Multiple fractions of sodium exchange in human lymphocytes

Human lymphocytes contain a large, saturable fraction of K⁺ that exchanges slowly with K⁺ in the external medium, and a small non-saturable fraction that exchanges rapidly. We determined whether or not Na⁺ exchanges in a similar manner with external Na⁺. Cells were pre-equilibrated to ensure absence of net ion movements. Efflux was studied by loading with ²²Na and transferring without washing to a non-labeled medium. Influx was studied by transferring to labeled medium and separating large samples of cells at 6,000g. There are fast, intermediate, and slow fractions of Na⁺ exchange, with half-times of 2, 14, and 120 minutes. At normal external K⁺, most cell Na⁺ exchanges rapidly, while at lower external K⁺ the Na⁺ that replaces cell K⁺ exchanges slowly. Parallel sources of fast and slow fractions, such as extracellular ones and subpopulations of cells, were ruled out by simultaneous ⁴²K and ²²Na fluxes and by a quantitative analysis of the combined K⁺ and Na⁺ content and flux data over a range of external K⁺ and Na⁺ levels. Five possible models of ion fluxes occurring in series were considered. Surface matrix, surface binding sites, and cytoplasmic channels with rapid nuclea exchange were eliminated as sources of the fast fractions. Therefore, the fast fractions of K⁺ and Na⁺ must reflect the permeability of the surface membrane. This left only two possible sources of the slow fractions. One, a subcellular compartment (e.g., nucleus), was eliminated by the combined content and flux data. We conclude that the slow fractions of ion flux are rate-limited by adsorption onto and desorption from cellular macromolecules. The data support the association-induction hypothesis and are understood by reference to two fundamental concepts: that of rapid solute exclusion from cell water existing in a polarized state; and that of solute accumulation limited by adsorption onto fixed anionic sites within the cell.     

Ouabain-resistant Na+, K+ transport system in mouse NIH 3T3 cells

Summary It is shown that the ouabain-resistant (OR) furosemide-sensitive K⁺(Rb⁺) transport system performs a net efflux of K⁺ in growing mouse 3T3 cells. This conclusion is based on the finding that under the same assay conditions the furosemidesensitive K⁺(Rb⁺) efflux was found to be two- to threefold higher than the ouabain-resistant furosemide-sensitive K⁺(Rb⁺) influx. The oubain-resistant furosemide-sensitive influxes of both²²Na and⁸⁶Rb appear to be Cl^– dependent, and the data are consistent with coupled unidirectional furosemide-sensitive influxes of Na⁺, K⁺ and Cl^– with a ratio of 1 1 2. However, the net efflux of K⁺ performed by this transport system cannot be coupled to a ouabain-resistant net efflux of Na⁺ since the unidirectional ouabain-resistant efflux of Na⁺ was found to be negligible under physiological conditions. This latter conclusion was based on the fact that practically all the Na⁺ efflux appears to be ouabainsensitive and sufficient to balance the Na⁺ influx under such steady-state conditions. Therefore, it is suggested that the ouabain-resistant furosemide-sensitive transport system in growing cells performs a facilitated diffusion of K⁺ and Na⁺, driven by their respective concentration gradients: a net K⁺ efflux and a net Na⁺ influx.     

Cation Metabolism in Relation to Cell Size in Synchronously Grown Tissue Culture Cell

In randomly grown tissue culture cells (mouse leukemic lymphoblast, L5178Y) the number, volume, and Na⁺ and K⁺ content increase as an exponential function with a doubling time of 11.3 hr. In synchronously grown cells the volume increase of the population and of single cells follows the same exponential function as in randomly grown cells. In contrast, the cation content fluctuates during a single cell cycle. About 1½ hr after the cell division burst (at the beginning of the S period), a net loss of K⁺ occurs for a period of about 1 hr amounting to about 20% of the total K. Over the next 5 to 6 hr, the deficit in K⁺ is eliminated. The Na⁺ content shows a double fluctuation. It falls during the cell division burst, rises when the K⁺ content decreases, falls again when K⁺ content rises, and then increases again before the next cell division burst. The net fluxes of both Na⁺ and K⁺ are very small compared to the unidirectional fluxes (less than 5%), thus small changes in the balance of influx and efflux account for the changes in cation content during the growth cycle. Both unidirectional fluxes increase dramatically (by a factor of two) about 2 hr after the cell division burst, and then remain constant until after the next cell division. The pattern of electrolyte regulation during cell division does not follow a simple function such as cell number, cell surface, or cell volume, but must be related to specific internal events in the cell.     

