Affinity For Na Research Articles

THU578 Concept: Role Of Spiral Steroids (SS) During Pregnancy

Abstract Disclosure: F.I. Chasalow: None. Background: Earlier, my laboratory reported 3 novel endogenous steroids (SS) that cross react with digoxin-specific antibodies (DLM). The SS are readily distinguished from all other steroids by 3 characteristics: [1] each is a phosphocholine diester, [2] each has more than 21 carbon atoms in the steroid fragment and [3] the extra carbons form an E-ring with a cyclic lactone. The discovery included a biosynthetic scheme with known enzymes and no need for a ‘magic’ step. Function: NaK-ATPase has 3 subunits, designated FXYD, α, and β. Affinity for Na+ and K+ ions is regulated by FXYD proteins. Each SS matches a tissue-specific α-subunit. Each α-subunit has 2 different binding sites for steroids. One site binds a SS; the ligand of the other site is unknown. For example, Ionotropin is specific for the α subunit present in cardiac and renal tissue. Kaleotropin is isolated from gonads, but the α subunit which matches each specific ovarian cell type is unknown. Galotropin (G-trop): One of the SS, G-trop, was detected as a DLM in human breast cyst fluids, specifically cysts with high K+ ion levels. Solubility properties distinguished it from previously known cardiac glycosides and, like other DLM, it inhibited NaK-ATPase. G-trop was also detected in milk from cows, sheep and goats. Ions derived from Ionotropin (m/z=341 Da) or Kaleotropin (m/z=369 Da) were not present in milk extracts. On LC-MS, G-trop fragmented to an ion at m/z= 381 Da. Trial and error analysis suggests C25O4H34. No other formula is consistent with m/z=381 Da. G-trop: G-trop wasn’t present in serum from children. At 22-26 weeks of gestation, whether or not a pregnant woman had pre-eclampsia, serum had low levels of G-trop. Maternal G-trop increases during the 3rd trimester, coincidentally with initiation of milk production; simultaneously, K+ ion levels in milk increased. Pre-eclampsia. In a pilot study, 65% of patients (n=20) with pre-eclampsia had elevated levels of Ionotropin precursor and 30% had elevated levels of Kaleotropin precursor. 25% of patients with pre-eclampsia develop life-threatening hypertension or seizures, typical of the expected response to elevated Ionotropin levels. Therapy includes immediate delivery of the infant at 30-36 weeks of gestation. Maternal G-trop synthesis has not yet begun and mother’s milk has low K+ ion levels. Elevated maternal Kaleotropin is not the cause of maternal hypertension, suggesting a specific gestational maternal α-subunit is its target. Kaleotropin from placenta may be the signal to respond to maternal hypokalemia. Necrotizing enterocolitis (NEC):In utero the fetus does not absorb K+ from the GI tract because fetal need for K+ ion is provided via the placenta. Concept: After premature delivery, without G-trop in milk, the infant can’t absorb K+ from the GI tract; the GI tract fails to grow and tight junctions do not form. As a result, infants are at high risk for developing NEC. Presentation Date: Thursday, June 15, 2023

Ammonium Recovery from Synthetic Wastewaters by Using Zeolitic Mixtures: A Desorption Batch-Study

Resource recovery from wastewater is very important in view of a circular economy approach in the water field. Among the different technologies applied to realize circular economy, an attractive option is the use of nutrient-enriched media that can be utilized as slow-release fertilizers. Zeolites have been re-discovered for their key role in ammonium (NH4+) adsorption from treated wastewater. Although many studies have been carried out to assess the ability of zeolites to adsorb NH4+, only few papers concerning NH4+ desorption from zeolites are available in the literature. Therefore, this study investigated NH4+ desorption from mineralogically different zeolites, before (ZNS and ZNC) and after (ZSS and ZSC) their treatment with sodium chloride. The zeolites differed in mordenite content. The amount of the desorbed NH4+ varied from 78 to 84% of the total NH4+ adsorbed. In particular, the NaCl-treated materials showed the largest desorption (27.6 ± 0.2 mg L−1, and 27.9 ± 0.7 mg L−1, ZSS, and ZSC, respectively) as compared to the untreated zeolites (22.9 ± 0.3 mg L−1, and 24.2 ± 0.3 mg L−1, ZNS, and ZNC, respectively) because of the different affinity of the cations for the zeolite surface. A monomodal pseudo-first-order model best approximated the desorption kinetics, suggesting only one mechanism of NH4+ desorption from zeolites. Such a mechanism is based on the ion exchange between dissolved Na+ and adsorbed NH4+. The desorption kinetics also showed that NH4+ desorbed slower from the NaCl-treated zeolites than the untreated ones. This effect was explained by the different affinity of Na+ and NH4+ for the zeolite surfaces as due to the diverse sizes of the Na+ and NH4+ hydration spheres. By revealing the effect of zeolite mineralogy and surface treatments in the desorption of NH4+, this study can suggest new and effective synthetic strategies for the achievement of cheap new materials to be applied in environmental remediation within a circular economy perspective.

