Apparent Affinity For Na Research Articles

Sodium pump regulation in health and disease.

The sodium-potassium ATPase (NKA) is the ion motive ATP-dependent transporter that establishes Na+ and K+ gradients across the cell membrane, facilitating many physiological processes. In the heart, NKA activity is regulated by phospholemman (PLM), which PLM inhibits NKA by reducing its apparent affinity for Na+. In this study, we used time-correlated single-photon counting, FRET-microscopy, molecular dynamics simulations, and electrophysiology to investigate the stoichiometry of the native NKA-PLM regulatory complex. Our data suggest a stoichiometry of the NKA-PLM complex composed of two α subunits, two β subunits, and decorated with two PLM regulators. Protein-protein docking and molecular dynamics simulations generated a structural model of the regulatory complex. This model was characterized by contacts between cytoplasmic domains of α subunits, between β subunit extracellular domains, and between the highly-conserved α subunits’ M3 helix and the N-termini of opposing β subunit. The helix M3 shows significant movement during the pump's reaction cycle and mutations in M3 are associated with several diseases. Here, we studied the G301R mutant that is unable to correctly localize in the plasma membrane when expressed alone, but a milder G301A mutant localizes correctly. The G301R localization was improved in the presence of a WT pump suggesting a dominant negative effect of this mutation. Our simulations of the dimeric complexes revealed decreased affinity of the Na+ in position II, which is unbound during the simulated time window. Surprisingly, Na+ binding was not affected in the simulated WT or G301A/R monomers. Two-electrode voltage clamp experiments revealed decreased affinity of G301A for external Na+ and ion leak from G301R mutants supporting our simulation data. This combination of ex vivo and in silico experiments provides an insight into the quaternary structure of the NKA-PLM regulatory complex and helps to explain the mechanism of G301A/R mutation pathology.

Functional analysis of mosquito and Drosophila Na+‐dependent cation‐chloride cotransporters

Dipteran Na+‐dependent cation‐chloride cotransporters (CCCs) group into two clades. Drosophila melanogaster Ncc69 is a bona fide Na+‐K+‐Cl− cotransporter and has a single ortholog in Aedes aegypti (aeNKCC1). Drosophila Ncc83 has not been physiologically characterized and has two orthologs in Ae. aegypti (aeCCC2 and aeCCC3). Previous work showed that oocytes expressing aeCCC2 have higher Na+ conductances than controls. Moreover, in flux assays using Li+ as a Na+ tracer, Li+ transport by aeCCC2 was not dependent upon extracellular Cl− and not inhibited by the loop diuretics furosemide or bumetanide. These are surprising results, since previously characterized CCCs are electroneutral, Cl−‐dependent, and sensitive to the loop diuretics. We have further characterized aeCCC2 and performed an initial analysis of Ncc83, using two‐electrode voltage clamp. Following a hypotonic pre‐incubation, changes in membrane potential upon Na+ replacement were 20 ± 3 mV in Ncc83 and 26 ± 4 mV in aeCCC2, approximately two‐fold greater than before swelling. Na+‐dependent currents in oocytes expressing aeCCC2 depended on extracellular Na+ with saturable Michaelis‐Menton kinetics. The apparent affinity for Na+ was 4.6 ± 0.5 mM (mean, SEM, n=5). To characterize the function of Ncc83 in Drosophila Malpighian tubules (MT), we developed procedures to assess the cation concentrations of secreted fluid in Ramsay secretion assays using cation‐exchange chromatography. The physiological cations were readily detectable in 10,000–50,000 fold dilutions of secreted fluid, and Li+ and Rb+ were also detectable when added to the bathing saline as tracers for Na+ and K+. In w118‐control flies, fluid secreted by MT measured using these procedures had a [K+] of 104.1 ± 8.8 mM (mean, SEM, n=7) and [Na+] of 61.2 ± 7.8 mM. In preliminary experiments, [K+] was 112.7 ± 1.3 mM (n=4) and [Na+] was 48.2 ± 4.1 mM in fluid secreted by MTs of flies with MT‐specific knockdown of Ncc83 using the GAL4‐UAS RNAi system. Further work is needed to determine the physiological roles of Ncc83 and aeCCC2. Funding: American Physiological Society Research Career Enhancement Award, NIH F33 GM131599, and Kenyon College.Support or Funding InformationFunding: American Physiological Society Research Career Enhancement Award, NIH F33 GM131599, and Kenyon College.