Plasma membrane phosphate transport and extracellular phosphate concentration in the regulation of cellular respiration in isolated perfused rat heart

The role of extracellular P_i and transmembrane fluxes across the sarcolemma in the regulation of cellular respiration was studied in isolated Langendorff-perfused rat hearts. Extracellular phosphate did not significantly affect the oxygen consumption or cellular phosphorylation potential of the myocardium. K⁺-induced arrest was used to change the mechanical work load of the heart. Arresting the heart caused a rapid decrease in the unidirectional efflux of phosphate determined by in vitro prelabelling of the intracellular phosphate compounds with ³²P and determining the specific radioactivity of the γ-P of ATP, and the label appearance into the perfusion medium. At normal or elevated perfusate phosphate concentration there was a fairly slow net uptake of phosphate. The decrease in phosphate fluxes upon the K⁺-induced arrest was probably not due to a decrease in the transmembrane Na⁺ or K⁺ gradients because a further increase in the perfusate K⁺ concentration caused an increase in the K⁺ efflux to the levels observed in contracting hearts. The use of higher than normal concentrations of phosphate necessitated a lowering of the extracellular Ca²⁺ concentration, which caused a diminution of the oxygen consumption, accompanied by mitochondrial flavoprotein oxidation in the heart. This finding suggested that the extracellular Ca²⁺ concentration may be involved in the substrate level regulation of mitochondrial metabolism.     

Extracellular charge adsorption influences intracellular electrochemical homeostasis in amphibian skeletal muscle

The membrane potential measured by intracellular electrodes, E_m, is the sum of the transmembrane potential difference (E₁) between inner and outer cell membrane surfaces and a smaller potential difference (E₂) between a volume containing fixed charges on or near the outer membrane surface and the bulk extracellular space. This study investigates the influence of E₂ upon transmembrane ion fluxes, and hence cellular electrochemical homeostasis, using an integrative approach that combines computational and experimental methods. First, analytic equations were developed to calculate the influence of charges constrained within a three-dimensional glycocalyceal matrix enveloping the cell membrane outer surface upon local electrical potentials and ion concentrations. Electron microscopy confirmed predictions of these equations that extracellular charge adsorption influences glycocalyceal volume. Second, the novel analytic glycocalyx formulation was incorporated into the charge-difference cellular model of Fraser and Huang to simulate the influence of extracellular fixed charges upon intracellular ionic homeostasis. Experimental measurements of E_m supported the resulting predictions that an increased magnitude of extracellular fixed charge increases net transmembrane ionic leak currents, resulting in either a compensatory increase in Na⁺/K⁺-ATPase activity, or, in cells with reduced Na⁺/K⁺-ATPase activity, a partial dissipation of transmembrane ionic gradients and depolarization of E_m.     

Cation flux in the ehrlich ascites tumor cell evidence for Na-for-Na and K-for-K exchange diffusion

