Sodium, an Obligate Growth Requirement for Predominant Rumen Bacteria

Sodium is an obligate growth requirement for most currently recognized predominant species of rumen bacteria. The isoosmotic deletion of Na(+) from a nutritionally adequate defined medium completely eliminated growth of most species. Growth yields and rates were both a function of Na(+) concentration for Na(+)-requiring species, and Na(+) could not be replaced by Rb(+), Li(+), or Cs(+) when these ions were substituted for Na(+) at a concentration equivalent to an Na(+) concentration that supported abundant growth. Li(+), Cs(+), or Rb(+) was toxic at an Na(+)-replacing concentration (15 mM) but not at a K(+)-replacing concentration (0.65 mM). K(+) was also an obligate growth requirement for rumen bacteria in media containing Na(+) and K(+) as major monovalent cations, but K(+) could be replaced, for most species, by Rb(+). The quantities of Na(+) that support rapid and abundant growth of Na(+)-requiring rumen bacteria show that these organisms are slight halophiles. A growth requirement for Na(+) appears more frequent among nonmarine bacteria than has been previously believed.     

Requirement for sodium in the anaerobic growth of Aerobacter aerogenes on citrate

Anaerobic growth of Aerobacter aerogenes on citrate as a carbon source required the presence of Na(+). The growth rate increased with increasing Na(+) concentration and was optimal at 0.10 m Na(+). The requirement was specific for Na(+), which could not be replaced by K(+), NH(4) (+), Li(+), Rb(+), or Cs(+). K(+) was required for growth in the presence of Na(+), the optimal K(+) concentration being 0.15 mm. Enzyme profiles were determined on cells grown in three different media: (i) intermediate Na(+), high K(+) concentration, (ii) high Na(+), high K(+) concentration, and (c) high Na(+), low K(+) concentration. All cells contained the enzymes of the citrate fermentation pathway, namely, citritase and the Na(+)-requiring oxalacetate (OAA) decarboxylase. All of the enzymes of the citric acid cycle were present, except alpha-ketoglutarate dehydrogenase which could not be detected. The incomplete citric acid cycle was, in effect, converted into two biosynthetic pathways leading to glutamate and succinate, respectively. The specific activities of citritase and OAA decarboxylase were lowest in medium (i), and under these conditions the activity of OAA decarboxylase appeared to be limited in vivo by the availability of Na(+). Failure of A. aerogenes to grow anaerobically on citrate in the absence of Na(+) can be explained at the enzymatic level by the Na(+) requirement of the OAA decarboxylase step of the citrate fermentation pathway and by the absence of an alternate pathway of citrate catabolism.     

Inorganic and Metal-Organic Growth Requirements of the Genus Bacteroides

The inorganic and metal-organic growth requirements of ruminal and nonruminal Bacteroides species were compared. The heme requirement of many nonruminal Bacteroides species was similar to that of Bacteroides ruminicola subsp. ruminicola and was a general tetrapyrrole requirement. Some nonruminal Bacteroides species utilized succinate or alpha-ketoglutarate, as well as tetrapyrrole-containing compounds, in place of heme. Fe(+) as well as heme was required for maximal yields of some Bacteroides species. The divalent cation requirements of Bacteroides species are complex. Mg(2+) deletion from a medium containing Mg(2+), Ca(2+), Co(2+), and Mn(2+) reduced the yields of all isolates. Ca(2+) deletion from the same medium reduced the growth yields of Bacteroides fragilis, B. fundiliformis, and one strain of B. oralis. The effects of Mg(2+) and Ca(2+) on the growth of Bacteroides isolates was influenced by other divalent cations. Relatively large quantities of Na(+) were obligately required by all of the currently recognized predominant rumen Bacteroides species. Nonruminal Bacteroides species either did not require Na(+) or required only small amounts. The Na(+) requirement of some nonruminal Bacteroides species could be partially replaced by Li(+) or Cs(+). The Na(+) requirement of rumen Bacteroides species was absolute. The inorganic and metal-organic growth requirements of Bacteroides species appear useful as aids in species differentiation.     