β subunit affects Na+ and K+ affinities of Na+/K+-ATPase: Na+ and K+ affinities of a hybrid Na+/K+-ATPase composed of insect α and mammalian β subunits

The affinity for K+ of silkworm Na+/K+-ATPase, which is composed of α and β subunits, is remarkably lower than that of mammalian Na+/K+-ATPase, with a slightly higher affinity for Na+. Because the α subunit had more than 70% identity to the mammalian α subunit in the amino acid sequence, whereas the β subunit, a glycosylated protein, had less than 30% identity to the mammalian β subunit, it was suggested that the β subunit was involved in the affinities for Na+ and K+ of Na+/K+-ATPase. To confirm this hypothesis, we examined whether replacing the silkworm β subunit with the mammalian β subunit affected the affinities for Na+ and K+ of Na+/K+-ATPase. Cloned silkworm α and cloned rat β1 were co-expressed in BM-N cells, a cultured silkworm ovary-derived cell lacking endogenous Na+/K+-ATPase, to construct a hybrid Na+/K+-ATPase, in which the silkworm β subunit was replaced with the rat β1 subunit. The hybrid Na+/K+-ATPase increased the affinity for K+ by 4.1-fold and for Na+ by 0.65-fold compared to the wild-type one. Deglycosylation of the silkworm β subunit did not affect the K+ affinity. These results support the involvement of the β subunit in the Na+ and K+ affinities of Na+/K+-ATPase.

Efficient, Selective Sodium and Lithium Removal by Faradaic Deionization Using Symmetric Sodium Titanium Vanadium Phosphate Intercalation Electrodes.

NASICON (sodium superionic conductor) materials are promising host compounds for the reversible capture of Na+ ions, finding prior application in batteries as solid-state electrolytes and cathodes/anodes. Given their affinity for Na+ ions, these materials can be used in Faradaic deionization (FDI) for the selective removal of sodium over other competing ions. Here, we investigate the selective removal of sodium over other alkali and alkaline-earth metal cations from aqueous electrolytes when using a NASICON-based mixed Ti-V phase as an intercalation electrode, namely, sodium titanium vanadium phosphate (NTVP). Galvanostatic cycling experiments in three-electrode cells with electrolytes containing Na+, K+, Mg2+, Ca2+, and Li+ reveal that only Na+ and Li+ can intercalate into the NTVP crystal structure, while other cations show capacitive response, leading to a material-intrinsic selectivity factor of 56 for Na+ over K+, Mg2+, and Ca2+. Furthermore, electrochemical titration experiments together with modeling show that an intercalation mechanism with a limited miscibility gap for Na+ in NTVP mitigates the state-of-charge gradients to which phase-separating intercalation electrodes are prone when operated under electrolyte flow. NTVP electrodes are then incorporated into an FDI cell with automated fluid recirculation to demonstrate up to 94% removal of sodium in streams with competing alkali/alkaline-earth cations with 10-fold higher concentration, showing process selectivity factors of 3-6 for Na+ over cations other than Li+. Decreasing the current density can improve selectivity up to 25% and reduce energy consumption by as much as ∼50%, depending on the competing ion. The results also indicate the utility of NTVP for selective lithium recovery.