In a previous study, evidence was presented for an external Na⁺-dependent, ouabain-insensitive component of Na⁺ efflux and an external K⁺-dependent component of K⁺ efflux in the Ehrlich ascites tumor cell. Evidence is now presented that these components are inhibited by the diuretic furosemide and that under conditions of normal extracellular Na⁺ and K⁺ they represent Na⁺-for-Na⁺ and K-⁺for-K⁺ exchange mechanisms. Using ⁸⁶Rb to monitor K⁺ movements, furosemide is shown to inhibit an ouabain-insensitive component of Rb⁺ influx and a component of Rb⁺ efflux, both representing approx. 30% of the total fux. Inhibition of Rb⁺ efflux is greatly reduced by removal of extracellular K⁺. Furosemide does not alter steady-state levels of intracellular K⁺ and it does not prevent cells depleted of K⁺ by incubation in the cold from regaining K⁺ upon warming. Using ²²Na to monitor Na⁺ movements, furosemide is shown to inhibit an ouabain-insensitive component of unidirectional Na⁺ efflux which represents approx. 22% of total Na⁺ efflux. Furosemide does not alter steady-state levels of intracellular Na⁺ and does not prevent removal of intracellular Na⁺ upon warming from cells loaded with Na⁺ by preincubation in the cold. The ability of furosemide to affect unidirectional Na⁺ and K⁺ fluxes but not net fluxes is consistent with the conclusion that these components of cation movement across the cell membrane represent one-for-one exchange mechanisms. Data are also presented which demonstrate that the uptake of α-aminoisobutyrate is not affected by furosemide. This indicates that these components of cation flux are not directly involved in the Na⁺-dependent amino acid transport system A.     

The Third Sodium Binding Site of Na,K-ATPase Is Functionally Linked to Acidic pH-Activated Inward Current

Sodium- and potassium-activated adenosine triphosphatases (Na,K-ATPase) is the ubiquitous active transport system that maintains the Na⁺ and K⁺ gradients across the plasma membrane by exchanging three intracellular Na⁺ ions against two extracellular K⁺ ions. In addition to the two cation binding sites homologous to the calcium site of sarcoplasmic and endoplasmic reticulum calcium ATPase and which are alternatively occupied by Na⁺ and K⁺ ions, a third Na⁺-specific site is located close to transmembrane domains 5, 6 and 9, and mutations close to this site induce marked alterations of the voltage-dependent release of Na⁺ to the extracellular side. In the absence of extracellular Na⁺ and K⁺, Na,K-ATPase carries an acidic pH-activated, ouabain-sensitive “leak” current. We investigated the relationship between the third Na⁺ binding site and the pH-activated current. The decrease (in E961A, T814A and Y778F mutants) or the increase (in G813A mutant) of the voltage-dependent extracellular Na⁺ affinity was paralleled by a decrease or an increase in the pH-activated current, respectively. Moreover, replacing E961 with oxygen-containing side chain residues such as glutamine or aspartate had little effect on the voltage-dependent affinity for extracellular Na⁺ and produced only small effects on the pH-activated current. Our results suggest that extracellular protons and Na⁺ ions share a high field access channel between the extracellular solution and the third Na⁺ binding site.     

The α1 Na+−K+ pump of the Dahl salt-sensitive rat exhibits altered Na+ modulation of K+ transport in red blood cells