The effects of olive mill waste compost and poultry manure on the availability and plant uptake of nutrients in a highly saline soil

The effects of a compost (produced from by-products of the olive oil industry) and a poultry manure on mineral ion solubility and exchangeability in a highly saline agricultural soil (electrical conductivity for a 1:5 soil:water extract=1.85 dS m(-1)) from Murcia (SE Spain) were studied. The organic amendments did not change significantly the soil electrical conductivity or the soluble Na(+), Ca(2+) or Mg(2+). Only soluble K(+) increased, due to the K(+) supplied by the amendments. The cation exchange capacity increased in treated soils, the exchange complex being mainly saturated with Ca(2+), Mg(2+) and K(+). However, Na(+) was not retained in the exchange sites, and the sodium absorption ratio remained low. The compost and manure increased markedly the shoot growth of the salt-tolerant Beta maritima L. (sea beet) and Beta vulgaris L. (sugar beet). For B. maritima, this seemed to be related to decreases in the shoot concentrations of Na(+) and Cl(-) and increases in K(+) and H(2)PO(4)(-). In the case of B. vulgaris, increases in shoot H(2)PO(4)(-) and B and, for manure-treated soil, a decrease in shoot Na(+) may have been involved. Cultivation of tomato (Lycopersicon esculentum Mill. cv. Moneymaker) in the soil used previously for B. vulgaris indicated that the effects of the manure on tissue cation concentrations were longer-lasting than those of the compost.     

The Occlusion of Rb(+) in the Na(+)/K(+)-ATPase. II. The effects of Rb(+), Na(+), Mg2(+), or ATP on the equilibrium between free and occluded Rb(+).

We used the direct route of occlusion to study the equilibrium between free and occluded Rb(+) in the Na(+)/K(+)-ATPase, in media with different concentrations of ATP, Mg(2+), or Na(+). An empirical equation, with the restrictions imposed by the stoichiometry of ligand binding was fitted to the data. This allowed us to identify which states of the enzyme were present in each condition and to work out the schemes and equations that describe the equilibria between the ATPase, Rb(+), and ATP, Mg(2+), or Na(+). These equations were fitted to the corresponding experimental data to find out the values of the equilibrium constants of the reactions connecting the different enzyme states. The three ligands decreased the apparent affinity for Rb(+) occlusion without affecting the occlusion capacity. With [ATP] tending to infinity, enzyme species with one or two occluded Rb(+) seem to be present and full occlusion seems to occur in enzymes saturated with the nucleotide. In contrast, when either [Mg(2+)] or [Na(+)] tended to infinity no occlusion was detectable. Both Mg(2+) and Na(+) are displaced by Rb(+) through a process that seems to need the binding and occlusion of two Rb(+), which suggests that in these conditions Rb(+) occlusion regains the stoichiometry of the physiological operation of the Na(+) pump.     

Effects of nigericin and monactin on cation permeability of Streptococcus faecalis and metabolic capacities of potassium-depleted cells

At a concentration of 10(-6)m, nigericin and monactin inhibited growth of Streptococcus faecalis, and the inhibition was reversed by addition of excess K(+). In the presence of certain antibiotics, the cells exhibited increased permeability to certain cations; internal Rb(+) was rapidly lost by exchange with external H(+), K(+) Rb(+), and, more slowly, with Na(+) and Li(+). No effect was observed on the penetration of other small molecules. Cation exchanges induced by nigericin and monactin were metabolically passive and apparently did not involve the energy-dependent K(+) pump. When the cells were washed, the cytoplasmic membrane recovered its original impermeability to cations. By use of monactin, we prepared cells whose K(+) content had been completely replaced by other cations, and the metabolic characteristics of K(+)-depleted cells were studied. Cells containing only Na(+) glycolyzed almost as well as did normal ones and, under proper conditions, could accumulate amino acids and orthophosphate. These cells also incorporated (14)C-uracil into ribonucleic acid but incorporation of (14)C-leucine into protein was strictly dependent upon the addition of K(+). When K(+) or Rb(+) was added to sodium-loaded cells undergoing glycolysis, these ions were accumulated by stoichiometric exchange for Na(+). From concurrent measurements of the rate of glycolysis, it was calculated that one mole-pair of cations was exchanged for each mole of adenosine triphosphate produced.     