Determinants of Cation Permeation and Drug Sensitivity in Predicted Transmembrane Helix 9 and Adjoining Exofacial Re-entrant Loop 5 of Na+/H+ Exchanger NHE1

Mammalian Na(+)/H(+) exchangers (NHEs) regulate numerous physiological processes and are involved in the pathogenesis of several diseases, including tissue ischemia and reperfusion injuries, cardiac hypertrophy and failure, and cancer progression. Hence, NHEs are being targeted for pharmaceutical-based clinical therapies, but pertinent information regarding the structural elements involved in cation translocation and drug binding remains incomplete. Molecular manipulations of the prototypical NHE1 isoform have implicated several predicted membrane-spanning (M) helices, most notably M4, M9, and M11, as important determinants of cation permeation and drug sensitivity. Here, we have used substituted-cysteine accessibility mutagenesis and thiol-modifying methanethiosulfonate (MTS) reagents to further probe the involvement of evolutionarily conserved sites within M9 (residues 342-363) and the adjacent exofacial re-entrant loop 5 between M9 and M10 (EL5; residues 364-415) of a cysteine-less variant of rat NHE1 on its kinetic and pharmacological properties. MTS treatment significantly reduced the activity of mutants containing substitutions within M9 (H353C, S355C, and G356C) and EL5 (G403C and S405C). In the absence of MTS, mutants S355C, G403C, and S405C showed modest to significant decreases in their apparent affinities for Na(+) o and/or H(+) i. In addition, mutations Y370C and E395C within EL5, whereas failing to confer sensitivity to MTS, nevertheless, reduced the affinity for Na(+) o, but not for H(+) i. The Y370C mutant also exhibited higher affinity for ethylisopropylamiloride, a competitive antagonist of Na(+) o transport. Collectively, these results further implicate helix M9 and EL5 of NHE1 as important elements involved in cation transport and inhibitor sensitivity, which may inform rational drug design.

Conserved Asparagine Residue Located in Binding Pocket Controls Cation Selectivity and Substrate Interactions in Neuronal Glutamate Transporter

Transporters of the major excitatory neurotransmitter glutamate play a crucial role in glutamatergic neurotransmission by removing their substrate from the synaptic cleft. The transport mechanism involves co-transport of glutamic acid with three Na(+) ions followed by countertransport of one K(+) ion. Structural work on the archeal homologue Glt(Ph) indicates a role of a conserved asparagine in substrate binding. According to a recent proposal, this residue may also participate in a novel Na(+) binding site. In this study, we characterize mutants of this residue from the neuronal transporter EAAC1, Asn-451. None of the mutants, except for N451S, were able to exhibit transport. However, the K(m) of this mutant for l-aspartate was increased ∼30-fold. Remarkably, the increase for d-aspartate and l-glutamate was 250- and 400-fold, respectively. Moreover, the cation specificity of N451S was altered because sodium but not lithium could support transport. A similar change in cation specificity was observed with a mutant of a conserved threonine residue, T370S, also implicated to participate in the novel Na(+) site together with the bound substrate. In further contrast to the wild type transporter, only l-aspartate was able to activate the uncoupled anion conductance by N451S, but with an almost 1000-fold reduction in apparent affinity. Our results not only provide experimental support for the Na(+) site but also suggest a distinct orientation of the substrate in the binding pocket during the activation of the anion conductance.

