The Na(+)-translocating methyltransferase complex from methanogenic archaea

Methanogenic archaea are dependent on sodium ions for methane formation. A sodium ion-dependent step has been shown to be methyl transfer from N(5)-methyltetrahydromethanopterin to coenzyme M. This exergonic reaction (DeltaG degrees '=-30 kJ/mol) is catalyzed by a Na(+)-translocating membrane-associated multienzyme complex composed of eight different subunits, MtrA-H. Subunit MtrA harbors a cob(I)amide prosthetic group which is methylated and demethylated in the catalytic cycle, demethylation being sodium ion-dependent. Based on the finding that in the cob(II)amide oxidation state the corrinoid is bound in a base-off/His-on configuration it is proposed that methyl transfer from MtrA to coenzyme M is associated with a conformational change of the protein and that this change drives the electrogenic translocation of the sodium ions.     

Regulation of Na(+) reabsorption by the aldosterone-induced small G protein K-Ras2A

Xenopus laevis A6 cells were used as model epithelia to test the hypothesis that K-Ras2A is an aldosterone-induced protein necessary for steroid-regulated Na(+) transport. The possibility that increased K-Ras2A alone is sufficient to mimic aldosterone action on Na(+) transport also was tested. Aldosterone treatment increased K-Ras2A protein expression 2.8-fold within 4 h. Active Ras is membrane associated. After aldosterone treatment, 75% of K-Ras was localized to the plasma membrane compared with 25% in the absence of steroid. Aldosterone also increased the amount of active (phosphorylated) mitogen-activated protein kinase kinase likely through K-Ras2A signaling. Steroid-induced K-Ras2A protein levels and Na(+) transport were decreased with antisense K-ras2A oligonucleotides, showing that K-Ras2A is necessary for the natriferic actions of aldosterone. Aldosterone-induced Na(+) channel activity, was decreased from 0.40 to 0.09 by pretreatment with antisense ras oligonucleotide, implicating the luminal Na(+) channel as one final effector of Ras signaling. Overexpression of K-Ras2A increased Na(+) transport approximately 2.2-fold in the absence of aldosterone. These results suggest that aldosterone signals to the luminal Na(+) channel via multiple pathways and that K-Ras2A levels are limiting for a portion of the aldosterone-sensitive Na(+) transport.     

Effects of choline on Na(+)- and K(+)-interactions with the Na+/K(+)-ATPase.

Choline chloride, 100 mM, stimulates Na+/K(+)-ATPase activity of a purified dog kidney enzyme preparation when Na+ is suboptimal (9 mM Na+ and 10 mM K+) and inhibits when K+ is suboptimal (90 mM Na+ and 1 mM K+), but has a negligible effect at optimal concentrations of both (90 mM Na+ and 10 mM K+). Stimulation occurs at low Na+ to K+ ratios, but not at those same ratios when the actual Na+ concentration is high (90 mM). Stimulation decreases or disappears when incubation pH or temperature is increased or when Li+ is substituted for K+ or Rb+. Choline+ also reduces the Km for MgATP at the low ratio of Na+ to K+ but not at the optimal ratio. In the absence of K+, however, choline+ does not stimulate at low Na+ concentrations: either in the Na(+)-ATPase reaction or in the E1 to E2P conformational transition. Together, these observations indicate that choline+ accelerates the rate-limiting step in the Na+/K(+)-ATPase reaction cycle, K(+)-deocclusion; consequently, optimal Na+ concentrations reflect Na+ accelerating that step also. Thus, the observed K0.5 for Na+ includes high-affinity activation of enzyme phosphorylation and low-affinity acceleration of K(+)-deocclusion. Inhibition of Na+/K(+)-ATPase and K(+)-nitrophenylphosphatase reactions by choline+ increases as the K(+)-concentration is decreased; the competition between choline+ and K+ may represent a similar antagonism between conformations selected by choline+ and by K+.     

Kinetics of Na(+)-dependent conformational changes of rabbit kidney Na+,K(+)-ATPase.

