Charge translocation by the Na+/K+-ATPase investigated on solid supported membranes: rapid solution exchange with a new technique

Adsorption of Na+/K+-ATPase containing membrane fragments from pig kidney to lipid membranes allows the detection of electrogenic events during the Na+/K+-ATPase reaction cycle with high sensitivity and time resolution. High stability preparations can be obtained using solid supported membranes (SSM) as carrier electrodes for the membrane fragments. The SSMs are prepared using an alkanethiol monolayer covalently linked to a gold surface on a glass substrate. The hydrophobic surface is covered with a lipid monolayer (SAM, self-assembled monolayer) to obtain a double layer system having electrical properties similar to those of unsupported bilayer membranes (BLM). As we have previously shown (, Biophys. J. 64:384-391), the Na+/K+-ATPase on a SSM can be activated by photolytic release of ATP from caged ATP. In this publication we show the first results of a new technique which allows rapid solution exchange at the membrane surface making use of the high mechanical stability of SSM preparations. Especially for substrates, which are not available as a caged substance-such as Na+ and K+-this technique is shown to be capable of yielding new results. The Na+/K+-ATPase was activated by rapid concentration jumps of ATP and Na+ (in the presence of ATP). A time resolution of up to 10 ms was obtained in these experiments. The aim of this paper is to present the new technique together with the first results obtained from the investigation of the Na+/K+-ATPase. A comparison with data taken from the literature shows considerable agreement with our experiments.     

Kinetics of conformational changes associated with potassium binding to and release from Na+/K(+)-ATPase

Previous studies have demonstrated that embryonal carcinoma (EC) cells express both fibroblast growth factor-4 (FGF-4) and FGF receptors. It has also been established that differentiation of EC cells represses the expression of the FGF-4 gene. Currently, the role of FGF-4 in the growth and differentiation of EC cells is unclear. In this study, we examined whether the differentiation of EC cells requires the repression of FGF-4 expression. To address this and related questions, F9 EC cells were transfected with an expression vector that uses the human beta-actin promoter to drive the constitutive expression of recombinant FGF-4. Unlike their untransfected counterparts, F9 EC cells transfected with this plasmid continue to produce recombinant FGF-4 after they differentiate. However, constitutive expression of this growth factor does not block morphological differentiation of the cells, nor does it alter the expression of six genes regulated by the differentiation of EC cells. Constitutive expression of recombinant FGF-4 also did not noticeably alter the growth of the transfected F9 EC cells before or after differentiation. Furthermore, unlike immortalized fibroblasts, which are known to grow in soft agar after transfection with FGF-4 expression plasmids, continued expression of recombinant FGF-4 activity did not enhance the ability of the EC-derived differentiated cells to form colonies in soft agar. These findings argue that continuous expression of recombinant FGF-4 activity does not block the differentiation of EC cells and that repression of the FGF-4 gene after EC cells differentiate does not appear, on its own, to be responsible for the loss of tumorigenicity that accompanies the differentiation of EC cells.     

Fluoresceinyl-ethylenediamine-ouabain detects an acidic environment in the cardiac glycoside binding site of Na+/K+-ATPase

To probe the pH value in the microenvironment of the cardiac glycoside-binding site of Na+/K+-ATPase, pH-sensitive fluorescent derivatives of ouabain were synthesized. The fluoresceinyl derivative of ethylenediamino-ouabain (FEDO) had a pKs of 6.0 and showed a H+-dependent fluorescence change, when its ratio of excitation at 490 nm/450 nm was recorded at 530 nm. Binding of FEDO inactivated Na+/K+-ATPase at 37 degrees C and pH 7.25 in a slow time-dependent process under the conditions of backdoor phosphorylation with k(on) of 891 s(-1) M(-1). The complex dissociated with k(on) of 0.35 x 10(-3) s(-1) resulting in a Kd value of 0.4 microM for the FEDO x enzyme complex. Binding of FEDO was associated with a decrease of the excitatory fluorescence ratio at 490 nm/450 nm which could be used to convert this change into a pH value. A pH value of 5.1 +/- 0.2 was calculated to exist in the microenvironment of the FEDO x enzyme complex. This pH value was independent of the pH of the incubation medium used to form the FEDO x enzyme complex. Analysis of the accessibility of the fluorophore in the FEDO x enzyme complex to the dynamic quencher potassium iodide detected a decrease of the Stern-Volmer constant from 6.2 mM(-1) (free FEDO) to 1.5 mM(-1) (FEDO x enzyme complex) indicating thereby a limited accessibility of the fluorophore to anions. Analysis of the microenvironment of the fluorescein residue of the FEDO x enzyme complex by measurements of the anisotropy and the fluorescence half-life time revealed that both processes differed significantly when H2O was replaced by D2O. We conclude, therefore, that a pH of 5.1 +/- 0.2 exists in the vicinity of ouabain that is hidden in the depth of the receptor site when the ouabain receptor complex has been formed.     

