Ion binding to cytochrome c studied by nuclear magnetic quadrupole relaxation.

The enhancement of the 35Cl- transverse relaxation rate on binding of chloride ions to oxidized and reduced cytochrome c has been studied under conditions of variable sodium chloride concentration, temperature, pH, sodium phosphate, iron hexacyanide, and sodium cyanide concentration. The results revealed the presence of a strong binding site(s) for chloride in both oxidized and reduced cyt c, with a higher affinity in ferrocytochrome c. Competition experiments suggest that these sites also bind iron hexacyanide and phosphate. Cyanide binding to the iron in ferricytochrome c at alkaline and neutral pH was shown to decrease the binding of chloride. The pH dependence of the 35Cl- relaxation rate has been fitted by using literature pK values for ionizable groups. No indications of Na+ binding to oxidized and reduced cytochrome c have been observed by using 23Na+ NMR. Our results suggest that chloride is bound near the exposed heme edge and that the surface structure or dynamics in this region are different in the two oxidation states.     

Interaction of alkali metal ions with Ca2+-binding center of Ca2+- ATPase of the sarcoplasmic reticulum in skeletal muscles

Sodium ion interaction with sarcoplasmic reticulum (SR) membranes leads to considerable alterations of the [23Na]NMR lineshape. Na+ binding to SR in the presence of Ca2+ and H+ is well described by a model which postulates a competitive ion binding to high and low affinity sites of Ca2+-ATPase. The dissociation constant, Kd, for high and low affinity sites is 5 and 10 mM, respectively, for Na+ and (3-5).10(-8) and 1.5.10(-3) M, respectively, for Ca2+. The pK value for high and low affinity sites is 7.3 and 6.1, respectively. Other alkaline metal ions compete with Na+ for the low affinity sites of Ca2+-ATPase; their affinities decrease in the following order: Na+ = K+ greater than Rb+ greater than Cs greater than Li+. Some of the Na+ binding sites (approximately 10%) do not interact with Ca2+.     

Alkali cation exchangers: roles in cellular homeostasis and stress tolerance

Uptake and translocation of cations play essential roles in plant nutrition, signal transduction, growth, and development. Among them, potassium (K+) and sodium (Na+) have been the focus of numerous physiological studies because K+ is an essential macronutrient and the most abundant inorganic cation in plant cells, whereas Na+ toxicity is a principal component of the deleterious effects associated with salinity stress. Although the homeostasis of these two ions was long surmised to be fine tuned and under complex regulation, the myriad of candidate membrane transporters mediating their uptake, intracellular distribution, and long-distance transport is nevertheless perplexing. Recent advances have shown that, in addition to their function in vacuolar accumulation of Na+, proteins of the NHX family are endosomal transporters that also play critical roles in K+ homeostasis, luminal pH control, and vesicle trafficking. The plasma membrane SOS1 protein from Arabidopsis thaliana, a highly specific Na+/H+ exchanger that catalyses Na+ efflux and that regulates its root/shoot distribution, has also revealed surprising interactions with K+ uptake mechanisms by roots. Finally, the function of individual members of the large CHX family remains largely unknown but two CHX isoforms, AtCHX17 and AtCH23, have been shown to affect K+ homeostasis and the control of chloroplast pH, respectively. Recent advances on the understanding of the physiological processes that are governed by these three families of cation exchangers are reviewed and discussed.     

Specificity of Na+ binding to phosphatidylserine vesicles from a 23Na NMR relaxation rate study.

23Na NMR relaxation rate measurements show that Na+ binds specifically to phosphatidylserine vesicles and is displaced partially from the binding site by K+ and Ca2+ but to a considerably less extent by tetraethylammonium ion. The data indicate that tetraethylammonium ion affects the binding of Na+ only slightly, by affecting the surface potential through its presence in the double layer, without competing for a phosphatidylserine binding site. Values for the intrinsic binding constant for the Na+-phosphatidylserine complex that would be consistent with the competition experiments (and the dependence of the relaxation rate on concentration of free Na+) fall in the range 0.4--1.2 M-1 with a better fit towards the higher values. We conclude that in the absence of competing cations in solution an appreciable fraction of the phosphatidylserine sites could be associated with bound Na+ at 0.1 M Na+ concentration.     