Post-transcriptional Control of Na,K-ATPase Activity and Cell Growth by a Splice Variant of FXYD2 Protein with Modified mRNA

In kidney, FXYD proteins regulate Na,K-ATPase in a nephron segment-specific way. FXYD2 is the most abundant renal FXYD but is not expressed in most renal cell lines unless induced by hypertonicity. Expression by transfection of FXYD2a or FXYD2b splice variants in NRK-52E cells reduces the apparent Na(+) affinity of the Na,K-ATPase and slows the cell proliferation rate. Based on RT-PCR, mRNAs for both splice variants were expressed in wild type NRK-52E cells as low abundance species. DNA sequencing of the PCR products revealed a base alteration from C to T in FXYD2b but not FXYD2a from both untreated and hypertonicity-treated NRK-52E cells. The 172C→T sequence change exposed a cryptic KKXX endoplasmic reticulum retrieval signal via a premature stop codon. The truncation affected trafficking of FXYD2b and its association with Na,K-ATPase and blocked its effect on enzyme kinetics and cell growth. The data may be explained by altered splicing or selective RNA editing of FXYD2b, a supplementary process that would ensure that it was inactive even if transcribed and translated, in these cells that normally express only FXYD2a. 172C→T mutation was also identified after mutagenesis of FXYD2b by error-prone PCR coupled with a selection for cell proliferation. Furthermore, the error-prone PCR alone introduced the mutation with high frequency, implying a structural peculiarity. The data confirm truncation of FXYD2b as a potential mechanism to regulate the amount of FXYD2 at the cell surface to control activity of Na,K-ATPase and cell growth.

Increase in ouabain-sensitive K +-ATPase activity in hepatocellular carcinoma by overexpression of Na +,K +-ATPase α3-isoform

Na +,K +-ATPase is a housekeeping pump in virtually all animal cells. Recently, cardiac glycosides that inhibit Na +,K +-ATPase have been reported to be candidate for novel anticancer drug. Here, we investigated clinical significance of Na +,K +-ATPase α1-isoform (α1NaK), α2-isoform (α2NaK) and α3-isoform (α3NaK) in hepatocellular carcinoma (HCC). Interestingly, the expression levels of α3NaK protein in HCC tissues were significantly higher than those in the accompanying non-tumor tissues, whereas no significant increases in expression of α1NaK and α2NaK proteins were observed in HCC compared to non-tumor tissues. The ouabain (10 μM)-sensitive K +-ATPase activities (Na +,K +-ATPase activities) in the membrane fraction from HCC tissue were significantly higher than those from non-tumor tissues. The Na +,K +-ATPase activity was positively and significantly correlated with the expression level of α3NaK. Apparent affinity for Na + in the Na +,K +-ATPase activity in HCC tissues was significantly lower than that in non-tumor tissues, consistent with an elevated expression of α3NaK relative to α1NaK. Our results suggest that overexpression of α3NaK increases the Na +,K +-ATPase activity of HCC cells.

FXYD2 and Na,K-ATPase Expression in Isolated Human Proximal Tubular Cells: Disturbed Upregulation on Renal Hypomagnesemia?

Autosomal dominant renal hypomagnesemia (OMIM 154020), associated with hypocalciuria, has been linked to a 121G to A mutation in the FXYD2 gene. To gain insight into the molecular mechanisms linking this mutation to the clinical phenotype, we studied isolated proximal tubular cells from urine of a patient and a healthy subject. Cells were immortalized and used to assess the effects of hypertonicity-induced overexpression of FXYD2 on amount, activity and apparent affinities for Na+, K+ and ATP of Na,K-ATPase. Both cell lines expressed mRNA for FXYD2a and FXYD2b, and patient cells contained both the wild-type and mutated codons. FXYD2 protein expression was lower in patient cells and could be increased in both cell lines upon culturing in hyperosmotic medium but to a lesser extent in patient cells. Similarly, hyperosmotic culturing increased Na,K-ATPase protein expression and ATP hydrolyzing activity but, again, to a lesser extent in patient cells. Apparent affinities of Na,K-ATPase for Na+, K+ and ATP did not differ between patient and control cells or after hyperosmotic induction. We conclude that human proximal tubular cells respond to a hyperosmotic challenge with an increase in FXYD2 and Na,K-ATPase protein expression, though to a smaller absolute extent in patient cells.