The kinetics of Na(+)-dependent partial reactions of the Na+,K(+)-ATPase from rabbit kidney were investigated via the stopped-flow technique, using the fluorescent labels N-(4-sulfobutyl)-4-(4-(p-(dipentylamino)phenyl)butadienyl)py ridinium inner salt (RH421) and 5-iodoacetamidofluorescein (5-IAF). When covalently labeled 5-IAF enzyme is mixed with ATP, the two labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau 1 approximately 155 s-1 for 5-IAF-labeled enzyme and 1/tau 1 approximately 200 s-1 for native enzyme (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3-->E2P(Na+)3 + ADP). The smaller amplitude slow phase, 1/tau 2 = 30-45 s-1, is attributed to the relaxation of the dephosphorylation/rephosphorylation equilibrium in the absence of K+ ions (E2P<==>E2). The Na+ concentration dependence of 1/tau 1 showed half-saturation at a Na+ concentration of 6-8 mM, with positive cooperatively involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high-affinity ATP-binding site determined from the ATP concentration dependence of 1/tau 1 was 8.0 (+/- 0.7) microM. It was found that P3-1-(2-nitrophenyl)ethyl ATP, tripropylammonium salt (NPE-caged ATP), at concentrations in the hundreds of micromolar range, significantly decreases the value of 1/tau 1, observed. This, as well as the biexponential nature of the kinetic traces, can account for previously reported discrepancies in the rates of the reactions investigated.     

Divergent homeobox gene hex regulates promoter of the Na(+)-dependent bile acid cotransporter

The divergent homeobox gene Hex is expressed in both developing and mature liver. A putative Hex binding site was identified in the promoter region of the liver-specific Na(+)-bile acid cotransporter gene (ntcp), and we hypothesized that Hex regulates the ntcp promoter through this site. Successive 5'-deletions of the ntcp promoter in a luciferase reporter construct transfected into Hep G2 cells confirmed a Hex response element (HRE) within the ntcp promoter (nt -733/-714). Moreover, p-CMHex transactivated a heterologous promoter construct containing HRE multimers (p4xHRELUC), whereas a 5-bp mutation of the core HRE eliminated transactivation. A dominant negative form of Hex (p-Hex-DN) suppressed basal luciferase activity of p-4xHRELUC and inhibited activation of this construct by p-CMHex. Interestingly, p-CMHex transactivated the HRE in Hep G2 cells but not in fibroblast-derived COS cells, suggesting the possibility that Hex protein requires an additional liver cell-specific factor(s) for full activity. Electrophoretic mobility shift assays confirmed that liver and Hep G2 cells contain a specific nuclear protein that binds the native HRE. We have demonstrated that the liver-specific ntcp gene promoter is the first known target of Hex and is a useful tool for evaluating function of the Hex protein.     

Thrombin is a Na(+)-activated enzyme.

The amidase activity of human alpha-thrombin has been studied at steady state as a function of the concentration of several chloride salts, at a constant ionic strength I = 0.2 M. All kinetic steps of the catalytic mechanism of the enzyme have been solved by studies conducted as a function of relative viscosity of the solution. Among all monovalent cations, Na+ is the most effective in activating thrombin catalysis. This effect is observed with different amide substrates and also with gamma-thrombin, a proteolytic derivative of the native enzyme which has little clotting activity but retains amidase activity toward small synthetic substrates. The specific effects observed as a function of Na+ concentration are indicative of a binding interaction of this monovalent cation with the enzyme. The basis of this interaction has been explored by measurements of substrate hydrolysis collected in a three-dimensional matrix of substrate concentration, relative viscosity, and Na+ concentration, keeping the ionic strength constant with an inert cation such as choline or tetraethylammonium. The data have globally been analyzed in terms of a kinetic linkage scheme where Na+ plays the role of an allosteric effector. The properties of the enzyme change drastically upon binding of Na+, with substrate binding and dissociation, as well as deacylation, occurring on a time scale which is 1 order of magnitude faster. The apparent association constants for Na+ binding to the various intermediate forms of the enzyme have all been resolved from analysis of experimental data and are in the range of 50-100 M-1 at 25 degrees C. Studies conducted at different temperatures, in the range 15-35 degrees C, have revealed the enthalpic and entropic components of Na+ binding to the enzyme. The results obtained from steady-state measurements are supported by independent measurements of the intrinsic fluorescence of the enzyme as a function of Na+ concentration at a constant ionic strength I = 0.2 M, over the temperature range 15-35 degrees C. These measurements are indicative of a drastic conformational change of the enzyme upon Na+ binding to a single site. The energetics of Na+ binding derived from analysis of fluorescence measurements agree very well with those derived independently from steady-state determinations. It is proposed that thrombin exists in two conformations, slow and fast, and that the slow-->fast transition is triggered by binding of a monovalent cation. The high specificity in thrombin activation found in the case of Na+ is the result of its higher affinity compared to all other monovalent cations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Aurora-B regulates RNA methyltransferase NSUN2