Ligand-sensitive interactions among the transmembrane helices of Na+/K+-ATPase

An extensively trypsin-digested Na+/K+-ATPase, which retains the ability to bind Na+, K+, and ouabain, consists of four fragments of the alpha-subunit that contain all 10 transmembrane alpha domains, and the beta-subunit, a fraction of which is cleaved at Arg142-Gly143. In previous studies, we solubilized this preparation with a detergent and mapped the relative positions of several transmembrane helices of the subunits by chemical cross-linking. To determine if these detected helix-helix proximities were representative of those existing in the bilayer prior to solubilization, we have now done similar studies on the membrane-bound preparation of the same digested enzyme. After oxidative sulfhydryl cross-linking catalyzed by Cu2+-phenanthroline, two prominent products were identified by their mobilities and the analyses of their N termini. One was a dimer of a 11-kDa alpha-fragment containing the H1-H2 helices and a 22-kDa alpha-fragment containing the H7-H10 helices. This dimer seemed to be the same as that obtained in the solubilized preparation. The other product was a trimer of the above two alpha-fragments and that fraction of beta whose extracellular domain was cleaved at Arg142-Gly143. This product was different from a similar one of the solubilized preparation in that the latter contained the predominant fraction of beta without the extracellular cleavage. The cross-linking reactions of the membrane preparation, but not those of the solubilized one, were hindered specifically by Na+, K+, and ouabain. These findings indicate that (a) the H1-H2 transmembrane helices of alpha are adjacent to some of its H7-H10 helices both in solubilized and membrane-bound states, (b) the alignment of the residues of the single transmembrane helix of beta with the interacting H1-H2 and H7-H10 helices of alpha is altered by detergent solubilization and by structural changes in the extracellular domain of beta, and (c) the three-dimensional packing of the interacting transmembrane helices of alpha and beta are regulated by the specific ligands of the enzyme.     

Amyloid beta-peptides inhibit Na+/K+-ATPase: tissue slices versus primary cultures

BACKGROUND: Urinary bladder augmentation is gaining popularity for the treatment of dysfunctional bladders in renal transplant patients. Although reported cases of adult and pediatric transplants into the augmented bladder have been favorable, the potential risk of urinary tract infection and graft failure under immunosuppression is still disputable. We report our experiences with 4 patients who underwent renal transplantation into an augmented bladder. METHODS: Between 1971 and 1996, 1275 renal transplants were performed at our institution. Of these transplants, 4 patients underwent renal transplantation into an augmented urinary bladder. Augmentation cystoplasty was performed before transplantation in 3 patients and 7 years after transplantation in the other patient. The bladder was augmented with an ileal segment in 3 patients and a ureter in the fourth patient. Graft function was assessed by the serum creatinine level. Fluorocystometrograms were performed in all patients at fixed intervals. RESULTS: Posttransplant renal function was satisfactory overall and no patient exhibited proteinuria. All patients except 1 acquired a large capacity low pressure bladder and remained continent with clean intermittent catheterization. One patient who underwent ureterocystoplasty is still incontinent because of his relatively small bladder capacity. Posttransplant pyelonephritis was documented in 3 patients during the follow-up period, but no other complications were observed. CONCLUSIONS: Our study demonstrates that renal transplantation into extensively reconstructed bladders can be safely performed with good success. Although urinary tract infection is a major consideration, we recommend pretransplant reconstruction not only to preserve graft function, but also to achieve urinary continence.     