23Na-NMR studies of Na+ interaction with human red cell membranes from normotensives and hypertensives

Na+ interaction with unsealed human red cell ghosts has been studied by 23Na-NMR relaxation rate (R1) measurements. Data on a total of nine subjects including seven volunteer normotensives (NBP) and two untreated hypertensives (HBP) are presented. Qualitative treatment of the data gives information on the dynamic behavior of Na+ undergoing fast exchange between the free and bound states. The excess longitudinal relaxation rate (delta R)-1 plotted against total [Na+], known as the James-Noggle plot, exhibits different behavior for NBP and HBP ghosts, with a relatively low binding constant of approx. 100 M-1 for HBP (p less than 0.025) compared to a high constant of 500-1000 M-1 for NBP. To associate our NMR data with membrane-bound (Na+ + K+)-ATPase, 23Na relaxation rates were measured in the presence of 5 mM ouabain. James-Noggle plots constructed for ouabain-sensitive excess relaxation rates show the binding for NBP to be even high affinity (greater than 10(3) M-1) but low capacity. These data may suggest that for a given amount of intracellular Na+, the binding affinity could determine the distribution of Na+ between the membrane and cytoplasm, and that the (Na+ + K+)-ATPase which is primarily responsible for the Na+ affinity might assume an abnormal transport mechanism in HBP membranes.     

Effects of ATP on the intermediary steps of the reaction of the (Na+ + K+)-ATPase. IV. Effect of ATP on K0.5 for Na+ and on hydrolysis at different pH and temperature.

The pH optimum for (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) depends on the combination of monovalent cations, on the ATP concentration and on temperature. ATP decreases the Na+ concentration necessary for half maximum activation, K0.5 for Na+ (Na+ + K+ = 150 mM), and the effect is pH and temperature dependent. At a low ATP concentration a decrease in pH leads to an increase in K0.5 for Na+, while at the high ATP concentration it leads to a decrease. K0.5 for ATP for hydrolysis decreases with an increase in pH. The fractional stimulation by K+ in the presence of Na+ decreases with the ATP concentration, and at a low ATP concentration K+ becomes inhibitory, this being most pronounced at 0 degrees C. The results suggest that (a) ATP at a given pH has two different effects: it increases the Na+ relative to K+ affinity on the internal site (K0.5 for ATP at pH 7.4, 37 degrees C, is less than 10 microM); it increases the molar activity in the presence of Na+ + K+ (K0.5 for ATP at pH 7.4, 37 degrees , is 127 microM), (b) binding of the cations to the external as well as the internal sites leads to pK changes (Bohr effect) which are different for Na+ and for K+, i.e. the selectivity for Na+ relative to K+ depends both on ATP and on the degree of protonation of certain groups on the system, (c) ATP involves an extra dissociable group in the determination of the selectivity of the internal site, and thereby changes the effect of an increase in protonation of the system from a decrease to an increase in selectivity for Na+ relative to K+.     

Phosphate-dependent sodium transport in S. faecalis investigated by 23Na and 31P NMR

Na+ movements in S. faecalis were studied by 23Na NMR. They proved to be dependent on phosphate concentration in the buffer during the de-energization step. K+ and H+ were also studied respectively by potentiometry and 31P NMR and were shown not to be implicated. For de-energized cells the internal phosphate concentration, on the contrary, was directly linked to the external phosphate contained in the buffer. The experiments showed a Na+/Pi dependence in this prokaryote so far known only in eukaryotes.     