Truncation Of The Na/K Pump's C-terminus Attenuates Voltage-dependent Binding Of External Na+ By Destabilizing Na+ Occlusion

In the recent Na/K pump crystal structure the C-terminus directly contacts the transmembrane region, and deletion of its last five residues (KETYY in pig Na/K pumps) strongly lowered (26-fold) apparent affinity for Na+-dependent phosphorylation, which reflects cytoplasmic-side Na+ binding (Morth et al., 2007). Here we use two-microelectrode voltage-clamp recordings in Xenopus oocytes to investigate the consequence of this C-terminal truncation on the external Na+ dependence of steady-state, and transient, Na/K pump-mediated currents. The corresponding deletion (ΔKESYY in Xenopus α1) was introduced in Xenopus α1β3 pumps made ouabain resistant by either Q120R-N131D (RD) or C113Y (C-Y) mutations. All extracellular solutions then contained 1 μM ouabain to silence the endogenous Xenopus Na/K pumps, and the mutant pump currents were determined by addition of 10 mM ouabain. Inhibition of steady outward Na/K pump current (at 15 mM external K+) by 125 mM extracellular Na+ at negative voltages was weaker in both RD and C-Y pumps than in wild-type Xenopus Na/K pumps, but was almost completely absent (at -120 mV) in RD-ΔKESYY or C-Y-ΔKESYY pumps. Transient, external Na+-dependent, pump currents were similarly measured as 10 mM ouabain-sensitive currents, but in K+-free solution. The voltage dependence of these transient charge movements was shifted to more negative potentials for RD-ΔKESYY and C-Y-ΔKESYY pumps compared to RD or C-Y pumps, similar to the shift caused in wild-type pumps by decreasing the extracellular Na+ concentration. Together, these results suggest that the C-terminal deletion lowers the apparent affinity with which extracellular Na+ ions bind to the Na/K pump to reverse Na+-ion deocclusion. The C-terminus therefore seems important for stabilizing the occluded conformation of the phosphorylated Na/K pump containing 3 bound Na+ ions. [NIH HL36783]

Role of Ser-340 and Thr-341 in Transmembrane Domain IX of the Na+/Proline Transporter PutP of Escherichia coli in Ligand Binding and Transport

The Na+/solute symporter family comprises more than 400 members of pro- and eukaryotic origin. Using the Na+/proline transporter PutP of Escherichia coli as a model, the role of two conserved residues, Ser-340 and Thr-341, is investigated to obtain insights into the mechanism of transport catalyzed by members of this family. Substitution of these amino acids alters the transport kinetics of cells and proteoliposomes containing the PutP variants significantly. In particular, the apparent affinities for Na+ and Li+ are reduced by 2 orders of magnitude or more. Also proline binding is affected, albeit to a lesser extent than ion binding. Thereby, the presence of a hydroxyl group at position 341 is essential for high affinity ligand binding. Furthermore, Cys placed at position 340 or 341 reacts with sulfhydryl reagents of different polarity, indicating accessibility from the water phase. In addition, Cys cross-linking suggests proximity of the residues to other amino acids previously shown to be crucial for ligand binding. For these reasons it is suggested that Ser-340 and Thr-341 are located in a ligand translocation pathway. Furthermore, it is proposed that the side chain of Thr-341 directly participates in Na+ binding.

Evidence for allosteric regulation of pH-sensitive System A (SNAT2) and System N (SNAT5) amino acid transporter activity involving a conserved histidine residue.

System A and N amino acid transporters are key effectors of movement of amino acids across the plasma membrane of mammalian cells. These Na+-dependent transporters of the SLC38 gene family are highly sensitive to changes in pH within the physiological range, with transport markedly depressed at pH 7.0. We have investigated the possible role of histidine residues in the transporter proteins in determining this pH-sensitivity. The histidine-modifying agent DEPC (diethyl pyrocarbonate) markedly reduces the pH-sensitivity of SNAT2 and SNAT5 transporters (representative isoforms of System A and N respectively, overexpressed in Xenopus oocytes) in a concentration-dependent manner but does not completely inactivate transport activity. These effects of DEPC were reversed by hydroxylamine and partially blocked in the presence of excess amino acid substrate. DEPC treatment also blocked a reduction in apparent affinity for Na+ (K0.5Na+) of the SNAT2 transporter at low external pH. Mutation of the highly conserved C-terminal histidine residue to alanine in either SNAT2 (H504A) or SNAT5 (H471A) produced a transport phenotype exhibiting reduced, DEPC-resistant pH-sensitivity with no change in K0.5Na+ at low external pH. We suggest that the pH-sensitivity of these structurally related transporters results at least partly from a common allosteric mechanism influencing Na+ binding, which involves an H+-modifier site associated with C-terminal histidine residues.

