic DASS family members. The broader range of substrates carried by SdcL may provide insight into domains of the protein that allow a more flexible or larger substrate binding pocket.     

Respiratory-driven Na+ electrical potential in the bacterium Vitreoscilla

Vitreoscilla is a Gram-negative bacterium with unique respiratory physiology in which Na+ was implicated as a coupling cation for the generation of a transmembrane electrical gradient (delta psi). Thus, cells respiring in the presence of 110 mM Na+ generated a delta psi of -142 mV compared to only -42 and -56 mV for Li+ and choline, respectively, and even the -42 and -56 mV were insensitive to the protonophore 3,5-di-tert-butyl-4-hydroxybenzaldehyde (DTHB). The kinetics of delta psi formation and collapse correlated well with the kinetics of Na+ fluxes but not with those of H+ fluxes. Cyanide inhibited respiration, Na+ extrusion, and delta psi formation 81% or more, indicating that delta psi formation and Na+ extrusion were coupled to respiration. Experiments were performed to distinguish among three possible transport systems for this coupling: (1) a Na(+)-transporting ATPase; (2) an electrogenic Na+/H+ antiport system; (3) a primary Na+ pump directly driven by the free energy of electron transport. DCCD and arsenate decreased cellular ATP up to 86% but had no effect on delta psi, evidence against a Na(+)-transporting ATPase. Low concentrations of DTHB had no effect on delta psi; high concentrations transiently collapsed delta psi, but led to a stimulation of Na+ extrusion, the opposite of that expected for a Na+/H+ antiport system. Potassium ion, which collapses delta psi, also stimulated Na+ extrusion. The experimental evidence is against Na+ extrusion by mechanisms 1 and 2 and supports the existence of a respiratory-driven primary Na+ pump for generating delta psi in Vitreoscilla.