Furosemide-sensitive salt transport in the Madin-Darby canine kidney cell line. Evidence for the cotransport of Na+, K+, and Cl-

Confluent monolayer cultures of the Madin-Darby canine kidney (MDCK) cell line have been shown to possess a furosemide and bumetanide-sensitive (Na+,K+)-cotransport system. We have studied the effect of anion substitutions on (Na+,K+)-cotransport. In Na+-depleted cells, bumetanide-sensitive uptake of 22Na+ or 86Rb+ exhibited an absolute requirement for extracellular Cl-. Chloride could be replaced in the buffers by Br-, but not by F-, I-, acetate, nitrate, thiocyanate, sulfate, or gluconate. The effect of Cl- was saturating, and Na+-stimulated 86RB+ uptake as well as K+-stimulated 22Na+ uptake was shown to be dependent on the square of the Cl- concentration. The concentration of Cl- which gave half-maximal stimulation of cation cotransport varied between 58 and 70 mM. There was a small degree of cooperativity between the binding affinities for Cl- and K+ at constant Na+ concentrations. Bumetanide-sensitive 36Cl- uptake could be demonstrated when extracellular Na+ and K+ were present simultaneously. Uptake through this system was unaffected by changes in the membrane potential or by the imposition of pH gradients. Together these data strongly suggest that the bumetanide-sensitive transport system in Madin-Darby canine kidney cells co-transports Na+, K+, and Cl- in a ratio of 1:1:2.     

Cis-allosteric effects of cytoplasmic Na+/K+ discrimination at varying pH. Low-affinity multisite inhibition of cytoplasmic K+ in reconstituted Na+/K(+)-ATPase engaged in uncoupled Na(+)-efflux.

In liposomes with reconstituted shark Na+/K(+)-ATPase the effect of cytoplasmic K+ was investigated in the absence of extracellular alkali ions. During such conditions the Na+/K(+)-ATPase is engaged in the so called uncoupled Na+ efflux mode in which cytoplasmic Na+ activates and binds to the enzyme and becomes translocated without countertransport of K+ as in the physiological Na+/K+ exchange mode. In this uncoupled flux mode only low-affinity inhibition by K+cyt is found to be present. The inhibition pattern is consistent with a model in which cytoplasmic K+ exhibit mixed inhibition of Na+ activation, probably by binding at the three cytoplasmic loading sites on E1ATP (E1A). With determined intrinsic binding constants for cytoplasmic Na+ to this form of KS1, KS2, KS3 = 40 mM, 2 mM, 2 mM the inhibition pattern can be simulated assuming three K+cyt sites with equal affinity for Ki = 40 mM, similar to KS1 for the first Na+cyt site. The discrimination between cytoplasmic Na+ and K+ is therefore enhanced by allosteric interaction initiated from the cis-side due to binding of the first Na+, as opposed to K+, which induces the positive cooperatively in the successive Na+ bindings. pH is found to influence the pattern of K+cyt inhibition: A lowering of the pH potentiates the K+cyt inhibition, whereas at increased pH the inhibition is decreased and transformed into a pure competitive competition.     

Characteristics of glutamic acid transport by rabbit intestinal brush-border membrane vesicles. Effects of Na+-, K+- and H+-gradients

In the presence of a Na+-gradient (out greater than in), L-glutamic acid and L-and D-aspartic acids were equally well concentrated inside the vesicles, while no transport above simple diffusion levels was seen by replacement of Na+ by K+. Equilibrium uptake values were found inversely proportional to the medium osmolarity, thus demonstrating uptake into an osmotically sensitive intravesicular space. The extrapolation of these lines to infinite medium osmolarity (zero space) showed only a small binding component in acidic amino-acid transport. When the same experiment was performed at saturating substrate concentrations, linear relationships extrapolating through the origin but showing smaller slope values were recorded, thus indicating that the binding component could be more important than suspected above. However, binding to the membrane was neglected in our studies as it was absent from initial rate measurements. Na+-dependent uphill transport of L-glutamic acid was stimulated by K+ present on the intravesicular side only but maximal stimulation was recorded under conditions of an outward K+-gradient (in greater than out). Quantitative and qualitative differences in the K+ effect were noted between pH 6.0 and 8.0. Initial uptake rates showed pH dependency in Na+-(out greater than in) + K+-(in greater than out) gradient conditions only with a physiological pH optimum between 7.0 and 7.5. It was also found that a pH-gradient (acidic outside) could stimulate both the Na+-gradient and the Na+ + K+-gradient-dependent transport of L-glutamic acid. However, pH- or K+-gradient alone were ineffective in stimulating uptake above simple diffusion level. Finally, it was found that increased rates of efflux were always observed with an acidic pH outside, whatever the conditions inside the vesicles. From these results, we propose a channel-type mechanism of L-glutamic acid transport in which Na+ and K+ effects are modulated by the surrounding pH. The model proposes a carrier with high or low affinity for Na+ in the protonated or unprotonated forms, respectively. We also propose that K+ binding occurs only to the unprotonated carrier and allows its fast recycling as compared to the free form of the carrier. Such a model would be maximally active and effective in the intestine in the in vivo physiological situations.     