Carboxyl-terminal hydrophilic tail of a NhaP type Na +/H + antiporter from cyanobacteria is involved in the apparent affinity for Na + and pH sensitivity

Little information is available on the C-terminal hydrophilic tails of prokaryotic Na +/H + antiporters. To address functional properties of the C-terminal tail, truncation mutants in this domain were constructed. Truncation of C-terminal amino acid residues of NhaP1 type antiporter from Synechocystis PCC6803 (SynNhaP1) did not change the V max values, but increased the K m values for Na + and Li + about 3 to 15-fold. Truncation of C-terminal tail of a halotolerant cyanobacterium Aphanothece halophytica (ApNhaP1) significantly decreased the V max although it did not alter the K m values for Na +. The C-terminal part of SynNhaP1 was expressed in E. coli and purified as a 16 kDa soluble protein. Addition of purified polypeptide to the membrane vesicles expressing the C-terminal truncated SynNhaP1 increased the exchange activities. Change of Glu519 and Glu521 to Lys in C-terminal tail altered the pH dependence of Na +/H + and Li +/H + exchange activities. These results indicate that the specific acidic amino acid residues at C-terminal domain play important roles for the K m and the pH dependence of the exchange activity.

Thr-774 (Transmembrane Segment M5), Val-920 (M8), and Glu-954 (M9) Are Involved in Na+ Transport, and Gln-923 (M8) Is Essential for Na,K-ATPase Activity

The highly conserved amino acids of rat Na,K-ATPase, Thr-774 in the transmembrane helices M5, Val-920 and Gln-923 in M8, and Glu-953 and Glu-954 in M9, the side chains of which appear to be in close proximity, were mutated, and the resulting proteins, T774A, E953A/K, and E954A/K, V920E and Q923N/E/D/L, were expressed in HeLa cells. Ouabain-resistant cell lines were obtained from T774A, V920E, E953A, and E954A, whereas Q923N/E/D/L, E953K, and E954K could only be transiently expressed as fusion proteins with an enhanced green fluorescent protein. The apparent K0.5 values for Na+, as estimated by the Na+-dependent phosphoenzyme formation (K0.5(Na,EP)) or Na,K-ATPase activity (K(0.5)(Na,ATPase)), were increased by around 2 approximately 8-fold in the case of T774A, V920E, and E954A. The apparent K0.5 values for K+, as estimated by the Na,K-ATPase (K0.5(K,ATPase)) or p-nitrophenylphosphatase activity (K0.5(K,pNPPase)), were affected only slightly by the 3 mutations, except that V920E showed a 1.7-fold increase in the K0.5(K,ATPase). The apparent K0.5 values for ATP (K0.5(EP)), as estimated by phosphorylation (a high affinity ATP effect), were increased by 1.6 approximately 2.6-fold in the case of T774A, V920E, and E954A. Those estimated by Na,K-ATPase activity (K0.5(ATPase)) and ATP-induced inhibition (K(i,0.5)(pNPPase)) of K-pNPPase activity (low affinity ATP effects) were, respectively, increased by 1.8-fold and unchanged in the case of T774A but decreased by 2- and 4.8-fold in the case of V920E and were slightly changed and increased by 1.7-fold in the case of E954A. The E953A showed little significant change in the apparent affinities. These results suggest that Gln-923 in M8 is crucial for the active transport of Na+ and/or K+ across membranes and that the side chain oxygen atom of Thr-774 in M5, the methyl group(s) of Val-920 in M8, and the carboxyl oxygen(s) of Glu-954 in M9 mainly play some role in the transport of Na+ and also in the high and low affinity ATP effects rather than the transport of K+.