Sequence dependence of the conformational transitions of DNA

The sequence-dependent conformational transitions of DNA with the increase of Guanine (G) base content in physiological environment are studied by performing molecular dynamics simulation, and the interaction differences between Na+ and K+ ion with DNAs are compared. We find that the conformation of DNA shows the sequence-dependent change, undergoing B- to A-form transition, with increasing the content of G base.The conformational flexibility of DNA is caused mainly by the base composition, Na+ and K+ contribution to DNA conformational change shows no relation to their nature. However, the electrostatic interaction of ions with DNA displays sequence-dependent binding as the content of G is increased, and the affinity of Na+ to DNA is stronger than that of K+. Na+ has more high molarity condensing around DNA than K+, largely because Cl- ions weaken K+–DNA interaction due to easily form K+–Cl- clusters. Na+ binds to the electronegative sites of DNA bases in the major groove and the phosphate, while K+ interacts preferentially with the major groove. In the major groove, Na+ tends to bind to the adenine base, while K+ prefers to the guanine base. With increasing the content of G, the attraction of the hydrophobic groups between bases are weakened and the negatively charged atoms on the bases are exposed, leading to the electrostatic attraction of the major groove being enhanced. As a result, the ion occupancy and molarity distribution in the major groove are increased, but decreased around the phosphate groups.

Binding of Ouabain, Digoxin, or Marinobufagenin Induces Different Conformational Changes in Kidney α1-Na+,K+-ATPase Isoforms, Resistant and Sensitive to Cardiotonic Steroids

The affinity of rodent Na+,K+-ATPase α1-subunit to cardiotonic steroids (CTS) is known to be approximately 1000-fold less than the affinity of Na+,K+-ATPase α1-subunit from other mammals. The CTS-resistant isoform of Na+,K+-ATPase α1-subunit (α1R) is expressed in rodent cells, in contrast to the CTS-sensitive isoform of the α1-subunit (α1S), which is expressed in cells of other mammals. Earlier we have established that incubation with ouabain in concentrations that completely suppressed the activity of Na+,K+-ATPase α1-isoform (α1S-Na+,K+-ATPase) led to a death of human endothelial and smooth muscle cells but did not affect the survival of rat cells expressing α1R-Na+,K+-ATPase. Conformational transitions that are induced by CTS binding to Na+,K+-ATPase play a key role in the process of cell survival. To reveal differences in the CTS-induced conformational changes of α1R- and α1S-Na+,K+-ATPase isoforms, we used three different CTS (two cardenelids, ouabain and digoxin, and one bufadienolid, marinobufagenin) and analyzed the trypsinolysis products of α1-subunits in two main conformations of Na+,K+-ATPase (E1 and E2-P). The treatment of the pig kidney α1S-Na,K-ATPase in E1 conformation by trypsin results in a significant decrease of the amount of α1S-subunit and in the appearance of protein fragments with molecular masses of about 40, 35, 23, and 19 kDa. Preincubation of Na+,K+-ATPase in E1 conformation with ouabain or with digoxin (1 mM) leads to a decrease of the amount of α1S-subunit, increase of the amount of polypeptide fragment with molecular mass of about 40 kDa, and a significant rise of the amount of fragment with molecular mass of about 35.5 kDa, which was not found after the preincubation of the Na+,K+-ATPase in E1 conformation with marinobufagenin (1 mM). In the absence of CTS the trypsinolysis of α1S-Na+,K+-ATPase in E2-P conformation results in a decrease of the amount of α1S-subunit and an increase of the amount of proteolytic products with molecular mass of about 40 and 35.5 kDa. Preincubation of the Na+,K+-ATPase in E2-P conformation in the presence of any of the CTS studied induces the appearance of big amount of an additional peptide fragment with molecular mass of about 45 kDa. Preincubation of α1R-Na+,K+-ATPase from rat kidney with any of the CTS does not change the composition of proteolytic products and their molecular masses in either E1 or E2-P conformation. The results suggest that the structure of the CTS-binding site and a conformational response of α1-Na+,K+-ATPase to binding of CTS is mainly determined by the primary structure of the CTS-resistant and CTS-sensitive α1-subunits of the Na+,K+-AТРase.