Disassembly of the nucleolus during mitosis is driven by phosphorylation of nucleolar proteins. RNA processing stops until completion of nucleolar reformation in G(1) phase. Here, we describe the RNA methyltransferase NSUN2, a novel substrate of Aurora-B that contains an NOL1/NOP2/sun domain. NSUN2 was concentrated in the nucleolus during interphase and was distributed in the perichromosome and cytoplasm during mitosis. Aurora-B phosphorylated NSUN2 at Ser139. Nucleolar proteins NPM1/nucleophosmin/B23 and nucleolin/C23 were associated with NSUN2 during interphase. In mitotic cells, association between NPM1 and NSUN2 was inhibited, but NSUN2-S139A was constitutively associated with NPM1. The Aurora inhibitor Hesperadin induced association of NSUN2 with NPM1 even in mitosis, despite the silver staining nucleolar organizer region disassembly. In vitro methylation experiments revealed that the Aurora-B-phosphorylation and the phosphorylation-mimic mutation (S139E) suppressed methyltransferase activities of NSUN2. These results indicate that Aurora-B participates to regulate the assembly of nucleolar RNA-processing machinery and the RNA methyltransferase activity of NSUN2 via phosphorylation at Ser139 during mitosis.     

Bradykinin acutely inhibits activity of the epithelial Na+ channel in mammalian aldosterone-sensitive distal nephron

Activation of the renal kallikrein-kinin system results in natriuresis and diuresis, suggesting its possible role in renal tubular sodium transport regulation. Here, we used patch-clamp electrophysiology to directly assess the effects of bradykinin (BK) on the epithelial Na(+) channel (ENaC) activity in freshly isolated split-opened murine aldosterone-sensitive distal nephrons (ASDNs). BK acutely inhibits ENaC activity by reducing channel open probability (P(o)) in a dose-dependent and reversible manner. Inhibition of B2 receptors with icatibant (HOE-140) abolished BK actions on ENaC. In contrast, activation of B1 receptors with the selective agonist Lys-des-Arg(9)-BK failed to reproduce BK actions on ENaC. This is consistent with B2 receptors playing a critical role in mediating BK signaling to ENaC. BK has little effect on ENaC P(o) when G(q/11) was inhibited with Gp antagonist 2A. Moreover, inhibition of phospholipase C (PLC) with U73122, but not saturation of cellular cAMP levels with the membrane-permeable nonhydrolysable cAMP analog 8-cpt-cAMP, prevents BK actions on ENaC activity. This argues that BK stimulates B2 receptors with subsequent activation of G(q/11)-PLC signaling cascade to acutely inhibit ENaC activity. Activation of BK signaling acutely depletes apical PI(4,5)P(2) levels. However, inhibition of Ca(2+) pump SERCA of the endoplasmic reticulum with thapsigargin does not prevent BK signaling to ENaC. Furthermore, caffeine, while producing a similar rise in [Ca(2+)](i) as in response to BK stimulation, fails to recapitulate BK actions on ENaC. Therefore, we concluded that BK acutely inhibits ENaC P(o) in mammalian ASDN via stimulation of B2 receptors and following depletion of PI(4,5)P(2), but not increases in [Ca(2+)](i).     

Tunicamycin reduces Na(+)-K(+)-pump expression in cultured skeletal muscle.

The purpose of this study was to examine effects of tunicamycin (TM), which inhibits core glycosylation of the beta-subunit, on functional expression of the Na(+)-K+ pump in primary cultures of embryonic chick skeletal muscle. Measurements were made of specific-[3H]-ouabain binding, ouabain-sensitive 86Rb uptake, resting membrane potential (Em), and electrogenic pump contribution to Em (Ep) of single myotubes with intracellular microelectrodes. Growth of 4-6-day-old skeletal myotubes in the presence of TM (1 microgram/ml) for 21-24 hr reduced the number of Na(+)-K+ pumps to 60-90% of control. Na(+)-K+ pump activity, the level of resting Em and Ep were also reduced significantly by TM. In addition, TM completely blocked the hyperpolarization of Em induced in single myotubes by cooling to 10 degrees C and then re-warming to 37 degrees C. Effects of tunicamycin were compared with those of tetrodotoxin (TTX; 2 x 10(-7) M for 24 hr), which blocks voltage-dependent Na+ channels. TM produced significantly greater decreases in ouabain-binding and Em than did TTX, findings that indicate that reduced Na(+)-K+ pump expression was not exclusively secondary to decreased intracellular Na+, the primary regulator of pump synthesis in cultured muscle. Similarly, effects of TM were significantly greater than those of cycloheximide, which inhibits protein synthesis by 95%. These findings demonstrate that effects were not due to inhibition of protein synthesis. We conclude that glycosylation of the Na(+)-K+ pump beta-subunit is required for full physiological expression of pump activity in skeletal muscle.     