FXYD3 (Mat-8), a New Regulator of Na,K-ATPase

Four of the seven members of the FXYD protein family have been identified as specific regulators of Na,K-ATPase. In this study, we show that FXYD3, also known as Mat-8, is able to associate with and to modify the transport properties of Na,K-ATPase. In addition to this shared function, FXYD3 displays some uncommon characteristics. First, in contrast to other FXYD proteins, which were shown to be type I membrane proteins, FXYD3 may have a second transmembrane-like domain because of the presence of a noncleavable signal peptide. Second, FXYD3 can associate with Na,K- as well as H,K-ATPases when expressed in Xenopus oocytes. However, in situ (stomach), FXYD3 is associated only with Na,K-ATPase because its expression is restricted to mucous cells in which H,K-ATPase is absent. Coexpressed in Xenopus oocytes, FXYD3 modulates the glycosylation processing of the beta subunit of X,K-ATPase dependent on the presence of the signal peptide. Finally, FXYD3 decreases both the apparent affinity for Na+ and K+ of Na,K-ATPase.

Residues within transmembrane domains 4 and 6 of the Na,K-ATPase alpha subunit are important for Na+ selectivity.

The Na,K- and H,K-ATPases are plasma membrane enzymes responsible for the active exchange of extracellular K(+) for cytoplasmic Na(+) or H(+), respectively. At present, the structural determinants for the specific function of these ATPases remain poorly understood. To investigate the cation selectivity of these ATPases, we constructed a series of Na,K-ATPase mutants in which residues in the membrane spanning segments of the alpha subunit were changed to the corresponding residues common to gastric H,K-ATPases. Thus, mutants were created with substitutions in transmembrane domains TM1, TM4, TM5, TM6, TM7, and TM8 independently or together (designated TMAll). The function of each mutant was assessed after coexpression with the beta subunit in Sf-9 cells using baculoviruses. The enzymatic properties of TM1, TM7, and TM8 mutants were similar to the wild-type Na,K-ATPase, and while TM5 showed modest changes in apparent affinity for Na(+), TM4, TM6, and TMAll displayed an abnormal activity. This resulted in a Na(+)-independent hydrolysis of ATP, a 2-fold higher K(0.5) for Na(+) activation, and the ability to function at low pH. These results suggest a loss of discrimination for Na(+) over H(+) for the enzymes. In addition, TM4, TM6, and TMAll mutants exhibited a 1.5-fold lower affinity for K(+) and a 4-5-fold decreased sensitivity to vanadate. Altogether, these results provide evidence that residues in transmembrane domains 4 and 6 of the alpha subunit of the Na,K-ATPase play an important role in determining the specific cation selectivity of the enzyme and also its E1/E2 conformational equilibrium.

Local conformational changes in the Vibrio Na+/galactose cotransporter.

Na(+) and sugar transport by cotransporters (symporters) is thought to occur as a series of ordered ligand-induced conformational changes. To localize these conformational changes in a bacterial Na(+)/galactose cotransporter, we have employed a combination of cysteine-scanning and fluorescence techniques. Single or pairs of cysteine residues were introduced into the external face of a cysteine-less Vibrio parahaemolyticus sodium/glucose cotransporter for expression in Escherichia coli, and each transporter was purified using affinity chromatography. All the mutant proteins retained transport activity in bacteria and proteoliposomes. Each mutant was exposed to two different fluorescence reagents, ThioGlo3 or pyrene maleimide, that are essentially nonfluorescent until they react with a thiol. Fluorescence was recorded as a function of time and ligand concentrations. The reagents specifically labeled six of the seven cysteine mutants, but only in Cysteine 423 was the fluorescence affected by ligands. The rate of labeling of Cys423 by ThioGlo3 or pyrene maleimide was reduced by D-galactose in Na(+) buffer. Furthermore, the fluorescence of Thioglo3-labeled Cys423 was quenched by D-galactose, but only in the presence of Na(+). This quench was not accompanied by a Stokes shift and was not produced by nontransported sugars, e.g., L-glucose. Reducing the sodium concentration from 200 to 10 mM decreased the apparent affinity for d-galactose without altering the maximum quench with saturating D-galactose. Reducing the galactose concentration from 20 to 0.5 mM reduced both the apparent affinity for Na(+) and the maximum quench at saturating Na(+). These results suggest an ordered reaction scheme with Na(+) binding first. The fluorescence results with ThioGlo3-labeled Cys423 indicate that conformational changes underlying Na(+)/galactose cotransport occur at or near the extracellular domain between transmembrane helices 10 and 11.