Further studies on the binding of divalent cations to the phosphoglycoprotein phosvitin.

Dialysis equilibrium measurements at 25 degrees indicate that, at pH 6.8 and at a concentration of 1.0 times 10(-10) 3 M MnC12 or CoC12, phosvitin binds 113 Mn2+ and 120 Co2+. The binding is cooperative at low cation concentrations. The number of Mg2+, Ca2+, Mn2+, and Co2+ bound is not affected by temperatures of up to 60 degrees; however, the cooperactivity is enhanced. Optical rotatory dispersion and circular dichroism studies indicate that a conformational change occurs on binding of Mn2+ and Co2+ which parallels the one produced by Ca2+ and reported elsewhere [Grizzuti, K., and Perlmann, G.E. (1973), Biochemistry 12, 4399]. The conformational changes induced by Mg2+ and Mn2+ follow different paths. Upon binding of Mn2+ and Co2+ the intrinsic viscosity, [eta], of phosvitin decreases from about 0.5 to 0.03 dl/g, while Mg2+ and Ca2+ decrease [eta] to 0.048 dl/g. The ultraviolet absorption spectrum of phosvitin is altered upon binding of Ca2+, Mn2+, and Co2+, but not upon binding of Mg2+; an increase of the temperature to 60% has no further effect on the spectra.     

(Na+, K+)-activated adenosinetriphosphatase of axonal membranes, cooperativity and control. Steady-state analysis.

1. The ATP sites. Homotropic interactions between ATP sites have been studied in a very large range of Na+ and K+ concentrations. The ( Na+, K+)-activated ATPase displays Michaelis-Menten kinetics for ATP under standard concentration conditions of Na+ (100 mM) and K+ (10 mM). The steady-state kinetics behavior changes at very low concentrations of K+ where negative cooperativity is observed. The existence of a high affinity and a low affinity site for ATP was clearly demonstrated from the study of the ATP stimulated hydrolysis of p-nitrophenylphosphate in the presence of Na+ and K+. The ratio of apparent affinities of high and low affinity sites for ATP is 86 at pH 7.5. 2. The Na+ sites. The binding of Na+ to its specific stimulatory sites (internal sites) is characterized by positive cooperativity with a Hill coefficient n(H(Na+))=2.0. Homotropic interactions between Na+ sites are unaffected by variations of the K+ concentration. 3. The K+ sites. (a) Binding of K+ to the (external) stimulatory site of the ATPase has been analyzed by following the (Na+, K+)-ATPase activity as well as the p-nitrophenylphosphatase activity in the presence of Na+ and K+ (with or without ATP). Binding is characterized by a Hill coefficient of 1.0 and a K(0.5(K+))=0.1 to 0.8 mM. The absence of positive or negative cooperativity persists between 5 mM and 100 mM Na+. (b) The analysis of the p-nitrophenylphosphatase or of the 2, 4 dinitrophenylphosphatase activity in the presence of K+ alone indicates the existence of low affinity sites for K+ with positive homotropic interactions. The characteristics of stimulation in that case are, K(0.5)=5 mM, n(H)=1.9. The properties of this family of site(s) are the following: firstly, saturation of the low affinity site(s) by K+ prevents ATP binding to its high affinity internal site. Secondly, saturation of the low affinity sites for K+ prevents binding of Na+ to its internal sites. Thirdly, this family of sites disappears in the presence of ATP, p-nitrophenylphosphate or of both substrates, when Na+ binds to its internal sites. Na+ binding to its specific stimulatory sites provokes the formation of the high affinity type of site for K+. 4. Mg2+ stimulation of the (Na+, K+)-ATPase is characterized by a Hill coefficient n(H(Mg2+))=1.0 and a K(0.5(Mg2+))=1 mM stimulation is essentially a V effect. Heterotropic effects between binding of Mg2+ and substrate to their respective sites are small. Heterotropic interactions between the Ms2+, Na+ and K+ sites are also small. 5. The fluidity of membrane lipids also controls the (Na+, K+)-ATPase activity. Phase transitions or separations in the membrane hardly affect recognition properties of substrates, Na+, K+ and Mg2+ for their respective sites on both sides of the membrane. Only the rate of the catalytic transformation is affected.     