Role of sodium in determining alternate pathways of aerobic citrate catabolism in Aerobacter aerogenes

In contrast to the absolute Na(+) requirement for anaerobic growth of Aerobacter aerogenes on citrate as sole carbon source, aerobic growth of this microorganism did not require the presence of Na(+). However, Na(+) (optimal concentration, 10 mm) did increase the maximal amount of aerobic growth by 60%, even though it did not change the rate of growth. This increase in growth was specifically affected by Na(+), which could not be replaced by K(+), NH(4) (+), Li(+), Rb(+), or Cs(+). Enzyme profiles were determined in A. aerogenes cells grown aerobically on citrate in media of varying cationic composition. Cells grown in Na(+)-free medium possessed all the enzymes of the citric acid cycle including alpha-ketoglutarate dehydrogenase, which is repressed by anaerobic conditions of growth. The enzymes of the anaerobic citrate fermentation pathway, citritase and oxalacetate decarboxylase, were also present in these cells, but this pathway of citrate catabolism was effectively blocked by the absence of Na(+), which is essential for the activation of the oxalacetate decarboxylase step. Thus, in Na(+)-free medium, aerobic citrate catabolism proceeded solely via the citric acid cycle. Addition of 10 mm Na(+) to the aerobic citrate medium resulted in the activation of oxalacetate decarboxylase and the repression of alpha-ketoglutarate dehydrogenase, thereby diverting citrate catabolism from the (aerobic) citric acid cycle mechanism to the fermentation mechanism characteristic of anaerobic growth. The further addition of 2% potassium acetate to the medium caused repression of citritase and derepression of alpha-ketoglutarate dehydrogenase, switching citrate catabolism back into the citric acid cycle.     

Enzymatic analysis of the requirement for sodium in aerobic growth of Salmonella typhimurium on citrate

Na(+) was required for the aerobic growth of Salmonella typhimurium on citrate, but not on l-malate, glucose, or glycerol. The maximal growth rate and the maximal total growth occurred with 6 to 7 mm Na(+). Na(+) could not be replaced by K(+), NH(4) (+), Li(+), Rb(+), or Cs(+). Sonically treated extracts of citrate-grown cells contained the enzymes of the citrate fermentation pathway (citritase and oxalacetate decarboxylase) and all of the enzymes of the citric acid cycle. Thus, two separate routes of citrate catabolism appeared to be operational in the cells. Two discrete oxalacetate (OAA) decarboxylases were also demonstrated. One was of the "classic" type, being activated by Mn(++) and inhibited by ethylenediaminetetracetate (EDTA). It was present in the cell sap. The second decarboxylase closely resembled the Na(+)-activated OAA decarboxylase of citrate-grown Aerobacter aerogenes, whose growth also requires, or is increased, by Na(+). This decarboxylase was EDTA-insensitive, specifically activated by Na(+) and inhibited by avidin, and it had a high affinity for OAA. It was induced by growth on citrate, but not l-malate or glycerol. It is suggested that the Na(+) requirement for growth reflects the need to activate this OAA decarboxylase as a component of the citrate fermentation pathway and that citrate catabolism via the citric acid cycle, which should be independent of Na(+), is somehow dependent upon the activity of the Na(+)-activated enzyme.     