Nucleotide binding to IAF-labelled Na+/K(+)-ATPase measured by steady state fluorescence quenching by TNP-ADP

Nucleotide binding to 5-iodoacetamidofluorescein (IAF) labelled Na+/K(+)-ATPase was measured by steady state fluorescence quenching of the fluorescein label via energy transfer to trinitrophenyl (TNP) labelled nucleotide. TNP-nucleotides are valuable probes of nucleotide binding to ATPases. Interpretation of these and other experiments in our laboratory using TNP-nucleotides with the Na+/K(+)-ATPase rely on having a good model for the interaction of TNP-nucleotide with the enzyme. Sets of fluorescence quenching curves obtained by titrating the enzyme with TNP-ADP in the presence of various concentrations of ADP could not be adequately modelled using a simple model with a single nucleotide binding site. Therefore, we compare various models which allow for additional TNP-nucleotide binding to the enzyme. In the two-site model, the additional binding is to a second specific site for which TNP-nucleotide and unlabelled nucleotide compete. In two other models, the additional binding (in one case saturable, and in the other case non-saturable) of TNP-nucleotide is not blocked by or affected by unlabelled nucleotide, and is, therefore, referred to as non-specific binding of the TNP-nucleotide. The goal of this work is to determine which of the distinctly different physical pictures associated with these models most accurately describes the interaction of TNP-nucleotide with the enzyme. We find that the interaction of TNP-ADP with IAF-labelled Na+/K(+)-ATPase is best described by a model in which there are two classes of binding: TNP-ADP and ADP compete for a specific binding site with dissociation binding constants of 0.13 microM for TNP-ADP and 2.0 microM for ADP; and non-saturable non-specific binding of TNP-ADP.     

Cooperative binding of the organophosphate paraoxon to the (Na+ + K+)-ATPase

Paraoxon, the main active metabolite of the organophosphorus insecticide parathion, exerted a dose-dependent inhibitory effect on the activity of pig kidney (Na+ + K+)-ATPase contained in microsomal fraction and purified from it. Substrate kinetics studies revealed the existence of two active sites with high and low affinity to ATP. The Dixon analysis of the mode of the inhibition indicated its noncompetitive character. The purified enzyme was more affected than enzyme contained in the microsomal fraction. The inhibition constant Ki ranged from 73 to 245 microM depending on the type of preparation. The Hill coefficient (n) fulfilled the relationship 1 < n < 3. These properties of the interaction suggest the cooperative binding of paraoxon to the enzyme. An indirect mechanism of the interaction was proposed: paraoxon could inhibit the activity of the (Na+ + K+)-ATPase by excluding the enzyme protein from its normal lipid milieu.     

E31-K352, the minimal cation binding moiety of Na+,K(+)-ATPase

While examining an imported Indonesian mangrove monitor, Varanus indicus (Reptilia: Sauria: Varanidae), for helminths, a new species of Hastospiculum was collected and is described as Hastospiculum spiralis n. sp. This species differs from all other members of the genus in caudal papillae number and arrangement, a pair of large cephalic papillae, and a spirally twisted left spicule in males. Additionally, H. spiralis n. sp. differs from certain Hastospiculum species by the right and left spicule lengths, egg shape, and the final host.     

Inhibition of rat Na+/K+-ATPase isoforms by endogenous digitalis extracts from neonatal human plasma

An unique endogenous digitalis-like factor (EDLF) has been previously purified from human newborn cord plasma and its differential effects tested on the three well defined functional isoforms (alpha1, alpha2 and alpha3) of the alpha subunits of Na+/K+-ATPase in rat. EDLF specifically inhibits the enzymatic activity. It differs from ouabain by three criteria: a preincubation with the membranes is required for full activity, no effect on the rat cerebral alpha3 isoform and a steep dose-response curve with the same apparent potency for rat alpha2 and alpha1 isoforms of high (10(-7) M) and low affinity (3 x 10(-5) M) for ouabain. These results indicate that the Na+/K+-ATPase inhibitor involved in the regulation of sodium and body fluid volume and present in neonate and adult human plasmas is distinct from ouabain.     