Sodium transport and phosphorus metabolism in sodium-loaded yeast: simultaneous observation with sodium-23 and phosphorus-31 NMR spectroscopy in vivo

Simultaneous 23Na and 31P NMR spectra were obtained from a number of yeast suspensions. Prior to NMR spectroscopy, the yeast cells were Na-loaded: this replaced some of the intracellular K+ with Na+. These cells were also somewhat P-deficient in that they had no polyphosphate species visible in the 31P NMR spectrum. In the NMR experiments, the Na-loaded cells were suspended in media which contained inorganic phosphate, very low Na+, and a shift reagent for the Na+ NMR signal. The media differed as to whether dioxygen, glucose, or K+ was present individually or in combinations and as to whether the medium was buffered or not. The NMR spectra revealed that the cells always lost Na+ and gained phosphorus. However, the nature of the Na+ efflux time course and the P metabolism differed depending on the medium. The Na+ efflux usually proceeded linearly until the amount of Na+ extruded roughly equalled the amount of NH4+ and orthophosphate initially present in the medium (external phosphate was added as NH4H2PO4). Thus, we presume this first phase reflects a Na+ for NH4+ exchange. The Na+ efflux then entered a transition phase, either slowing, ceasing, or transiently reversing, before resuming at about the same value as that of the first phase. We presume that this last phase involves the simultaneous extrusion of intracellular anions as reported in the literature. The phosphorus metabolism was much more varied. In the absence of exogenous glucose, the P taken up accumulated first as intracellular inorganic phosphate; otherwise, it accumulated first in the "sugar phosphate" pool. In most cases, at least some of the P left the sugar phosphate pool and entered the polyphosphate reservoir in the vacuole. However, this never happened until the phase probably representing Na+ for NH4+ exchange was completed, and the P in the polyphosphate pool never remained there permanently but always eventually reverted back to the sugar phosphate pool. These changes are interpreted in terms of hierarchical energy demands on the cells under the different conditions. In particular, the energy for the Na+ for NH4+ exchange takes precedence over that required to produce and store polyphosphate. This conclusion is supported by the fact that when the cells are "forced" to exchange K+, as well as NH4+, for Na+ (by the addition of 5 times as much K+ to the NH4+-containing medium), polyphosphates are never significantly formed, and the initial linear Na+ efflux phase persists possibly 6 times as long.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of intracellular pH in ligand internalization

Internalization of EGF and transferrin measured as the rate of uptake of 125I-labeled ligands was compared in the cell line CCL39 and a mutant derivative, PS-120, lacking the Na+/H+ antiport system. No significant alteration was detected between the two cell lines. In contrast, pretreatment of the mutant cells PS-120 with 20 mM NH4Cl for 30 min to decrease persistently intracellular pH resulted in an increase in 125I-EGF and 125I-transferrin uptake by 60% and 25%, respectively. However, similar NH4Cl pretreatment of the parental cell line, CCL-39, which only affected intracellular pH very transiently did not cause an increase of ligand uptake. The binding of 125I-EGF to CCL-39 and PS-120 cells with or without NH4Cl pretreatment showed that NH4Cl pretreatment did not affect EGF binding in either CCL-39 or PS-120 cells. Since cells regulate intracellular pH by ion transport systems, we also examined the role of Na+, K+-ATPase. Ouabain, an inhibitor of Na+, K+-ATPases, showed no effect on 125I-EGF uptake in either of the cell types with or without NH4Cl pretreatment. Taken together, these results suggest that the plasma membrane-bound Na+/H+ antiport, a major pHi-regulating system in vertebrates, indirectly plays a role in ligand internalization through regulation of intracellular pH.     

