NMR Evidence for Complexing of Na+ in Muscle, Kidney, and Brain, and by Actomyosin. The Relation of Cellular Complexing of Na+ to Water Structure and to Transport Kinetics

The nuclear magnetic resonance (NMR) spectrum of Na⁺ is suitable for qualitative and quantitative analysis of Na⁺ in tissues. The width of the NMR spectrum is dependent upon the environment surrounding the individual Na⁺ ion. NMR spectra of fresh muscle compared with spectra of the same samples after ashing show that approximately 70% of total muscle Na⁺ gives no detectable NMR spectrum. This is probably due to complexation of Na⁺ with macromolecules, which causes the NMR spectrum to be broadened beyond detection. A similar effect has been observed when Na⁺ interacts with ion exchange resin. NMR also indicates that about 60% of Na⁺ of kidney and brain is complexed. Destruction of cell structure of muscle by homogenization little alters the per cent complexing of Na⁺. NMR studies show that Na⁺ is complexed by actomyosin, which may be the molecular site of complexation of some Na⁺ in muscle. The same studies indicate that the solubility of Na⁺ in the interstitial water of actomyosin gel is markedly reduced compared with its solubility in liquid water, which suggests that the water in the gel is organized into an icelike state by the nearby actomyosin molecules. If a major fraction of intracellular Na⁺ exists in a complexed state, then major revisions in most theoretical treatments of equilibria, diffusion, and transport of cellular Na⁺ become appropriate.     

Competitive membrane adsorption of Na+, K+, and Ca2+ in smooth muscle cells

Summary A theory for Na⁺, K⁺ and Ca²⁺ competitive adsorption to a charged membrane is used to explain a number of experimental observations in smooth muscle. Adsorption is described by Langmuir isotherms for mono- and divalent cations which in turn are coupled in a self-consistent way to the bulk solution through the diffuse double layer theory and the Boltzman equations. We found that the dissociation constants for binding of Na⁺, K⁺ and Ca²⁺ in guinea pig taenia coli areca. 0.009, 1.0, and 4×10^–8 m, respectively. Furthermore, the effect of a Ca²⁺ pump that maintains free surface Ca²⁺ concentration constant is investigated. A decrease in intracellular Na⁺ content results in an increased Ca²⁺ uptake; part of this uptake is due to an increase in surface-bound Ca²⁺ in an intracellular compartment which is in contact with the myofilaments. Variations in the amount of charge available to bind Ca²⁺ and the surface charge density are studied and their effect interpreted in terms of different pharmacological agents.     

Energy metabolism of reticulocytes: Two different sources of energy for Na+K+-ATPase activity

Total energy production in rabbit reticulocytes amounted to 136·52 ± 6·50μmol ATP h^?1ml^?1 of reticulocytes: 88·3 per cent was provided by oxidative phosphorylation, whereas only 11·7 per cent by aerobic glycolysis. Na⁺K⁺-ATPase accounted for 23 per cent, i.e. 27·65 ± 2·55μmol ATP h^?1ml^?1 of reticulocytes, in the overall energy consumption in reticulocytes of rabbits. Under basal conditions ATP for Na⁺K⁺-ATPase activity was derived exclusively from oxidative phosphorylation. However, when the activity of Na⁺K⁺-ATPase was increased due to the stimulation of adenylate cyclase by (?)-isoprenaline, the additional energy required was provided by aerobic glycolysis. These results indicate that two different compartments, one cytosolic and the other mitochondrial, provide energy for Na⁺K⁺-ATPase activity in reticulocytes.     

Na+-sensitive component of 3-O-methylglucose uptake in frog skeletal muscle

Summary A Na⁺-sensitive uptake of 3-O-methylglucose (3-O-MG), a nonmetabolized sugar, was characterized in frog skeletal muscle. A removal of Na⁺ from the bathing solution reduced 3-O-MG uptake, depending on the amount of Na⁺ removed. At a 3-O-MG concentration of 2mm, the Na⁺-sensitive component of uptake in Ringer's solution was estimated to be about 26% of the total uptake. The magnitude of Na⁺-sensitive component sigmoidally increased with an increase of 3-O-MG in bathing solution, whereas in Na⁺-free Ringer's solution the uptake was proportional to the concentration. The half saturation of the Na⁺-sensitive component was at a 3-O-MG concentration of about 13mm, and the Hill coefficient was 1.4 to 1.6. Phlorizin (5mm), a potent inhibitor specific for Na⁺-coupled glucose transport, reduced the uptake in a solution containing Na⁺ to the level in Na⁺-free Ringer's solution. Glucose of concentrations higher than 20mm suppressed 3-O-MG uptake to a level slightly lower than that in Na⁺-free Ringer's solution. These observations indicate that there are Na⁺-coupled sugar transport systems in frog skeletal muscle which are shared by both glucose and 3-O-MG.     