The properties of the α1 Na⁺-K⁺ pump were compared in Dahl salt-sensitive (DS) and salt-resistant (DR) strains by measuring ouabain-sensitive luxes (mmol/liter cell x hr = FU, Mean ± se) in red blood cells (RBCs) and varying internal ( _i ) and external ( _o ) Na⁺ and K⁺ concentrations. Kinetic parameters of several modes of operation, i.e., Na⁺/ K⁺, K⁺/K⁺, Na⁺/Na⁺ exchanges, were characterized and analyzed for curve-fitting using the Enzfitter computer program. In unidirectional flux studies (n=12 rats of each strain) into fresh cells incubated in 140 mm Na⁺ + 5 mm K⁺, ouabain-sensitive K⁺ influx was substantially lower in the DS than in DR RBCs, while ouabain-sensitive Na⁺ efflux and Na _i were similar in both strains. Thus, the coupling ratio between unidirectional Na⁺∶K⁺ fluxes was significantly higher in DS than in DR cells at similar RBC Na⁺ content. In the presence of 140 mm Na _o , activation of ouabain-sensitive K⁺ influx by K _o had a lower K _m and V _max in DS as estimated by the Garay equation (N=2.70 ± 0.33, K _m 0.74 ± 0.09 mm; V _max 2.87 ± 0.09 FU) than in DR rats (N=1.23 ± 0.36, K _m 2.31 ± 0.16 mm; v _max 5.70 ± 0.52 FU). However, the two kinetic parameters were similar following Na _o removal. The activation of ouabain-sensitive K⁺ influx by Na _i had significantly lower V _max in DS (9.3 ± 0.4 FU) than in DR (14.5 ± 0.6 FU) RBCs but similar K _m. These data suggest that the low K⁺ influx in DS cells is caused by a defect in modulation by Na _o and Na _i . Na⁺ efflux showed no differences in Na _i activation or trans effects by Na _o and K _o , thus accounting for the different Na⁺∶K⁺ coupling ratio in the Dahl strains. Further evidence for the differences in the coupling of ouabain-sensitive fluxes was found in studies of net Na⁺ and K⁺ fluxes, where the net ouabain-sensitive Na⁺ losses showed similar magnitudes in the two Dahl strains while the net ouabainsensitive K⁺ gains were significantly greater in the DR than the DS RBCs. Ouabain-sensitive Na⁺ influx and K⁺ efflux were also measured in these rat RBCs. The inhibition of ouabain-sensitive Na⁺ influx by K _o was fully competitive for the DS but not for the DR pumps. Thus, for DR pumps, K _o could activate higher K⁺ influx in DR pumps without a complete inhibition of ouabain-sensitive Na⁺ influx. This behavior is consistent with K _o interaction with distinct Na⁺ and K⁺ transport sites. In addition, the inhibition of K⁺ efflux by Na, was different between Dahl strains. Ouabain-sensitive K⁺ efflux at Na _i level of 4.6 mmol/liter cell, was significantly higher in DS (3.86 ± 0.67 FU) than in DR (0.86 ± 0.14 FU) due to a threefold higher K₅₀ for Na _i -inhibition 9.66 ± 0.41 vs. 3.09 ± 0.11 mmol/liter cell. This finding indicates that Na⁺ modulation of K⁺ transport is altered at both sides of the membrane. The dissociation of Na⁺ modulatory sites of K⁺ transport from Na⁺ transport sites observed in RBCs of Dahl strains suggests that K⁺ transport by the Na⁺-K⁺ pump is controlled by Na⁺ allosteric sites different from the Na⁺ transport sites. The alterations in K⁺ transport may be related to the amino acid substitution (Leu/Gln₂₇₆) reported for the cDNA of the α1 subunit of the Na⁺-K⁺ pump in the DS strain or to post-translational modifications during RBC maturation. These studies were supported by the following grants: NIH (HL-35664, HL-42120, HL-18318, HL-39267, HL-01967). J.R.R. is a Ford Foundation Predoctoral Fellow. A preliminary report of this work was presented at the International Conference on the Na⁺-K⁺ pump and 44th Annual Meeting of the Society of General Physiologists held at Woods Hole, MA, September 5–9, 1990, and published as an abstract in the J. Gen. Physiol. 96:70a, 1990.     

Variable affinity of the (Na+ + K+)-dependent adenosine triphosphatase for potassium: Studies using beryllium inactivation

Inactivation of the (Na⁺ + K⁺)-dependent ATPase by 50 μm BeCl₂ occurred during brief incubations in the presence of both Mg²⁺ and K⁺. Inactivation followed, initially, a first-order time course, with rate constants sensitive to the concentration of K⁺ (other components held constant). From these data dissociation constants can be calculated for K⁺ binding to sites controlling inactivation. Comparisons of relative affinities for K⁺ analogs (T1⁺ and NH₄⁺), and of sensitivity to reagents altering K⁺ activation (phlorizin and dimethylsulfoxide) indicate that the same K⁺ sites operate for both Be²⁺ inactivation and enzyme activation. With 3 mm MgCl₂ the dissociation constant, K_D, for K⁺ was 1.4 mm, but decreased 20-fold on addition of both Na⁺ and CTP. Alone, Na⁺ increased the apparent K_D for K⁺, either by direct competition or indirectly from its own site, with a K_D of 7 mm. The data suggest a model for K⁺ transport with K⁺ sites on the outer membrane surface that increase in affinity after formation of the phosphorylated enzyme intermediate, sufficiently to bind K⁺ in a high Na⁺ environment. Translocation may occur by an “oscillating pore” mechanism discharging K⁺ at the inner surface, while leaving demonstrable sites of moderate affinity at the outer end of the pore (which preclude attempts to document low-affinity discharge sites).     