Cation transport by the neuronal K(+)-Cl(-) cotransporter KCC2: thermodynamics and kinetics of alternate transport modes

Both Cs(+) and NH(4)(+) alter neuronal Cl(-) homeostasis, yet the mechanisms have not been clearly elucidated. We hypothesized that these two cations altered the operation of the neuronal K(+)-Cl(-) cotransporter (KCC2). Using exogenously expressed KCC2 protein, we first examined the interaction of cations at the transport site of KCC2 by monitoring furosemide-sensitive (86)Rb(+) influx as a function of external Rb(+) concentration at different fixed external cation concentrations (Na(+), Li(+), K(+), Cs(+), and NH(4)(+)). Neither Na(+) nor Li(+) affected furosemide-sensitive (86)Rb(+) influx, indicating their inability to interact at the cation translocation site of KCC2. As expected for an enzyme that accepts Rb(+) and K(+) as alternate substrates, K(+) was a competitive inhibitor of Rb(+) transport by KCC2. Like K(+), both Cs(+) and NH(4)(+) behaved as competitive inhibitors of Rb(+) transport by KCC2, indicating their potential as transport substrates. Using ion chromatography to measure unidirectional Rb(+) and Cs(+) influxes, we determined that although KCC2 was capable of transporting Cs(+), it did so with a lower apparent affinity and maximal velocity compared with Rb(+). To assess NH(4)(+) transport by KCC2, we monitored intracellular pH (pH(i)) with a pH-sensitive fluorescent dye after an NH(4)(+)-induced alkaline load. Cells expressing KCC2 protein recovered pH(i) much more rapidly than untransfected cells, indicating that KCC2 can mediate net NH(4)(+) uptake. Consistent with KCC2-mediated NH(4)(+) transport, pH(i) recovery in KCC2-expressing cells could be inhibited by furosemide (200 microM) or removal of external [Cl(-)]. Thermodynamic and kinetic considerations of KCC2 operating in alternate transport modes can explain altered neuronal Cl(-) homeostasis in the presence of Cs(+) and NH(4)(+).     

Monovalent cation activation in Escherichia coli inosine 5'-monophosphate dehydrogenase

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate with the concomitant reduction of NAD to NADH. Escherichia coli IMPDH is activated by K(+), Rb(+), NH(+)(4), and Cs(+). K(+) activation is inhibited by Li(+), Na(+), Ca(2+), and Mg(2+). This inhibition is competitive versus K(+) at high K(+) concentrations, noncompetitive versus IMP, and competitive versus NAD. Thus monovalent cation activation is linked to the NAD site. K(+) increases the rate constant for the pre-steady-state burst of NADH production, possibly by increasing the affinity of NAD. Three mutant IMPDHs have been identified which increase the value of K(m) for K(+): Asp13Ala, Asp50Ala, and Glu469Ala. In contrast to wild type, both Asp13Ala and Glu469Ala are activated by all cations tested. Thus these mutations eliminate cation selectivity. Both Asp13 and Glu469 appear to interact with the K(+) binding site identified in Chinese hamster IMPDH. Like wild-type IMPDH, K(+) activation of Asp50Ala is inhibited by Li(+), Na(+), Ca(2+), and Mg(2+). However, this inhibition is noncompetitive with respect to K(+) and competitive with respect to both IMP and NAD. Asp50 interacts with residues that form a rigid wall in the IMP site; disruption of this wall would be expected to decrease IMP binding, and the defect could propagate to the proposed K(+) site. Alternatively, this mutation could uncover a second monovalent cation binding site.     