Chemiosmotic systems in bioenergetics: H(+)-cycles and Na(+)-cycles.

The development of membrane bioenergetic studies during the last 25 years has clearly demonstrated the validity of the Mitchellian chemiosmotic H+ cycle concept. The circulation of H+ ions was shown to couple respiration-dependent or light-dependent energy-releasing reactions to ATP formation and performance of other types of membrane-linked work in mitochondria, chloroplasts, some bacteria, tonoplasts, secretory granules and plant and fungal outer cell membranes. A concrete version of the direct chemiosmotic mechanism, in which H+ potential formation is a simple consequence of the chemistry of the energy-releasing reaction, is already proved for the photosynthetic reaction centre complexes. Recent progress in the studies on chemiosmotic systems has made it possible to extend the coupling-ion principle to an ion other than H+. It was found that, in certain bacteria, as well as in the outer membrane of the animal cell, Na+ effectively substitutes for H+ as the coupling ion (the chemiosmotic Na+ cycle). A precedent is set when the Na+ cycle appears to be the only mechanism of energy production in the bacterial cell. In the more typical case, however, the H+ and Na+ cycles coexist in one and the same membrane (bacteria) or in two different membranes of one and the same cell (animals). The sets of delta mu H+ and delta mu Na+ generators as well as delta mu H+ and delta mu Na+ consumers found in different types of biomembranes, are listed and discussed.     

NKS1, Na(+)- and K(+)-sensitive 1, regulates ion homeostasis in an SOS-independent pathway in Arabidopsis

An Arabidopsis thaliana mutant, nks1-1, exhibiting enhanced sensitivity to NaCl was identified in a screen of a T-DNA insertion population in the genetic background of Col-0 gl1sos3-1. Analysis of the genome sequence in the region flanking the T-DNA left border indicated two closely linked mutations in the gene encoded at locus At4g30996. A second allele, nks1-2, was obtained from the Arabidopsis Biological Resource Center. NKS1 mRNA was detected in all parts of wild-type plants but was not detected in plants of either mutant, indicating inactivation by the mutations. Both mutations in NKS1 were associated with increased sensitivity to NaCl and KCl, but not to LiCl or mannitol. NaCl sensitivity was associated with nks1 mutations in Arabidopsis lines expressing either wild type or null alleles of SOS1, SOS2 or SOS3. The NaCl-sensitive phenotype of the nks1-2 mutant was complemented by expression of a full-length NKS1 allele from the CaMV35S promoter. When grown in medium containing NaCl, nks1 mutants accumulated more Na(+) than wild type and K(+)/Na(+) homeostasis was perturbed. It is proposed NKS1, a plant-specific gene encoding a 19kDa endomembrane-localized protein of unknown function, is part of an ion homeostasis regulation pathway that is independent of the SOS pathway.     

A marine algal Na(+)-activated ATPase possesses an immunologically identical epitope to Na+,K(+)-ATPase.

Immunological homology was investigated between Heterosigma akashiwo (a marine algae) Na(+)-activated ATPase and animal Na+,K(+)-ATPase. The former polypeptide [(1989) Plant Cell Physiol. 30, 923-928] reacted with anti-serum raised against the amino-terminal half of the pig kidney Na+,K(+)-ATPase alpha subunit. It is suggested that the Na+,K(+)-ATPase epitope within the amino-terminal region is conserved in the plant Na(+)-activated ATPase, and the region containing the epitope may be important for Na ion transport.     

Electrogenicity of Na(+)-coupled bile acid transporters.

The Na(+)-bile acid cotransporters NTCP and ASBT are largely responsible for the Na(+)-dependent bile acid uptake in hepatocytes and intestinal epithelial cells, respectively. This review discusses the experimental methods available for demonstrating electrogenicity and examines the accumulating evidence that coupled transport by each of these bile acid transporters is electrogenic. The evidence includes measurements of transport-associated currents by patch clamp electrophysiological techniques, as well as direct measurement of fluorescent bile acid transport rates in whole cell patch clamped, voltage clamped cells. The results support a Na+:bile acid coupling stoichiometry of 2:1.     

Human placental brush-border membrane Na(+)-pantothenate cotransport.