The effect of ultraviolet light on the sodium and potassium composition of resting yeast cells

The Na⁺ and K⁺ content of non-metabolizing yeast cells was determined before and after monochromatic ultraviolet (UV) irradiation. UV facilitated the uptake of Na⁺ into and the loss of K⁺ from the cells (net ion flux); the effect is greatest for the shortest wavelength employed (239 mµ) and is partly dependent upon the presence of oxygen. The UV effect on net ion flux persists for at least 90 minutes during which tests were made and it occurs following dosages which are without measurable effect on colony formation. The UV effect on net ion flux is decreased by acidity and promoted by alkalinity. Addition of calcium ions in sufficient amount prevents the usual net ion flux changes observed in irradiated yeast. Increase in concentration gradient between the inside and the outside of the cell increases the net ion flux of irradiated yeast, Na⁺ uptake leading K⁺ loss in all cases. UV appears to act by disorganizing the constituents of the cell surface, permitting K⁺ to leave the cell in exchange for Na⁺. At low intensities of UV this ionic exchange approaches equivalence, but at higher intensities more Na⁺ is taken up than K⁺ is lost. Some evidence suggests that the Na⁺ in excess over that exchanged for K⁺ is adsorbed to charged groups produced by the photochemical effect of UV on the cell surface.     

Rescue of Na+ Affinity in Aspartate 928 Mutants of Na+,K+-ATPase by Secondary Mutation of Glutamate 314

The Na⁺,K⁺-ATPase binds Na⁺ at three transport sites denoted I, II, and III, of which site III is Na⁺-specific and suggested to be the first occupied in the cooperative binding process activating phosphorylation from ATP. Here we demonstrate that the asparagine substitution of the aspartate associated with site III found in patients with rapid-onset dystonia parkinsonism or alternating hemiplegia of childhood causes a dramatic reduction of Na⁺ affinity in the α1-, α2-, and α3-isoforms of Na⁺,K⁺-ATPase, whereas other substitutions of this aspartate are much less disruptive. This is likely due to interference by the amide function of the asparagine side chain with Na⁺-coordinating residues in site III. Remarkably, the Na⁺ affinity of site III aspartate to asparagine and alanine mutants is rescued by second-site mutation of a glutamate in the extracellular part of the fourth transmembrane helix, distant to site III. This gain-of-function mutation works without recovery of the lost cooperativity and selectivity of Na⁺ binding and does not affect the E₁-E₂ conformational equilibrium or the maximum phosphorylation rate. Hence, the rescue of Na⁺ affinity is likely intrinsic to the Na⁺ binding pocket, and the underlying mechanism could be a tightening of Na⁺ binding at Na⁺ site II, possibly via movement of transmembrane helix four. The second-site mutation also improves Na⁺,K⁺ pump function in intact cells. Rescue of Na⁺ affinity and Na⁺ and K⁺ transport by second-site mutation is unique in the history of Na⁺,K⁺-ATPase and points to new possibilities for treatment of neurological patients carrying Na⁺,K⁺-ATPase mutations.     