31P and 23Na nuclear magnetic resonance studies of resting and stimulated mast cells

Exocytosis induced by crosslinking the type I receptor for Fc epsilon domains present on rat mucosal mast cells (RBL-2H3-line) requires the influx of Ca2+ ions and is markedly influenced by the concentration of monovalent cations (K+, Na+ and protons) in their medium. We investigated the role of these ions in coupling the immunological stimulus to secretion using NMR spectroscopy to monitor simultaneously intracellular pH, ATP and Na+ concentrations and the secretory response of living adherent mast cells. Using this methodology we observed that: (i) ATP concentration and intracellular pH are highly regulated and no changes could be resolved in them upon stimulation and during exocytosis. (ii) In the absence of potassium ions in the cells' medium, a decrease is observed in the intracellular pH and ATP concentration and an increase in the Na+ concentration. (iii) From the influx of extracellular Na+ following inhibition of the Na+, K(+)-ATPase by ouabain, we estimated the inward Na+ current of resting cells to 5 x 10(7) ions/(cell.s). This value does not vary by more than 10% during exocytosis.     

Facilitation of ouabain binding to (Na+ + K+)-ATPase by vanadate at in vivo concentrations.

In the presence of Mg2+ vanadate was shown to facilitate ouabain binding to (Na+ + K+)-ATPase in much the same way as Pi does. Thus the hypothesis that vanadate interacts with the phosphate site of the enzyme seems to be supported by ouabain binding experiments. At given ouabain concentrations maximum binding is achieved at microM concentrations of vanadate whereas mM concentrations of Pi are needed. Na+ as well as K+ counteract ouabain binding but some cardiac glycoside binding is still possible at in vivo concentrations of these cations. A minor contamination of the enzyme preparations with vanadate could explain the in vitro binding of ouabain that can be obtained with Mg2+ and in the absence of Pi.     

Simultaneous bindings of ATP and vanadate to (Na+ + K+)-ATPase. Implications for the reaction mechanism of the enzyme

Inhibition of (Na+ + K+)-dependent adenosine triphosphatase phosphatase by vanadate is thought to occur through the tight binding of vanadate to the same site from which Pi is released. To see if ATP binds to [48V] vanadate-enzyme complex, just as it does to the phosphoenzyme, the effects of Na+, K+, and ATP on the dissociation rate of the complex at 10 degrees C were studied. The rate constant was increased by Na+, and this increase was blocked by K+, indicating that either Na+ or K+ binds to the complex. ATP alone, or in combination with K+, had no effect on the rate constant. In the presence of Na+, however, ATP caused a further increase in the rate constant. The value of K0.5 of Na+ was the same in the presence or absence of ATP; K0.5 of ATP (0.2 mM) did not seem to change significantly when Na+ concentration was varied, and K0.5 of K+, at a constant Na+ concentration, was the same in the presence or absence of ATP. The data indicate that ATP binds to the enzyme-vanadate complex regardless of the presence or absence of Na+ or K+, but it affects the dissociation rate only when Na+ is bound simultaneously. The value of K0.5 of Na+ decreased as pH was increased in the range of 6.5-7.8, but K0.5 of ATP was independent of pH. Demonstration of ATP binding to the enzyme-vanadate complex provides further support for the suggestion that the oligomeric enzyme contains a low-affinity regulatory site for ATP that is distinct from the interacting high-affinity catalytic sites.     