Structure-dynamic and functional relationships in a Li+-transporting sodium‑calcium exchanger mutant

The cell membrane (NCX) and mitochondrial (NCLX) Na+/Ca2+ exchangers control Ca2+ homeostasis. Eleven (out of twelve) ion-coordinating residues are highly conserved among eukaryotic and prokaryotic NCXs, whereas in NCLX, nine (out of twelve) ion-coordinating residues are different. Consequently, NCXs exhibit high selectivity for Na+ and Ca2+, whereas NCLX can exchange Ca2+ with either Na+ or Li+. However, the underlying molecular mechanisms and physiological relevance remain unresolved. Here, we analyzed the NCX_Mj-derived mutant NCLX_Mj (with nine substituted residues) imitating the ion selectivity of NCLX. Site-directed fluorescent labeling and ion flux assays revealed the nearly symmetric accessibility of ions to the extracellular and cytosolic vestibules in NCLX_Mj (Kint = 0.8–1.4), whereas the extracellular vestibule is predominantly accessible to ions (Kint = 0.1–0.2) in NCX_Mj. HDX-MS (hydrogen-deuterium exchange mass-spectrometry) identified symmetrically rigidified core helix segments in NCLX_Mj, whereas the matching structural elements are asymmetrically rigidified in NCX_Mj. The HDX-MS analyses of ion-induced conformational changes and the mutational effects on ion fluxes revealed that the “Ca2+-site” (SCa) of NCLX_Mj binds Na+, Li+, or Ca2+, whereas one or more additional Na+/Li+ sites of NCLX_Mj are incompatible with the Na+ sites (Sext and Sint) of NCX_Mj. Thus, the replacement of ion-coordinating residues in NCLX_Mj alters not only the ion selectivity of NCLX_Mj, but also the capacity and affinity for Na+/Li+ (but not for Ca2+) binding, bidirectional ion-accessibility, the response of the ion-exchange to membrane potential changes, and more. These structure-controlled functional features could be relevant for differential contributions of NCX and NCLX to Ca2+ homeostasis in distinct sub-cellular compartments.

Selective exclusion and selective binding both contribute to ion selectivity in KcsA, a model potassium channel

The selectivity filter in potassium channels, a main component of the ion permeation pathway, configures a stack of binding sites (sites S1-S4) to which K+ and other cations may bind. Specific ion binding to such sites induces changes in the filter conformation, which play a key role in defining both selectivity and permeation. Here, using the potassium channel KcsA as a model, we contribute new evidence to reinforce this assertion. First, ion binding to KcsA blocked by tetrabutylammonium at the most cytoplasmic site in the selectivity filter (S4) suggests that such a site, when in the nonconductive filter conformation, has a higher affinity for cation binding than the most extracellular S1 site. This filter asymmetry, along with differences in intracellular and extracellular concentrations of K+versus Na+ under physiological conditions, should strengthen selection of the permeant K+ by the channel. Second, we used different K+ concentrations to shift the equilibrium between nonconductive and conductive states of the selectivity filter in which to test competitive binding of Na+ These experiments disclosed a marked decrease in the affinity of Na+ to bind the channel when the conformational equilibrium shifts toward the conductive state. This finding suggested that in addition to the selective binding of K+ and other permeant species over Na+, there is a selective exclusion of nonpermeant species from binding the channel filter, once it reaches a fully conductive conformation. We conclude that selective binding and selective exclusion of permeant and nonpermeant cations, respectively, are important determinants of ion channel selectivity.

Substitutions in the cardenolide binding site and interaction of subunits affect kinetics besides cardenolide sensitivity of insect Na,K-ATPase

Substitutions within the cardenolide target site of several insects' Na,K-ATPase α-subunits may confer resistance against toxic cardenolides. However, to which extent these substitutions alter the Na,K-ATPase's kinetic properties and how they interact with different β-subunits is not clear. The cardenolide-adapted milkweed bug Oncopeltus fasciatus possesses three paralogs of the α-subunit (A, B, and C) that differ in number and identity of resistance-conferring substitutions. We introduced these substitutions into the α-subunit of Drosophila melanogaster and combined them with the β-subunits Nrv2.2 and Nrv3. The substitutions Q111T-N122H-F786N-T797A (A-copy mimic) and Q111T-N122H-F786N (B-copy mimic) mediated high insensitivity to ouabain, yet they drastically lowered ATPase activity. Remarkably, the identity of the β-subunit was decisive and all α-subunits were less active when combined with Nrv3 than when combined with Nrv2.2. Both the substitutions and the co-expressed β-subunit strongly affected the enyzme's affinity for Na+ and K+. Na+ affinity was considerably higher for all enzymes expressed with nrv3 while expression with nrv2.2 mostly increased K+ affinity. Our results provide the first evidence that resistance against cardenolides comes at the cost of significantly altered kinetic properties of the Na,K-ATPase. The β-subunit can strongly modulate these properties but cannot fully compensate for the effect of the substitutions.