The Na+, K+, and Cl- Content of Goose Salt Gland Slices and the Effects of Acetylcholine and Ouabain

In goose salt gland slices incubated in bicarbonate-buffered medium which contained 170 mEq of Na⁺/liter, net total tissue Na⁺, expressed as milliequivalents per kilogram, was, in the presence of either acetylcholine (plus eserine) or ouabain, significantly higher than that of the bathing fluid. Acetylcholine caused an increase in the tissue Na⁺ content as compared with untreated slices; there was an approximately equivalent decrease in K⁺ and a significant decrease in Cl^-. The calculated net intracellular concentrations of Na⁺, expressed as milliequivalents per liter of intracellular water, in unstimulated, acetylcholine-stimulated, and ouabain-treated slices were 2.1, 3.1, and 2.7 times higher, respectively, than the concentration of Na⁺ in the bathing fluid. The net intracellular concentration of Na⁺, expressed as milliequivalents per liter of intracellular water, in slices incubated in the presence of acetylcholine was 531 mEq/liter; this is approximately the same as the concentration of Na⁺ in the secreted fluid of the goose salt gland (515 mEq/liter). The results indicate that the main concentration gradient for Na⁺ could be established across the basal membrane. The data do not indicate whether this involves active transport of Na⁺ per se. A second stage which might involve Na-K ATPase activity at the luminal membrane is discussed. The sum of the total tissue Na⁺ and K⁺ was approximately 250 mEq/kg, whereas the Cl^- content was only approximately 130 mEq/kg.     

Simultaneous net accumulation of both K+ and Na+ by lymphocytes at 0°C

Human lymphocytes at 0°C in low Na⁺ medium accumulate both K⁺ and Na⁺ to levels higher than in the external medium. This is not due to an impermeable compartment or a Donnan equilibrium, and is incompatible with the membrane Na⁺-pump concept. In contrast, it supports prior evidence that ion exchange in lymphocytes is mediated by adsorption onto and desorption from fixed anionic sites within the cell. Additional aspects of ion and water contents of cells in low Na⁺ medium are described and are explained by this concept.     

Characterization of cell ion exchange in the sea anemoneCondylactis gigantea

Summary Maintenance of intracellular ion contents and their relations to transmembrane potential were studied in tentacles ofCondylactis gigantea. Tentacles leached at 2°C in 10 mMK⁺ and 2 mM K⁺ artificial seawaters (10K ASW and 2K ASW) with and without 2 MM ouabain, and in 0K ASW, lost cell K⁺ and gained Na⁺. Rewarming to 25°C in 10K ASW resulted in a marked accumulation of K⁺ and extrusion of Na⁺ in tentacles leached in 10K ASW and in 0K ASW. Initial rate of Na⁺ extrusion was twice the initial rate of K⁺ accumulation, suggesting a pump coupling ratio of 2. In tentacles leached and rewarmed in 2K ASW, no net reaccumulation of K⁺ and little net extrusion of Na⁺ was observed; i.e., the pump just kept pace with the leaks. Ouabain inhibited K⁺ reaccumulation and Na⁺ extrusion. This effect was less marked in 10K ASW than in 2K ASW confirming, in anemone tentacles, the well documented ouabain-K⁺ antagonism observed in other systems. In no case did K _i ⁺ /K ₀ ⁺ equal Cl ₀ ^– /Cl _i ^– ; therefore, the distribution of these ions did not fit a Donnan distribution. Transmembrane potential difference was –22±3 mV in 10K ASW at 25°C. It fitted a modified Nernst equation which includes the pump coupling ratio and a Na⁺ to K⁺ permeability ratio of 0.31.A moderately high permeability of the cell membrane to Na⁺, and a ouabain and K⁺ sensitive ion pump, exchanging 2 Na⁺ for 1 K⁺, appear to be responsible for the observed ionic distribution and transmembrane potential in anemone cells.     