Residues in the Extracellular Loop 4 Are Critical for Maintaining the Conformational Equilibrium of the γ-Aminobutyric Acid Transporter-1

We mutated residues Met345 and Thr349 in the rat gamma-aminobutyric acid transporter-1 (GAT-1) to histidines (M345H and T349H). These two residues are located four amino acids apart at the extracellular end of transmembrane segment 7 in a region of GAT-1 that we have previously suggested undergoes conformational changes critical for the transport process. The two single mutants and the double mutant (M345H/T349H) were expressed in Xenopus laevis oocytes, and their steady-state and presteady-state kinetics were examined and compared with wild type GAT-1 by using the two-electrode voltage clamp method. Oocytes expressing M345H showed a decrease in apparent GABA affinity, an increase in apparent affinity for Na+, a shift in the charge/voltage (Q/Vm) relationship to more positive membrane potentials, and an increased Li+-induced leak current. Oocytes expressing T349H showed an increase in apparent GABA affinity, a decrease in apparent Na+ affinity, a profound shift in the Q/Vm relationship to more negative potentials, and a decreased Li+-induced leak current. The data are consistent with a shift in the conformational equilibrium of the mutant transporters, with M345H stabilized in an outward-facing conformation and T349H in an inward-facing conformation. These data suggest that the extracellular end of transmembrane domain 7 not only undergoes conformational changes critical for the translocation process but also plays a role in regulating the conformational equilibrium between inward- and outward-facing conformations.

Influence of ionic strength on sodium-calcium exchange of two temperate climate soils

Salt-affected soils have been studied extensively with respect to their Na–Ca exchange properties. These studies have focused on soil environments of the arid West. However, because of irrigation and oil well brine discharges in the temperate region of the U.S. there is need to understand sodicity behavior of such soils. In this study, two Kentucky soils (Pembroke and Uniontown) at the 0–10 cm depth were studied to evaluate the influence of ionic strength (I) and sodium adsorption ratio (SAR) on cation selectivity coefficients. The data showed that both soils exhibit at least two classes of exchange sites and in general the apparent affinity for Na+ increased when solution ionic strength increased. Furthermore, both soils under all three ionic strengths tested showed greater affinity for Na+ than the average agricultural saline soil of the arid West. The data suggested the need for establishing critical salt dispersion thresholds for temperate climate soils and developing effective brine management approaches.

Replacement of Several Single Amino Acid Side Chains Exposed to the Inside of the ATP-binding Pocket Induces Different Extents of Affinity Change in the High and Low Affinity ATP-binding Sites of Rat Na/K-ATPase

To investigate the relationship between the high and the low affinity ATP-binding site, which appears during the Na(+)/K(+)-ATPase reaction, four amino acids were mutated, the side chains of which are exposed to inside of the ATP-binding pocket. Six mutants, F475Y, K480A, K480E, K501A, K501E, and R544A, where the numbers correspond to the pig Na(+)/K(+)-ATPase alpha-chain, were expressed in HeLa cells. The apparent affinities were determined by high affinity ATP-dependent phosphorylation and by the low affinity activation of Na(+)/K(+)-ATPase or low affinity ATP inhibition of K(+)-para-nitrophenylphosphatase (pNPPase). For the mutants K480A and K501A, little affinity change was detected for either the high affinity or the low affinity effect. In contrast, the other four mutants reduced both apparent affinities. Strikingly, R544A had a 30-fold greater effect on the high affinity ATP site than the low affinity site. For the F475Y mutant, it is likely that there was a greater effect on the low affinity site than the high affinity site, but for both F475Y and K480E the affinity for the low affinity ATP effect was reduced so much that it was not possible to estimate a K(0.5). However, both the affinities for the K480E were reduced to approximately 1/20. The turnover number of the Na(+)/K(+)-ATPase and the apparent affinity for Na(+) and pNPP was reduced slightly or not at all for these mutants, but the turnover number of K(+)-pNPPase and the apparent affinity for K(+) were increased. These and other data suggest the presence of only one ATP-binding site, which can change its conformation to accept ATP with a high and low affinity. The requirement of Arg-544 and possibly Lys-501 is more important in forming a high affinity ATP binding conformation, and Phe-475 and possibly Lys-480 are more important in forming the low affinity ATP binding conformation.