The synaptic vesicle protein synaptophysin: purification and characterization of its channel activity

The synaptic vesicle protein synaptophysin was solubilized from rat brain synaptosomes with a relatively low concentration of Triton X-100 (0.2%) and was highly purified (above 95%) using a rapid single chromatography step on hydroxyapatite/celite resin. Purified synaptophysin was reconstituted into a planar lipid bilayer and the channel activity of synaptophysin was characterized. In asymmetric KCl solutions (cis 300 mM/trans 100 mM), synaptophysin formed a fast-fluctuating channel with a conductance of 414 +/- 13 pS at +60 mV. The open probability of synaptophysin channels was decreased upon depolarization, and channels were found to be cation-selective. Synaptophysin channels showed higher selectivity for K(+) over Cl(-) (P(K(+))/P(Cl(-)) > 8) and preferred K(+) over Li(+), Na(+), Rb(+), Cs(+), or choline(+). The synaptophysin channel is impermeable to Ca(2+), which has no effect on its channel activity. This study is the second demonstration of purified synaptophysin channel activity, but the first biophysical characterization of its channel properties. The availability of large amounts of purified synaptophysin and of its characteristic channel properties might help to establish the role of synaptophysin in synaptic transmission.     

GerN, an endospore germination protein of Bacillus cereus, is an Na(+)/H(+)-K(+) antiporter

GerN, a Bacillus cereus spore germination protein, exhibits homology to a widely distributed group of putative cation transporters or channel proteins. GerN complemented the Na(+)-sensitive phenotype of an Escherichia coli mutant that is deficient in Na(+)/H(+) antiport activity (strain KNabc). GerN also reduced the concentration of K(+) required to support growth of an E. coli mutant deficient in K(+) uptake (strain TK2420). In a fluorescence-based assay of everted E. coli KNabc membrane vesicles, GerN exhibited robust Na(+)/H(+) antiport activity, with a K(m) for Na(+) estimated at 1.5 mM at pH 8.0 and 25 mM at pH 7.0. Li(+), but not K(+), served as a substrate. GerN-mediated Na(+)/H(+) antiport was further demonstrated in everted vesicles as energy-dependent accumulation of (22)Na(+). GerN also used K(+) as a coupling ion without completely replacing H(+), as indicated by partial inhibition by K(+) of H(+) uptake into right-side-out vesicles loaded with Na(+). K(+) translocation as part of the antiport was supported by the stimulatory effect of intravesicular K(+) on (22)Na(+) uptake by everted vesicles and the dependence of GerN-mediated (86)Rb(+) efflux on the presence of Na(+) in trans. The inhibitory patterns of protonophore and thiocyanate were most consistent with an electrogenic Na(+)/H(+)-K(+) antiport. GerN-mediated Na(+)/H(+)-K(+) antiport was much more rapid than GerN-mediated Na(+)/H(+) antiport.     

KcsA: it's a potassium channel

Ion conduction and selectivity properties of KcsA, a bacterial ion channel of known structure, were studied in a planar lipid bilayer system at the single-channel level. Selectivity sequences for permeant ions were determined by symmetrical solution conductance (K(+) > Rb(+), NH(4)(+), Tl(+) > Cs(+), Na(+), Li(+)) and by reversal potentials under bi-ionic or mixed-ion conditions (Tl(+) > K(+) > Rb(+) > NH(4)(+) > Na(+), Li(+)). Determination of reversal potentials with submillivolt accuracy shows that K(+) is over 150-fold more permeant than Na(+). Variation of conductance with concentration under symmetrical salt conditions is complex, with at least two ion-binding processes revealing themselves: a high affinity process below 20 mM and a low affinity process over the range 100-1,000 mM. These properties are analogous to those seen in many eukaryotic K(+) channels, and they establish KcsA as a faithful structural model for ion permeation in eukaryotic K(+) channels.     