Determination of the coupling ratio for Na+-H+ exchange in renal microvillus membrane vesicles

We evaluated the H⁺:Na⁺ coupling ratio of the Na⁺-H⁺ exchanger present in microvillus membrane vesicles isolated from the rabbit renal cortex. Our approach was to impose transmembrane Na⁺ and H⁺ gradients of varying magnitude and then to measure the net flux of Na⁺ over the subsequent 5-s period. The Na⁺-H⁺ exchanger was observed to be at equilibrium (i.e. no significant net Na⁺ flux) whenever [Na⁺]_i/[Na⁺]_o was equal to [H⁺]_i/[H⁺]_o. Moreover, under all conditions the magnitude and direction of net Na⁺ flux was independent of changes in the transmembrane electrical potential difference. These results are consistent with a value of 1.0 for the coupling ratio of Na⁺-H⁺ exchange in renal microvillus membrane vesicles.     

Studies on the glycoprotein component of (Na+-K+)ATPase from guinea pig brain

In this study an attempt was made to elucidate the possible mechanism of the brain microsomal (Na⁺-K⁺)ATPase inhibition based on the assumption that glycoprotein part of the enzyme is exposed on the outer membrane surface. In our experiments the modification with concanavalin A of sugar end groups exposed by neuraminidase treatment resulted in a significant decrease of the brain (Na⁺-K⁺)ATPase activity. The percentage of the enzyme inhibition by concanavalin A binding to the neuraminidase-treated preparation corresponds to the amount of liberated sialic acids. The modification of the glycoprotein part of the brain (Na⁺-K⁺)ATPase complex by neuraminidase and concanavalin A treatments did not affect K⁺-nitrophenylphosphatase activity.     

Kinetic studies on the (Na+ + K+)-dependent ATPase. Evidence for coexisting sites for Na+, K+ and Mg2+

Na⁺-ATPase activity of a dog kidney (Na⁺ + K⁺)-ATPase enzyme preparation was inhibited by a high concentration of NaCl (100 mM) in the presence of 30 μM ATP and 50 μM MgCl₂, but stimulated by 100 mM NaCl in the presence of 30 μM ATP and 3 mM MgCl₂. The $\text{K}{}_{0.5}$ for the effect of MgCl₂ was near 0.5 mM. Treatment of the enzyme with the organic mercurial thimerosal had little effect on Na⁺-ATPase activity with 10 mM NaCl but lessened inhibition by 100 mM NaCl in the presence of 50 μM MgCl₂. Similar thimerosal treatment reduced (Na⁺ + K⁺)-ATPase activity by half but did not appreciably affect the $\text{K}{}_{0.5}$ for activation by either Na⁺ or K⁺, although it reduced inhibition by high Na⁺ concentrations. These data are interpreted in terms of two classes of extracellularly-available low-affinity sites for Na⁺: Na⁺-discharge sites at which Na⁺-binding can drive E₂-P back to E₁-P, thereby inhibiting Na⁺-ATPase activity, and sites activating E₂-P hydrolysis and thereby stimulating Na⁺-ATPase activity, corresponding to the K⁺-acceptance sites. Since these two classes of sites cannot be identical, the data favor co-existing Na⁺-discharge and K⁺-acceptance sites. Mg²⁺ may stimulate Na⁺-ATPase activity by favoring E₂-P over E₁-P, through occupying intracellular sites distinct from the phosphorylation site or Na⁺-acceptance sites, perhaps at a coexisting low-affinity substrate site. Among other effects, thimerosal treatment appears to stimulate the Na⁺-ATPase reaction and lessen Na⁺-inhibition of the (Na⁺ + K⁺)-ATPase reaction by increasing the efficacy of Na⁺ in activating E₂-P hydrolysis.     

A cooperative transition theory applied to the kinetics of ionic exchanges in cells

Manifestations of a cooperative interaction between ion-adsorbing sites in cells include steep, sigmoidal equilibrium adsorption isotherms of K⁺ and Na⁺, critical temperature transitions of net exchanges of Na⁺ for K⁺, and the allosteric nature of the effects of ligands on cellular K⁺ and Na⁺. Cooperative ionic adsorption is described by a onedimensional Ising model. The experimentally-determined equilibrium parameters permit prediction of the kinetics of exchange of K⁺ for Na⁺ (the approach to equilibrium) by stochastic or hydrodynamic solutions of a time-dependent Ising model. Studies of the rates of self-exchange of adsorbed ions reveal properties of the cooperatively interacting adsorption sites and their dependence on temperature and chemical potential. High rates of isotopic exchanges of K⁺ and Na⁺ occur near the transition point. This is explained by the hypothesis of an increase in susceptibility of the ensemble to slight variations of {ie93-1} or {ie93-2} near the phase transition, which leads to an increase in microscopic fluctuations within the ensemble. It is suggested that the isotopic ionic exchange experiment may be a means to explore the microscopic states of the ensemble and their transition probabilities.     