Coexistence of light-driven Na+ and H+ transport in a microbial rhodopsin from Nonlabens dokdonensis

Ion pumping microbial rhodopsins are photochemically active membrane proteins, converting light energy into ion-motive-force for ATP synthesis. Nonlabens dokdonensis rhodopsin 2 (NdR2), was recently identified as a light-driven Na+ pump. However, few functional studies on NdR2 have been conducted to elucidate its mechanism of ion transport. By reconstituting NdR2 into liposomes, we proved that NdR2 functions as a light-driven Na+/H+ pump. As Na+ concentration increased, the dominant H+ pump activity switched to the Na+ pump activity at neutral pH. The inversion of pH change by the addition of CCCP at low Na+ further suggested that the transport of Na+ and H+ should coexist in NdR2. By increasing H+ concentration, the affinity for Na+ lowered, which was indicated by an increase in KM from ~31mM at pH ~7.5, to ~74mM at pH ~6.5. These results demonstrated that Na+ transport competed with H+ transport in NdR2, which was confirmed by the dominant H+ pump activity at pH ~5.7. Kinetic experiments using pyranine uncovered a transient H+ uptake, followed by an H+ release at the millisecond time scale in both Na+ and K+ solutions. Therefore, these NdR2 results may provide functional and kinetic insights into the ion transport mechanism in light-driven Na+ pumps.

Effects of ion interactions with a cholesterol-rich bilayer

Previous molecular dynamics (MD) simulations of ion-lipid interactions have focused on pure phospholipid bilayers. Many functional microdomains in membranes have a complex composition of cholesterol and phospholipids. Here, we reveal the distinctiveness of the interactions and the effects of the ions on a cholesterol-rich bilayer by performing MD simulations of a cholesterol-rich bilayer with a Na+/K+ mixture or a Na+/K+/Ca2+/Mg2+ mixture. The simulations reveal that Ca2+ maintains its dominant role in the interaction with the cholesterol-rich bilayer, but the binding affinity of Mg2+ to the cholesterol-rich bilayer is even weaker than the affinities of Na+ and K+, whereas its interaction with pure phospholipid bilayers is strong and is only slightly weaker than that of Ca2+. Additionally, it was found that the presence of additional divalent cations induces the headgroups of phospholipids to be more perpendicular to the membrane surface, reducing the lateral movement of lipids and slightly altering the ordering and packing of the cholesterol-rich bilayer, different from divalent cations, which strongly influence that ordering and packing of pure phospholipid bilayers. Therefore, this study indicates that cholesterol in the membrane could affect the interactions between membrane and cations. The findings could be helpful in understanding the biological processes relevant to regulation of cations in cholesterol-rich regions.

Heterogeneity of Signal Transduction by Na/K‐ATPase α Isoforms: Role of α3 Na/K‐ATPase

Na/K-ATPase is an ion transporting pump. The a1 subunit of Na/K-ATPase also forms a receptor complex by direct interaction with Src. Na/K-ATPase ligands like ouabain modulate this interaction and thereby activate various downstream protein and lipid kinases. The aim of this study was to test whether this signaling capability also exists in other a Na/K-ATPase isoforms in the absence of a1, focusing on α3 of Na/K-ATPase. To this end, a1 Na/K-ATPase knockdown pig kidney cells (PY-17) were transfected with α3 Na/K-ATPase to generate the first mammalian cell lines expressing virtually only α3 Na/K-ATPase (LM-α3-1). ATPase assay and Rb86+ uptake indicated that ion-pumping function in these cells is normal. These assays also showed that, compared to α1, α3 Na/K-ATPase has less affinity for Na+ (10.72± 3.1 and 27.7± 9.7 mM respectively) and has much more capacity to be activated when intracellular Na+ isincreased. This characteristic may explain the importance of α3 Na/K-ATPase in neuronal cells which are highly dependent on Na+ gradientfor action potential. In LM-α3-1, Na/K-ATPase–Src interaction was disrupted, and ouabain failed to stimulate Src. However, caveolin-1 expression was restored and ouabain-induced ERK and Akt activation was observed. Unlike in AAC-19 cells, ouabain-induced activation of ERK was PI3K- and PKC-dependent and not Src, as indicated by pharmacological inhibition studies. Finally, LM-α3-1 grew slower than AAC-19 but faster than PY-17 cells. Our results suggest that α3 Na/K-ATPase isoforms regulate signaling pathways differently from that of α1. Since α Na/K-ATPase isoforms have specific tissue-distributions, this may provide cell/tissue specific regulation. Figure 1. Possible signaling transduction mechanisms by isoforms of Na/K-ATPase