The influence of beta subunit structure on the stability of Na+/K(+)-ATPase complexes and interaction with K+

Heterologous expression of the beta subunit of H+/K(+)-ATPase (HK beta) with alpha subunits of Na+/K(+)-ATPase (NK alpha) in yeast leads to the formation of ouabain binding complexes, indicating assembly of the two subunits into active ion pumps (Eakle, K. A., Kim, K. S., Kabalin, M. A., and Farley, R. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2834-2838). Complexes of NK alpha and HK beta are less sensitive to inhibition of ouabain binding by K+, suggesting that HK beta lowers the affinity of K+ binding sites. This effect is particularly pronounced when HK beta is combined with the alpha 3 isoform of NK alpha. In this case, titration with K+ yields a biphasic curve, suggesting that there are two nonequivalent sites for K+ binding. Attempts at purifying complexes formed with either alpha 1 + HK beta or alpha 3 + HK beta using SDS extraction of microsomal membranes resulted in the loss of ouabain binding. Controls show that alpha 1 + beta 1 and alpha 3 + beta 1 complexes still retain ouabain binding after SDS extraction under the same conditions. This suggests that the HK beta subunit forms a less stable complex with NK alpha subunits. We have created chimeric beta subunits comprised of the amino-terminal cytoplasmic and transmembrane regions of HK beta combined with the carboxyl-terminal extracellular region of Na+/K(+)-ATPase beta 1 (HN beta 1) and the complementary chimera with amino-terminal cytoplasmic and transmembrane regions of beta 1 combined with the carboxyl-terminal extracellular region of HK beta (NH beta 1). When NH beta 1 is combined with either alpha 1 or alpha 3, the complexes show profiles of K+ inhibition of ouabain binding that are very similar to HK beta combined with either alpha 1 or alpha 3. The data suggest that the extracellular region of HK beta is primarily responsible for the effect on apparent K+ affinity. When the HN beta 1 subunit is expressed with the alpha 3 subunit, less than 5% of the amount of ouabain binding complexes are formed compared with HN beta 1 + alpha 1. This observation suggests that the HN beta 1 subunit either assembles poorly or forms an unstable complex with alpha 3. After SDS extraction, complexes of alpha 1 + NH beta 1 and alpha 3 + NH beta 1 retain ouabain binding, while alpha 1 + HN beta 1 complexes are sensitive to SDS extraction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Microenvironment of the high affinity ATP-binding site of Na+/K+-ATPase is slightly acidic

Fluorescein-5'-isothiocyanate (FITC) was used to study the high-affinity ATP-binding site of Na+/K+-ATPase. The molar ratio of specifically bound FITC per alpha-subunit of Na+/K+-ATPase was found to be 0.5 as followed from pretreatment experiments with another specific E1ATP-inhibitor Cr(H2O)4AdoPP[CH2]P. This indicated an existence of one high affinity ATP-binding site (E1ATP-binding site) in the native (alphabeta)2-diprotomer of Na+/K+-ATPase. Fluorescence dual-excitation ratio of specifically bound FITC revealed that at external pH 7.5, the pH value inside the E1ATP-binding site is 6.95 +/- 0.18. In addition, FITC fluorescence quenching by anti-fluorescein and by iodide choline indicated the limited access of water into the small pocket of the E1ATP-binding site.     

Binding of pyrene isothiocyanate to the E1ATP site makes the H4-H5 cytoplasmic loop of Na+/K+-ATPase rigid

1-Pyreneisothiocyanate was shown to be an inhibitor of Na+/K+-ATPase. Reverse-phase HPLC and activity studies indicated binding of 1-pyreneisothiocyanate at the H4-H5 loop of the alpha subunit and competition with the fluorescein 5'-isothiocyanate for the E1ATP site. While fluorescein 5'-isothiocyanate, the fluorescent ATP pseudo-analog, was shown to be immobilized at the E1ATP site, there was no possibility to draw any conclusion about the flexibility of the E1ATP site due to its short lifetime. Employing 1-pyreneisothiocyanate as a long-lived fluorophore and a label for the E1ATP site, we found that the ATP-binding site of Na+/K+-ATPase and, in fact, the whole large intracellularly exposed H4-H5 loop of the catalytic alpha subunit is rigid and rotationally immobilized. This has important consequences for the molecular mechanism of the transport function.     