Kinetic and thermodynamic analysis of the interaction of cations with dialkylglycine decarboxylase

Dialkylglycine decarboxylase (DGD) is a tetrameric pyridoxal phosphate (PLP)-dependent enzyme that catalyzes both decarboxylation and transamination in its normal catalytic cycle. Its activity is dependent on cations. Metal-free DGD and DGD complexes with seven monovalent cations (Li(+), Na(+), K(+), Rb(+), Cs(+), NH(4)(+), and Tl(+)) and three divalent cations (Mg(2+), Ca(2+), and Ba(2+)) have been studied. The catalytic rate constants for cation-bound enzyme (ck(cat) and ck(cat)/bK(AIB)) are cation-size-dependent, K(+) being the monovalent cation with the optimal size for catalytic activity. The divalent alkaline earth cations (Mg(2+), Ca(2+), and Ba(2+)) all give approximately 10-fold lower activity compared to monovalent alkali cations of similar ionic radius. The Michaelis constant for aminoisobutyrate (AIB) binding to DGD-PLP complexes with cations (bK(AIB)) varies with ionic radius. The larger cations (K(+), Rb(+), Cs(+), NH(4)(+), and Tl(+)) give smaller bK(AIB) ( approximately 4 mM), while smaller cations (Li(+), Na(+)) give larger values (approximately 10 mM). Cation size and charge dependence is also found with the dissociation constant for PLP binding to DGD-cation complexes (aK(PLP)). K(+) and Rb(+) possess the optimal ionic radius, giving the lowest values of aK(PLP). The divalent alkaline earth cations give aK(PLP) values approximately 10-fold higher than alkali cations of similar ionic radius. The cation dissociation constant for DGD-PLP-AIB-cation complexes (betaK(M)z+) was determined and also shown to be cation-size-dependent, K(+) and Rb(+) yielding the lowest values. The kinetics of PLP association and dissociation from metal-free DGD and its complexes with cations (Na(+), K(+), and Ba(2+)) were analyzed. All three cations tested increase PLP association and decrease PLP dissociation rate constants. Kinetic studies of cation binding show saturation kinetics for the association reaction. The half-life for association with saturating Rb(+) is approximately 24 s, while the half-life for dissociation of Rb(+) from the DGD-PLP-AIB-Rb(+) complex is approximately 12 min.     

Ion selectivity and current saturation in inward-rectifier K+ channels

We investigated the features of the inward-rectifier K channel Kir1.1 (ROMK) that underlie the saturation of currents through these channels as a function of permeant ion concentration. We compared values of maximal currents and apparent K(m) for three permeant ions: K(+), Rb(+), and NH(4)(+). Compared with K(+) (i(max) = 4.6 pA and K(m) = 10 mM at -100 mV), Rb(+) had a lower permeability, a lower i(max) (1.8 pA), and a higher K(m) (26 mM). For NH(4)(+), the permeability was reduced more with smaller changes in i(max) (3.7 pA) and K(m) (16 mM). We assessed the role of a site near the outer mouth of channel in the saturation process. This site could be occupied by either permeant ions or low-affinity blocking ions such as Na(+), Li(+), Mg(2+), and Ca(2+) with similar voltage dependence (apparent valence, 0.15-0.20). It prefers Mg(2+) over Ca(2+) and has a monovalent cation selectivity, based on the ability to displace Mg(2+), of K(+) > Li(+) ～ Na(+) > Rb(+) ～ NH(4)(+). Conversely, in the presence of Mg(2+), the K(m) for K(+) conductance was substantially increased. The ability of Mg(2+) to block the channels was reduced when four negatively charged amino acids in the extracellular domain of the channel were mutated to neutral residues. The apparent K(m) for K(+) conduction was unchanged by these mutations under control conditions but became sensitive to the presence of external negative charges when residual divalent cations were chelated with EDTA. The results suggest that a binding site in the outer mouth of the pore controls current saturation. Permeability is more affected by interactions with other sites within the selectivity filter. Most features of permeation (and block) could be simulated by a five-state kinetic model of ion movement through the channel.     