Membrane transport pathways for transplacental transfer of the water-soluble vitamin pantothenate were investigated by assessing the possible presence of a Na(+)-pantothenate cotransport mechanism in the maternal facing membrane of human placental epithelial cells. The presence of Na(+)-pantothenate cotransport was determined from radiolabeled tracer flux measurements of pantothenate uptake using preparations of purified brush-border membrane vesicles. Compared with other cations the imposition of an inward Na+ gradient stimulated vesicle uptake of pantothenate to levels approximately 40-fold greater than those observed at equilibrium. The observed stimulation of pantothenate uptake was not the result of indirect electrostatic coupling to an inside positive Na+ diffusion potential. In the absence of Na+ and pantothenate concentration gradients an inside negative voltage difference induced a Na(+)-dependent net influx of pantothenate, suggesting the presence of an electrogenic Na(+)-pantothenate cotransport mechanism. The effect of biotin on the kinetics of Na(+)-dependent pantothenate uptake and the effect of pantothenate on the kinetics of Na(+)-dependent biotin uptake suggested that placental absorption of biotin and pantothenate from the maternal circulation occurs by a common Na+ cotransport mechanism in apical brush-border membrane.     

Na+/K(+)-ATPase regulation by neurotransmitters.

A long period of experimental work has led to the conclusion that Na+/K(+)-ATPase is the enzymatic version of the Na+/K+ pump. This enzymatic system is in charge of various important cell functions. Among them cationic equilibrium and recovering of resting membrane potential in neurons is relevant. A tetrameric ensemble of peptides conform the system known as alpha and beta subunits. The alpha subunit is subdivided in alpha 1, alpha 2 and alpha 3, according to different location and properties. Regulatory factors intrinsic to the Na+/K(+)-ATPase system are: ATP, Na+ and Mg2+ concentrations inside the cell, and K+ outside. The enzyme activity is also regulated by extrinsic factors like some hormones (insulin and thyroxine). Induction of gene expression or post-translational modifications of the preexisting pool of the enzyme are the basic mechanisms of regulation proposed. Other extrinsic factors that seem to regulate the enzyme activity are some neurotransmitters. Among them the most extensively studied are catecholamines, mainly norepinephrine (NE) and lately serotonin (5-HT). The mechanism suggested for NE activation of the enzyme seems to involve specific receptors or a non-specific chelating action related to the catechol group that would relieve the inhibition by divalent cations. Another possibility is that NE removes an endogenous inhibitory factor present in the cytoplasm. The Na+/K(+)-ATPase is activated also by 5-HT. In vivo pharmacological and nutriological manipulations of brain 5-HT are accompanied by parallel responses of Na+/K(+)-ATPase activity. Serotonin agonists do activate the enzyme and antagonists neutralize the activation. In vitro there is a different dose dependent activation, according to the brain region. The mechanism involved seems to implicate a specific receptor system. Serotonin-Na+/K(+)-ATPase interaction in the rat brain is probably of functional relevance because it disappears in amygdaloid kindling. Also it seems to influence the ionic regulation of the pigment transport mechanism in crayfish photoreceptors. In relation to other neurotransmitters, a weak response to histamine was observed with acetylcholine, GABA and glutamic acid, the results were negative.     

Human placental brush-border membrane Na(+)-biotin cotransport.

Membrane transport pathways for transplacental transfer of the water-soluble vitamin biotin were investigated by assessing the possible presence of a Na(+)-biotin cotransport mechanism in the maternal-facing membrane of human placental epithelial cells. The presence of Na(+)-biotin cotransport was determined from radiolabeled tracer flux measurements of biotin uptake using preparations of purified brush-border membrane vesicles. The imposition of an inwardly directed Na+ gradient stimulated vesicle uptake of biotin to levels approximately 25-fold greater than those observed at equilibrium. The voltage sensitivity of Na+ gradient-driven biotin uptake suggested Na(+)-biotin cotransport is electrogenic occurring with net transfer of positive charge. A kinetic analysis of the activation of biotin uptake by increasing Na+ was most consistent with an interaction of Na+ at 2 sites in the transport protein. Static head determinations used to identify the magnitude of opposing driving forces bringing flux through the cotransport mechanism to equilibrium indicated a coupling ratio of 2 Na+ per biotin. Substrate specificity studies using chemical analogues of biotin suggested both the terminal carboxylic acid of the valeric acid side chain and a second nucleus of anionic charge were important determinants for substrate interaction with the cotransport protein. Initial rate determinations of biotin uptake indicate biotin interacts with a single saturable site (Km = 21 microM) with a maximal transport rate of 4.5 nmol/mg/min. The results of this study provide evidence for an electrogenic Na(+)-biotin cotransport mechanism in the maternal-facing membrane of human placental epithelial cells.