Carbachol-induced increase of Na+/H+ antiport and recruitment of Na+,K(+)-ATPase in rabbit lacrimal acini

Parallel arrays of Na+/H+ and Cl-/HCO3- antiporters are believed to catalyze the first step of transepithelial electrolyte secretion in lacrimal glands by coupling Na+ and Cl- influxes across acinar cell basolateral membranes. Tracer uptake methods were used to confirm the presence of Na+/H+ antiport activity in membrane vesicles isolated from rabbit lacrimal gland fragments. Outwardly-directed H+ gradients accelerated 22Na+ uptake, and amiloride inhibited 96% of the H+ gradient-dependent 22Na+ flux. Amiloride-sensitive 22Na+ influx was half-maximal at an extravesicular Na+ concentration of 14 mM. In vitro stimulation of isolated lacrimal acini with 10 microM carbachol for 30 min increased Na+/H+ antiport activity of a subsequently isolated basolateral membrane sample 2.5-fold, but it did not significantly affect Na+/H+ antiport activity measured in intracellular membrane samples. The same treatment increased basolateral membrane Na+,K(+)-ATPase activity 1.4-fold; this increase could be accounted for by decreases in the Na+,K(+)-ATPase activities of intracellular membranes. Thus, it appears that cholinergic stimulation causes recruitment of additional Na+,K(+)-ATPase pump units to the acinar cell basolateral plasma membrane. The mechanistic basis of the increase in basolateral membrane Na+/H+ antiport activity remains unclear.     

Role of the Na+/K+-ATPase in regulating the membrane potential in rat peritoneal mast cells

500 children with ages ranging between 2 and 13 years and their families joined the study. A detailed questionnaire and a 3-day food intake diary were evaluated to find the relationship between dental caries and dietary habits. It is concluded that the prevalence of caries increases by frequent and high sugar consumption. With lower age and lower caries experience followed a more balanced diet. There was an inverse relationship between caries prevalence and the mothers' educational level. The percentage of children who never or irregularly brushed their teeth was highest in the caries active group.     

Molecular distance measurements reveal an (alpha beta)2 dimeric structure of Na+/K+-ATPase. High affinity ATP binding site and K+-activated phosphatase reside on different alpha-subunits

ATP hydrolysis by Na+/K+-ATPase proceeds via the interaction of simultaneously existing and cooperating high (E1ATP) and low (E2ATP) substrate binding sites. It is unclear whether both ATP sites reside on the same or on different catalytic alpha-subunits. To answer this question, we looked for a fluorescent label for the E2ATP site that would be suitable for distance measurements by F?rster energy transfer after affinity labeling of the E1ATP site by fluorescein 5'-isothiocyanate (FITC). Erythrosin 5'-isothiocyanate (ErITC) inactivated, in an E1ATP site-blocked enzyme (by FITC), the residual activity of the E2ATP site, namely K+-activated p-nitrophenylphosphatase in a concentration-dependent way that was ATP-protectable. The molar ratios of FITC/alpha-subunit of 0.6 and of ErITC/alpha-subunit of 0.48 indicate 2 ATP sites per (alpha beta)2 diprotomer. Measurements of F?rster energy transfer between the FITC-labeled E1ATP and the ErITC-labeled or Co(NH3)4ATP-inactivated E2ATP sites gave a distance of 6.45 +/- 0.64 nm. This distance excludes 2 ATP sites per alpha-subunit since the diameter of alpha is 4-5 nm. F?rster energy transfer between cardiac glycoside binding sites labeled with anthroylouabain and fluoresceinylethylenediamino ouabain gave a distance of 4.9 +/- 0.5 nm. Hence all data are consistent with the hypothesis that Na+/K+-ATPase in cellular membranes is an (alpha beta)2 diprotomer and works as a functional dimer (Thoenges, D., and Schoner, W. (1997) J. Biol. Chem. 272, 16315-16321).     