The MgtC virulence factor of Salmonella enterica serovar Typhimurium activates Na(+),K(+)-ATPase

The mgtC gene of Salmonella enterica serovar Typhimurium encodes a membrane protein of unknown function that is important for full virulence in the mouse. Since mgtC is part of an operon with mgtB which encodes a Mg(2+)-transporting P-type ATPase, MgtC was hypothesized to function in ion transport, possibly in Mg(2+) transport. Consequently, MgtC was expressed in Xenopus laevis oocytes, and its effect on ion transport was evaluated using ion selective electrodes. Oocytes expressing MgtC did not exhibit altered currents or membrane potentials in response to changes in extracellular H(+), Mg(2+), or Ca(2+), thus ruling out a previously postulated function as a Mg(2+)/H(+) antiporter. However, addition of extracellular K(+) markedly hyperpolarized membrane potential instead of the expected depolarization. Addition of ouabain to block the oocyte Na(+),K(+)-ATPase completely prevented hyperpolarization and restored the normal K(+)-induced depolarization response. These results suggested that the Na(+),K(+)-ATPase was constitutively activated in the presence of MgtC resulting in a membrane potential largely dependent on Na(+),K(+)-ATPase. Consistent with the involvement of Na(+),K(+)-ATPase, oocytes expressing MgtC exhibited an increased rate of (86)Rb(+) uptake and had increased intracellular free [K(+)] and decreased free [Na(+)] and ATP. The free concentrations of Mg(2+) and Ca(2+) and cytosolic pH were unchanged, although the total intracellular Ca(2+) content was slightly elevated. These results suggest that the serovar Typhimurium MgtC protein may be involved in regulating membrane potential but does not directly transport Mg(2+) or another ion.     

Activation of AMP-activated protein kinase stimulates Na+,K+-ATPase activity in skeletal muscle cells

Contraction stimulates Na(+),K(+)-ATPase and AMP-activated protein kinase (AMPK) activity in skeletal muscle. Whether AMPK activation affects Na(+),K(+)-ATPase activity in skeletal muscle remains to be determined. Short term stimulation of rat L6 myotubes with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), activates AMPK and promotes translocation of the Na(+),K(+)-ATPase α(1)-subunit to the plasma membrane and increases Na(+),K(+)-ATPase activity as assessed by ouabain-sensitive (86)Rb(+) uptake. Cyanide-induced artificial anoxia, as well as a direct AMPK activator (A-769662) also increase AMPK phosphorylation and Na(+),K(+)-ATPase activity. Thus, different stimuli that target AMPK concomitantly increase Na(+),K(+)-ATPase activity. The effect of AICAR on Na(+),K(+)-ATPase in L6 myotubes was attenuated by Compound C, an AMPK inhibitor, as well as siRNA-mediated AMPK silencing. The effects of AICAR on Na(+),K(+)-ATPase were completely abolished in cultured primary mouse muscle cells lacking AMPK α-subunits. AMPK stimulation leads to Na(+),K(+)-ATPase α(1)-subunit dephosphorylation at Ser(18), which may prevent endocytosis of the sodium pump. AICAR stimulation leads to methylation and dephosphorylation of the catalytic subunit of the protein phosphatase (PP) 2A in L6 myotubes. Moreover, AICAR-triggered dephosphorylation of the Na(+),K(+)-ATPase was prevented in L6 myotubes deficient in PP2A-specific protein phosphatase methylesterase-1 (PME-1), indicating a role for the PP2A·PME-1 complex in AMPK-mediated regulation of Na(+),K(+)-ATPase. Thus contrary to the common paradigm, we report AMPK-dependent activation of an energy-consuming ion pumping process. This activation may be a potential mechanism by which exercise and metabolic stress activate the sodium pump in skeletal muscle.     