Ion transport by lasalocid A across red-blood-cell membranes. A multinuclear NMR study

Na+ and K+ fluxes mediated by lasalocid A across erythrocyte membranes have been determined from 23Na-NMR peak areas and chemical shifts, respectively. In similar experiments, Cl- transport has been monitored by NMR signal intensities. Taking into account the external pH variations, the results are readily explainable in terms of charge-balance conservation. The effect of disodium 4,4'-diisothiocyanostilbene-2,2'-disulfonate, an anion-exchange inhibitor, has also been studied.     

Multinuclear NMR studies of intracellular cations in perfused hypertensive rat kidney.

We have investigated hypertension-associated alterations in intracellular cations in the kidney by measuring intracellular pH, free Mg2+, free Ca2+, and Na+ concentrations in perfused normotensive and hypertensive rat (8-14 weeks old) kidneys using 31P, 19F, and double quantum-filtered (DQ) 23Na NMR. The effects of both anoxia and ischemia on the 23Na DQ signal confirmed its ability to detect changes in intracellular Na+. However, there was a sizable contribution of the extracellular Na+ to the 23Na DQ signal of the kidney. The intracellular free Ca2+ concentration, measured using 19F NMR and 5,5'difluoro-1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid, also increased dramatically during ischemia; the increase could be partly reversed by reperfusion. No significant differences were found between normotensive and hypertensive kidneys in the ATP level, intracellular pH, intracellular free Mg2+, and the 23Na DQ signal or in the extent of the extracellular contribution to the 23Na DQ signal. Oxygen consumption rates were also similar for the normotensive (5.02 +/- 0.46 mumol of O2/min/g) and hypertensive (5.47 +/- 0.42 mumol O2/min/g) rat kidneys. The absence of a significant difference in intracellular pH, Na+ concentration, and oxygen consumption between normotensive and hypertensive rat kidneys suggests that an alteration in the luminal Na+/H+ antiport activity in hypertension is unlikely. However, a highly significant increase (64%, p less than 0.01) in free Ca2+ concentration was found in perfused kidneys from hypertensive rats (557 +/- 48 nM, blood pressure = 199 +/- 5 mmHg, n = 6) compared with normotensive rats (339 +/- 21 nM, blood pressure = 134 +/- 6, n = 4) indicating altered renal calcium homeostasis in essential hypertension. An increase in intracellular free Ca2+ concentration without an accompanying change in the intracellular Na+ suggests, among many possibilities, that the Ca2+/Mg(2+)-ATPase may be inhibited in the hypertensive renal tissue.     

Kidney Na+,K(+)-ATPase is associated with moesin

Na+,K(+)-ATPase is a ubiquitous plasmalemmal membrane protein essential for generation and maintenance of transmembrane Na+ and K+ gradients in virtually all animal cell types. Activity and polarized distribution of renal Na+,(+)-ATPase appears to depend on connection of ankyrin to the spectrin-based membrane cytoskeleton as well as on association with actin filaments. In a previous study we showed copurification and codistribution of renal Na+,K(+)-ATPase not only with ankyrin, spectrin and actin, but also with two further peripheral membrane proteins, pasin 1 and pasin 2. In this paper we show by sequence analysis through mass spectrometry as well as by immunoblotting that pasin 2 is identical to moesin, a member of the FERM (protein 4.1, ezrin, radixin, moesin) protein family, all members of which have been shown to serve as cytoskeletal adaptor molecules. Moreover, we show that recombinant full-length moesin as well as its FERM domain bind to Na+,K(+)-ATPase and that this binding can be inhibited by an antibody specific for the ATPase activity-containing cytoplasmic loop (domain 3) of the Na+,K(+)-ATPase alpha-subunit. This loop has been previously shown to be a site essential for ankyrin binding. These observations indicate that moesin might not only serve as direct linker molecule of Na+,K(+)-ATPase to actin filaments but also modify ankyrin binding at domain 3 of Na+,K(+)-ATPase in a way similar to protein 4.1 modifying the binding of ankyrin to the cytoplasmic domain of the erythrocyte anion exchanger (AE1).