Effect of ENaC modulators on rat neural responses to NaCl.

The effects of small molecule ENaC activators N,N,N-trimethyl-2-((4-methyl-2-((4-methyl-1H-indol-3-yl)thio)pentanoyl)oxy)ethanaminium iodide (Compound 1) and N-(2-hydroxyethyl)-4-methyl-2-((4-methyl-1H-indol-3-yl)thio)pentanamide (Compound 2), were tested on the benzamil (Bz)-sensitive NaCl chorda tympani (CT) taste nerve response under open-circuit conditions and under ±60 mV applied lingual voltage-clamp, and compared with the effects of known physiological activators (8-CPT-cAMP, BAPTA-AM, and alkaline pH), and an inhibitor (ionomycin+Ca2+) of ENaC. The NaCl CT response was enhanced at −60 mV and suppressed at +60 mV. In every case the CT response (r) versus voltage (V) curve was linear. All ENaC activators increased the open-circuit response (ro) and the voltage sensitivity (κ, negative of the slope of the r versus V curve) and ionomycin+Ca2+ decreased ro and κ to zero. Compound 1 and Compound 2 expressed a sigmoidal-saturating function of concentration (0.25–1 mM) with a half-maximal response concentration (k) of 0.49 and 1.05 mM, respectively. Following treatment with 1 mM Compound 1, 8-CPT-cAMP, BAPTA-AM and pH 10.3, the Bz-sensitive NaCl CT response to 100 mM NaCl was enhanced and was equivalent to the Bz-sensitive CT response to 300 mM NaCl. Plots of κ versus ro in the absence and presence of the activators or the inhibitor were linear, suggesting that changes in the affinity of Na+ for ENaC under different conditions are fully compensated by changes in the apical membrane potential difference, and that the observed changes in the Bz-sensitive NaCl CT response arise exclusively from changes in the maximum CT response (rm). The results further suggest that the agonists enhance and ionomycin+Ca2+ decreases ENaC function by increasing or decreasing the rate of release of Na+ from its ENaC binding site to the receptor cell cytosol, respectively. Irrespective of agonist type, the Bz-sensitive NaCl CT response demonstrated a maximum response enhancement limit of about 75% over control value.

Desorption/ionization efficiencies of triacylglycerols and phospholipids via EASI‐MS

Knowledge of the major effects governing desorption/ionization efficiency is required for the development and application of ambient mass spectrometry. Although all triacylglycerols (TAG) have the same favorable protonation and cationization sites, their desorption/ionization efficiencies can vary dramatically during easy ambient sonic-spray ionization because of structural differences in the carbon chain. To quantify this somewhat surprising and drastic effect, we have performed a systematic investigation of desorption/ionization efficiencies as a function of unsaturation and length for TAG as well as for diacylglycerols, monoacylglycerols and several phospholipids (PL). Affinities for Na(+) as a function of unsaturation level have also been assayed via comprehensive metadynamics calculations to understand the influence of this phenomenon on the ionization efficiency. The results suggest that dipole-dipole interactions within a carbon chain tuned by unsaturation sites govern ionization efficiency of TAG and PL.