Extensive random mutagenesis analysis of the Na+/K+-ATPase alpha subunit identifies known and previously unidentified amino acid residues that alter ouabain sensitivity--implications for ouabain binding

Random mutagenesis with ouabain selection has been used to comprehensively scan the extracellular and transmembrane domains of the alpha1 subunit of the sheep Na+/K+-ATPase for amino acid residues that alter ouabain sensitivity. The four random mutant libraries used in this study include all of the transmembrane and extracellular regions of the molecule as well as 75% of the cytoplasmic domains. Through an extensive number of HeLa cell transfections of these libraries and subsequent ouabain selection, 24 ouabain-resistant clones have been identified. All previously described amino acids that confer ouabain resistance were identified, confirming the completeness of this random mutagenesis screen. The amino acid substitutions that confer the greatest ouabain resistance, such as Gln111-->Arg, Asp121-->Gly, Asp121-->Glu, Asn122-->Asp, and Thr797-->Ala were identified more than once in this study. This extensive survey of the extracellular and transmembrane regions of the Na+/K+-ATPase molecule has identified two new regions of the molecule that affect ouabain sensitivity: the H4 and the H10 transmembrane regions. The new substitutions identified in this study are Leu330-->Gln, Ala331-->Gly, Thr338-->Ala, and Thr338-->Asn in the H4 transmembrane domain and Phe982-->Ser in the H10 transmembrane domain. These substitutions confer modest increases in the concentration of cardiac glycoside needed to produce 50% inhibition of activity (IC50 values), 3.1-7.9-fold difference. The results of this extensive screening of the Na+/K+-ATPase alpha1 subunit to identify amino acids residues that are important in ouabain sensitivity further supports our hypothesis that the H1-H2 and H4-H8 regions represent the major binding sites for the cardiac glycoside class of drugs.     

An effect of voltage on binding of Na+ at the cytoplasmic surface of the Na(+)-K+ pump

This work utilizes proteoliposomes reconstituted with renal Na(+)-K(+)-ATPase to study effects of electrical potential (40-80 mV) on activation of pump-mediated fluxes of Na+ or Rb+ (K+) ions and on inhibitory effects of Rb+ ions or organic cations. The latter include guanidinium derivatives that are competitive Na(+)-like antagonists (David, P., Mayan, H., Cohen, H., Tal, D. M., and Karlish, S.J.D. (1992) J. Biol. Chem. 267, 1141-1149). Cytoplasmic side-positive diffusion potentials significantly decreased the K0.5 of Na+ at the cytoplasmic surface for activation of ATP-dependent Na(+)-K+ exchange but did not affect the inhibitory potency of Rb+ (K+) or any Na(+)-like antagonist. Diffusion potentials did not affect activation of Rb(+)-Rb+ exchange by Rb+ ions at the cytoplasmic surface and had only a minor effect on Rb+ activation at the extracellular surface. Previously, we proposed that the cation binding domain consists of two negatively charged sites, to which two K+ or two Na+ ions bind, and one neutral site for the third Na+ (Glynn, I. M., and Karlish, S.J.D. (1990) Annu. Rev. Biochem. 59, 171-205). The present experiments suggest that binding of a Na+ ion in the neutral site at the cytoplasmic surface is sensitive to voltage. By contrast, binding of Rb+ ions at the extracellular surface of renal pumps appears to be only weakly or insignificantly affected by voltage. Inferences on the identity of the charge-carrying steps, based on experiments using proteoliposomes, are discussed in relation to recent evidence that dissociation of Na+ or association of K+ ions, at the extracellular surface, represent the major charge-carrying steps.     

Na+,K+-ATPase inhibitors in rat urine: origins and physiological significance

To identify the origins and structures of mammalian tissue-derived Na+,K+-ATPase inhibitors, we investigated the tissue distribution of inhibitors in rats. Among many tissues tested, urine was found to contain high levels of many inhibitors. The structures of the two major inhibitors were identified as neoconvalloside and periplogenin monorhamnoside, which are derivatives of strophanthidin. Urinary levels of these inhibitors, however, decreased considerably after changing the diet from the regular diet to purified synthetic diet, suggesting that the majority of the urinary inhibitors are of dietary origin. Investigation of the ingredients of the diet further revealed that alfalfa meal and ground oats are the major sources of these cardiac glycosides. As to the physiological relevance of the cardiac glycosides, a low concentration (1-50 nM) of ouabain dose-dependently enhanced aldosterone secretion from adrenal glomerulosa cells by an increase in local renin release. Ouabain was also found to be involved in AT2 receptor-specific expression in rat PC12W cells through an increment in intracellular Na+. These results suggest that Na+,K+-ATPase inhibitors, regardless of the source, are involved in the regulation of blood pressure.