Characterization of Na+K+-ATPase in bovine sperm

Existing as a ubiquitous transmembrane protein, Na⁺K⁺-ATPase affects sperm fertility and capacitation through ion transport and a recently identified signaling function. Functional Na⁺K⁺-ATPase is a dimer of α and β subunits, each with isoforms (four and three, respectively). Since specific isoform pairings and locations may influence or indicate function, the objective of this study was to identify and localize subunits of Na⁺K⁺-ATPase in fresh bull sperm by immunoblotting and immunocytochemistry using antibodies against α1 and 3, and all β isoforms. Relative quantity of Na⁺K⁺-ATPase in head plasma membranes (HPM's) from sperm of different bulls was determined by densitometry of immunoblot bands, and compared to bovine kidney. Sperm and kidney specifically bound all antibodies at kDa equivalent to commercial controls, and to additional lower kDa bands in HPM. Immunofluorescence of intact sperm confirmed that all isoforms were present in the head region of sperm and that α3 was also uniformly distributed post-equatorially. Permeabilization exposing internal membranes typically resulted in an increase in fluorescence, indicating that some antibody binding sites were present on the inner surface of the HPM or the acrosomal membrane. Deglycosylation of β1 reduced the kDa of bands in sperm, rat brain and kidney, with the kDa of the deglycosylated bands differing among tissues. Two-dimensional blots of β1 revealed three distinct spots. Based on the unique quantity, location and structure Na⁺K⁺-ATPase subunits in sperm, we inferred that this protein has unique functions in sperm.     

High affinity k uptake in maize roots: a lack of coupling with h efflux

We report here on the putative coupling between a high affinity K⁺ uptake system which operates at low external K⁺ concentrations (K_m = 10-20 micromolar), and H⁺ efflux in roots of intact, low-salt-grown maize plants. An experimental approach combining electrophysiological measurements, quantification of unidirectional K⁺(⁸⁶Rb⁺) influx, and the simultaneous measurement of net K⁺ and H⁺ fluxes associated with individual cells at the root surface with K⁺- and H⁺-selective microelectrodes was utilized. A microelectrode system described previously (IA Newman, LV Kochian, MA Grusak, and WJ Lucas [1987] Plant Physiol 84: 1177-1184) was used to quantify net ion fluxes from the measurement of electrochemical potential gradients for K⁺ and H⁺ ions within the unstirred layer at the root surface. No evidence for coupling between K⁺ uptake and H⁺ efflux could be found based on: (a) extremely variable K⁺:H⁺ flux stoichiometries, with K⁺ uptake often well in excess of H⁺ efflux; (b) dramatic time-dependent variability in H⁺ extrusion when both fluxes were measured at a particular location along the root over time; and (c) a lack of pH sensitivity by the high affinity K⁺ uptake system (to changes in external pH) when net K⁺ uptake, unidirectional K⁺(⁸⁶Rb⁺) influx, and K⁺-induced depolarizations of the membrane potential were determined in uptake solutions buffered at pH values from pH 4 to 8. Based on the results presented here, we propose that high affinity active K⁺ absorption into maize root cells is not mediated by a K⁺/H⁺ exchange mechanism. Instead, it is either due to the operation of a K⁺-H⁺ cotransport system, as has been hypothesized for Neurospora, or based on the striking lack of sensitivity to changes in extracellular pH, uptake could be mediated by a K⁺-ATPase as reported for Escherichia coli and Saccharomyces.