Crystal structure of a Na+-bound Na+,K+-ATPase preceding the E1P state

Na(+),K(+)-ATPase pumps three Na(+) ions out of cells in exchange for two K(+) taken up from the extracellular medium per ATP molecule hydrolysed, thereby establishing Na(+) and K(+) gradients across the membrane in all animal cells. These ion gradients are used in many fundamental processes, notably excitation of nerve cells. Here we describe 2.8 Å-resolution crystal structures of this ATPase from pig kidney with bound Na(+), ADP and aluminium fluoride, a stable phosphate analogue, with and without oligomycin that promotes Na(+) occlusion. These crystal structures represent a transition state preceding the phosphorylated intermediate (E1P) in which three Na(+) ions are occluded. Details of the Na(+)-binding sites show how this ATPase functions as a Na(+)-specific pump, rejecting K(+) and Ca(2+), even though its affinity for Na(+) is low (millimolar dissociation constant). A mechanism for sequential, cooperative Na(+) binding can now be formulated in atomic detail.

Angiotensin II-induced hypertension increases plasma membrane Na pump activity by enhancing Na entry in rat thick ascending limbs

Thick ascending limbs (TAL) reabsorb 30% of the filtered NaCl load. Na enters the cells via apical Na-K-2Cl cotransporters and Na/H exchangers and exits via basolateral Na pumps. Chronic angiotensin II (ANG II) infusion increases net TAL Na transport and Na apical entry; however, little is known about its effects on the basolateral Na pump. We hypothesized that in rat TALs Na pump activity is enhanced by ANG II-infusion, a model of ANG II-induced hypertension. Rats were infused with 200 ng·kg(-1)·min(-1) ANG II or vehicle for 7 days, and TAL suspensions were obtained. We studied plasma membrane Na pump activity by measuring changes in 1) intracellular Na (Nai) induced by ouabain; and 2) ouabain-sensitive oxygen consumption (QO2). We found that the ouabain-sensitive rise in Nai in TALs from ANG II-infused rats was 12.8 ± 0.4 arbitrary fluorescent units (AFU)·mg(-1)·min(-1) compared with only 9.9 ± 1.1 AFU·mg(-1)·min(-1) in controls (P < 0.024). Ouabain-sensitive oxygen consumption was 17 ± 5% (P < 0.043) greater in tubules from ANG II-treated than vehicle rats. ANG II infusion did not alter total Na pump expression, the number of Na pumps in the plasma membrane, or the affinity for Na. When furosemide (1.1 mg·kg(-1)·day(-1)) was coinfused with ANG II, no increase in plasma membrane Na pump activity was observed. We concluded that in ANG II-induced hypertension Na pump activity is increased in the plasma membrane of TALs and that this increase is caused by the chronically enhanced Na entry occurring in this model.

Na+/K+ ATPase 1 and 3 Isoforms Are Differentially Expressed in - and -Motoneurons

The Na(+)/K(+) ATPase (NKA) is an essential membrane protein underlying the membrane potential in excitable cells. Transmembrane ion transport is performed by the catalytic α subunits (α1-4). The predominant subunits in neurons are α1 and α3, which have different affinities for Na(+) and K(+), impacting on transport kinetics. The exchange rate of Na(+)/K(+) markedly influences the activity of the neurons expressing them. We have investigated the distribution and function of the main isoforms of the α subunit expressed in the mouse spinal cord. NKAα1 immunoreactivity (IR) displayed restricted labeling, mainly confined to large ventral horn neurons and ependymal cells. NKAα3 IR was more widespread in the spinal cord, again being observed in large ventral horn neurons, but also in smaller interneurons throughout the dorsal and ventral horns. Within the ventral horn, the α1 and α3 isoforms were mutually exclusive, with the α3 isoform in smaller neurons displaying markers of γ-motoneurons and α1 in α-motoneurons. The α3 isoform was also observed within muscle spindle afferent neurons in dorsal root ganglia with a higher proportion at cervical versus lumbar regions. We confirmed the differential expression of α subunits in motoneurons electrophysiologically in neonatal slices of mouse spinal cord. γ-Motoneurons were excited by bath application of low concentrations of ouabain that selectively inhibit NKAα3 while α-motoneurons were insensitive to these low concentrations. The selective expression of NKAα3 in γ-motoneurons and muscle spindle afferents, which may affect excitability of these neurons, has implications in motor control and disease states associated with NKAα3 dysfunction.

Enthalpy-Entropy of Cation Association with the Acetate Anion in Water.