RGD-CAP ((beta)ig-h3) is expressed in precartilage condensation and in prehypertrophic chondrocytes during cartilage development

The TRK-HKT family of K(+) transporters mediates K(+) and Na(+) uptake in fungi and plants. In this study, we have investigated the molecular mechanism involved in the movement of alkali cations through the TRK1 transporter of Saccharomyces cerevisiae. The model that best explains the activity of ScTRK1 is a cotransport of two K(+) or Rb(+), both of which bind the two binding sites of ScTRK1 with very high affinities in K(+)-starved cells. Na(+) can be transported in the same way but it exhibits a much lower affinity for the second binding site. Therefore, only at critical concentration ratios between K(+) and Na(+), or Rb(+) and Na(+), the transporter takes up Na(+) together with K(+) or Rb(+). Mutation analyses suggest that the two binding sites are located in the P fragment of the first MPM motif of the transporter, and that Gln(90) is involved in these binding sites. ScTRK1 can be in two states, medium or high affinity, and we have found that Leu(949) is involved in the oscillation of the transporter between these two states. ScTRK1 mediates active K(+) uptake. This is not Na(+)-coupled and direct coupling of ScTRK1 to a source of chemical energy seems more probable than K(+)-H(+) cotransport.     

Selectivity of alkali cation influx across the plasma membrane of oat roots: cation specificity of the plasma membrane ATPase

Influx of alkali cations (Li(+), Na(+), K(+), Rb(+), Cs(+)) across plasma membranes of cells of excised roots of Avena sativa cv. Goodfield was selective, but different, in the absence and in the presence of 1 mm CaSO(4). Ca(2+) reduced the influx rates of all of the alkali cations-especially Na(+) and Li(+). Transport selectivity changed as the external concentrations of the alkali cations increased.Plasma membrane ATPase, purified from Avena sativa roots, was differentially stimulated by alkali cations. This specificity, however, was not altered by Ca(2+) or the external cation concentrations. A close correspondence existed between the relative influx rates of K(+), Rb(+), and Cs(+) and the relative stimulation of the ATPase by these cations. A similar correspondence did not occur for Na(+) and Li(+).Selective cation transport in oat roots could result, in part, from the specificity of the plasma membrane ATPase, but other factors such as specific carriers or porters or differential diffusion rates must also be involved.     

Evidence for tryptophan residues in the cation transport path of the Na(+),K(+)-ATPase

A family of aryl isothiouronium derivatives was designed as probes for cation binding sites of Na(+),K(+)-ATPase. Previous work showed that 1-bromo-2,4,6-tris(methylisothiouronium)benzene (Br-TITU) acts as a competitive blocker of Na(+) or K(+) occlusion. In addition to a high-affinity cytoplasmic site (K(D) < 1 microM), a low-affinity site (K(D) approximately 10 microM) was detected, presumably extracellular. Here we describe properties of Br-TITU as a blocker at the extracellular surface. In human red blood cells Br-TITU inhibits ouabain-sensitive Na(+) transport (K(D) approximately 30 microM) in a manner antagonistic with respect to extracellular Na(+). In addition, Br-TITU impairs K(+)-stimulated dephosphorylation and Rb(+) occlusion from phosphorylated enzyme of renal Na(+),K(+)-ATPase, consistent with binding to an extracellular site. Incubation of renal Na(+),K(+)-ATPase with Br-TITU at pH 9 irreversibly inactivates Na(+),K(+)-ATPase activity and Rb(+) occlusion. Rb(+) or Na(+) ions protect. Preincubation of Br-TITU with red cells in a K(+)-free medium at pH 9 irreversibly inactivates ouabain-sensitive (22)Na(+) efflux, showing that inactivation occurs at an extracellular site. K(+), Cs(+), and Li(+) ions protect against this effect, but the apparent affinity for K(+), Cs(+), or Li(+) is similar (K(D) approximately 5 mM) despite their different affinities for external activation of the Na(+) pump. Br-TITU quenches tryptophan fluorescence of renal Na(+),K(+)-ATPase or of digested "19 kDa membranes". After incubation at pH 9 irreversible loss of tryptophan fluorescence is observed and Rb(+) or Na(+) ions protect. The Br-TITU appears to interact strongly with tryptophan residue(s) within the lipid or at the extracellular membrane-water interface and interfere with cation occlusion and Na(+),K(+)-ATPase activity.