Influence of voltage and extracellular Na(+) on amiloride block and transport kinetics of rat epithelial Na(+) channel expressed in Xenopus oocytes.

We expressed the three subunits of the epithelial amiloride-sensitive Na(+) channel (ENaC) from rat distal colon heterologously in oocytes of Xenopus laevis and analysed blocker-induced fluctuations in current using conventional dual-microelectrode voltage-clamp. To minimize Na(+) accumulation we performed all experiments in low-Na(+) solutions (15 mM). Noise analysis revealed that control or ENaC-injected oocytes did not exhibit spontaneous relaxation noise. However, in ENaC-expressing oocytes, amiloride induced a distinct Lorentzian component in the power density spectra. With three amiloride concentrations and a linear analysis of the respective changes in the corner frequency f(c) (2 pi f(c) plot) we determined the rate constants k(on) and k(off) for the amiloride-ENaC interaction. At a clamp potential (V(m)) of -60 mV k(on) was 80.8 +/- 5.1 microM(-1) s(-1) and k(off) 15.4 +/- 4.2 s(-1). The half-maximal blocker concentration (K(mic,ami)) was 0.19 microM (V(m)=-60 mV). While k(on) was voltage-independent in the range -50 to -100 mV, k(off) and K(mic,ami) decreased significantly with increasing membrane hyperpolarization, resulting in an increased affinity of amiloride for its binding site on ENaC. Increasing extracellular [Na(+)] ([Na(+)](o)) led to saturation of ENaC. Subsequent noise analysis revealed that single-channel current increased non-linearly with [Na(+)](o) and that saturation was not due to a reduction in the number of open channels. The apparent affinity of Na(+) for its binding site on the channel was voltage dependent and increased with hyperpolarization. Noise analysis revealed that k(on) and k(off) for amiloride decreased with increasing [Na(+)](o), while the affinity of the amiloride-binding site did not change. These findings show that the affinity of rat intestinal ENaC for amiloride is voltage dependent and is influenced non-competitively by [Na(+)](o), indicating that Na(+) and amiloride do not compete for the same binding site at the channel.

Functional characterization of a Na+-phosphate cotransporter (NaPi-II) from zebrafish and identification of related transcripts.

1. We report the molecular identification of a Na+-Pi (inorganic phosphate) cotransport system of the NaPi-II protein family from zebrafish intestine. Following a PCR-related strategy, a DNA fragment from intestine-derived RNA was isolated. Rapid amplification of cDNA ends (3'- and 5'-RACE) resulted in the complete sequence (2607 bp) containing an open reading frame of 1893 bp. 2. The NaPi-II-related protein was expressed in Xenopus laevis oocytes and the resulting transport activity was analysed by electrophysiological means. The apparent Km for Pi was 250 microM (96 mM Na+, -60 mV), and voltage-dependent binding of Na+ exhibited a Km of 67.1 mM (1 mM Pi, -60 mV). 3. Interestingly, the overall transport activity was almost insensitive to changes in the holding potential. The apparent affinity for Na+ decreased under hyperpolarizing conditions, whereas Pi binding showed no voltage dependence. Transport activity was inhibited at low pH, which is characteristic for renal NaPi-II isoforms. 4. The expression of the NaPi-II-related isoform was addressed by reverse-transcription PCR. The mRNA could be detected in intestine, liver, eye and kidney. Unexpectedly, a second NaPi-II-related isoform was identified and found to be expressed in kidney, intestine, liver, brain, eye and prominently in testis. In addition, a shorter amplicon was demonstrated to be an antisense transcript related to the NaPi-II intestinal isoform.