Negatively charged carboxylate and phosphate groups on biomolecules have different affinity for Na(+) and K(+) ions. We performed molecular simulations and studied the pair potential of mean force between monovalent cations and the carboxylate group of the acetate anion in aqueous solution at 298 K. The simulations indicate that a larger affinity of Na(+) over K(+) in the contact ion pair (CIP) state is of entropic origin with the CIP state becoming increasingly populated at higher temperature. Differences between the osmotic activities of these two ions are however governed by interactions with acetate in the solvent-shared ion pair (SIP) state as was previously shown (Hess, B.; van der Vegt, N. F. A. Proc. Natl. Acad. Sci. U.S.A.2009, 106, 13296). SIP states with Na(+) are slightly more stable than SIP states with K(+), resulting in a smaller osmotic activity of sodium. We discuss the different affinities of Na(+) and K(+) in the SIP state in terms of an enthalpy-entropy reinforcement mechanism which involves a water-mediated hydrogen-bonding interaction between the oppositely charged ions. SIP states are enthalpically favorable and become decreasingly populated at higher temperature.

Kinetic Analysis of Gill (Na+,K+)-ATPase Activity in Selected Ontogenetic Stages of the Amazon River Shrimp, Macrobrachium amazonicum (Decapoda, Palaemonidae): Interactions at ATP- and Cation-Binding Sites

We investigated modulation by ATP, Mg²⁺, Na⁺, K⁺ and NH₄⁺ and inhibition by ouabain of (Na⁺,K⁺)-ATPase activity in microsomal homogenates of whole zoeae I and decapodid III (formerly zoea IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps, Macrobrachium amazonicum. (Na⁺,K⁺)-ATPase-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP (K(M) = 0.09 ± 0.01 mmol L⁻¹) of the decapodid III (Na⁺,K⁺)-ATPase, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na⁺,K⁺-ATPase activity by K⁺ also revealed a threefold greater affinity for K⁺ (K₀.₅ = 0.91 ± 0.04 mmol L⁻¹) in decapodid III than in other stages; NH₄⁺ had no modulatory effect. The affinity for Na⁺ (K₀.₅ = 13.2 ± 0.6 mmol L⁻¹) of zoea I (Na⁺,K⁺)-ATPase was fourfold less than other stages. Modulation by Na⁺, Mg²⁺ and NH₄⁺ obeyed cooperative kinetics, while K⁺ modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg²⁺ stimulation of ouabain-insensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg²⁺-stimulated ATPases other than (Na⁺,K⁺)-ATPase are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na⁺-ATPase may be involved in the ontogeny of osmoregulation in larval M. amazonicum. The NH₄⁺-stimulated, ouabain-insensitive ATPase activity seen in zoea I and decapodid III may reflect a stage-specific means of ammonia excretion since functional gills are absent in the early larval stages.

Influence of the ionic strength and solid/solution ratio on Ca(II)-for-Na+ exchange on montmorillonite. Part 1: Chemical measurements, thermodynamic modeling and potential implications for trace elements geochemistry

Na/Ca ion-exchange isotherms were performed on a Na-saturated montmorillonite for different constant normalities of the aqueous chloride solution and at two solid/solution ratios. The experimental data suggest that the affinity of Na(+) for the solid increases with total normality and m/V ratio and that a significant proportion of CaCl(+) is also sorbed. A thermodynamic modeling procedure with one sorption site and three sorbed cations (Na(+), Ca(2+) and CaCl(+)) was applied to interpret the data. We show that by accounting for the activities of aqueous species, the experimental data obtained at different total normalities for a given solid/solution ratio can be fitted using a unique set of selectivity coefficient values. However, when the m/V ratio is decreased from 25 to 2.5 g/L, an increase in the log Kc(Na(+)/Ca(2+)) of up to 0.4 ± 0.05 was required to interpret the data with a constant log Kc(Na(+)/CaCl(+)) value. The same behavior concerning the increase in the log Kc(Na(+)/Ca(2+)) with a m/V ratio decrease was observed when using a multi-site model taking into account the specific sorption of H(+). The results clearly indicate that the Na(+)/Ca(2+) selectivity coefficients strongly depend on the solid/solution ratio but are independent of the exchanger composition and the total normality of the solution. Such findings provide possible pitfalls when using selectivity coefficients obtained in diluted suspension to assess the sorption in compacted clayrocks and allow a better prediction of the sorption of trace elements in competition with major cations.