Ouabain switches Na+ transport to Na+/Na+ equivalent exchange in normal and apoptotic lymphoid cells

A theory of change of the ionic fluxes in the lymphoid cells in their transition from normal to apoptosis we have developed previously is applied to the analysis of Na⁺/Na⁺ exchange fluxes in human lymphoid cells U937 exposed to ouabain. We solve a system of equations describing changes in the intracellular concentrations of Na⁺, K⁺ and Cl^?, membrane potential and cell volume. It is shown that the Na⁺ influx (I _Na/Na) and output flux through the Na⁺/Na⁺ tract increased 4 times in 8 h after disconnecting Na⁺/K⁺-ATPase for normal cell U937. These fluxes increased 2.6 times for apoptotic cells. The value of I _Na/Na after 8 h off pump by ouabain is 97% of the total Na⁺ input for both cell types. It is concluded that ouabain not only inhibits the Na⁺/K⁺-ATPase, but also increases Na⁺ exchange fluxes through the Na⁺/Na⁺ tract, thereby switching sodium transport across the membrane of lymphoid cells to Na⁺/Na⁺ equivalent exchange.     

Glutathionylation-Dependence of Na+-K+-Pump Currents Can Mimic Reduced Subsarcolemmal Na+ Diffusion

The existence of a subsarcolemmal space with restricted diffusion for Na⁺ in cardiac myocytes has been inferred from a transient peak electrogenic Na⁺-K⁺ pump current beyond steady state on reexposure of myocytes to K⁺ after a period of exposure to K⁺-free extracellular solution. The transient peak current is attributed to enhanced electrogenic pumping of Na⁺ that accumulated in the diffusion-restricted space during pump inhibition in K⁺-free extracellular solution. However, there are no known physical barriers that account for such restricted Na⁺ diffusion, and we examined if changes of activity of the Na⁺-K⁺ pump itself cause the transient peak current. Reexposure to K⁺ reproduced a transient current beyond steady state in voltage-clamped ventricular myocytes as reported by others. Persistence of it when the Na⁺ concentration in patch pipette solutions perfusing the intracellular compartment was high and elimination of it with K⁺-free pipette solution could not be reconciled with restricted subsarcolemmal Na⁺ diffusion. The pattern of the transient current early after pump activation was dependent on transmembrane Na⁺- and K⁺ concentration gradients suggesting the currents were related to the conformational poise imposed on the pump. We examined if the currents might be accounted for by changes in glutathionylation of the β₁ Na⁺-K⁺ pump subunit, a reversible oxidative modification that inhibits the pump. Susceptibility of the β₁ subunit to glutathionylation depends on the conformational poise of the Na⁺-K⁺ pump, and glutathionylation with the pump stabilized in conformations equivalent to those expected to be imposed on voltage-clamped myocytes supported this hypothesis. So did elimination of the transient K⁺-induced peak Na⁺-K⁺ pump current when we included glutaredoxin 1 in patch pipette solutions to reverse glutathionylation. We conclude that transient K⁺-induced peak Na⁺-K⁺ pump current reflects the effect of conformation-dependent β₁ pump subunit glutathionylation, not restricted subsarcolemmal diffusion of Na⁺.     

Intracellular Na+ Regulates Epithelial Na+ Channel Maturation

Epithelial Na⁺ channel (ENaC) function is regulated by the intracellular Na⁺ concentration ([Na⁺]_i) through a process known as Na⁺ feedback inhibition. Although this process is known to decrease the expression of proteolytically processed active channels on the cell surface, it is unknown how [Na⁺]_i alters ENaC cleavage. We show here that [Na⁺]_i regulates the posttranslational processing of ENaC subunits during channel biogenesis. At times when [Na⁺]_i is low, ENaC subunits develop mature N-glycans and are processed by proteases. Conversely, glycan maturation and sensitivity to proteolysis are reduced when [Na⁺]_i is relatively high. Surface channels with immature N-glycans were not processed by endogenous channel activating proteases, nor were they sensitive to cleavage by exogenous trypsin. Biotin chase experiments revealed that the immature surface channels were not converted into mature cleaved channels following a reduction in [Na⁺]_i. The hypothesis that [Na⁺]_i regulates ENaC maturation within the biosynthetic pathways is further supported by the finding that Brefeldin A prevented the accumulation of processed surface channels following a reduction in [Na⁺]_i. Therefore, increased [Na⁺]_i interferes with ENaC N-glycan maturation and prevents the channel from entering a state that allows proteolytic processing.     

23Na multiple quantum filtered NMR characterisation of Na+ binding and dynamics in animal cells: a comparative study and effect of Na+/Li+ competition

Double quantum and triple quantum filtered ²³Na nuclear magnetic resonance techniques were used to characterise in detail the isotropic and anisotropic binding and dynamics of intra- and extracellular Na⁺ in different cellular systems, in the absence and presence of Li⁺. The kinetics of Li⁺ influx by different cell types was evaluated. At steady state, astrocytes accumulated more Li⁺ than red blood cells (RBCs), while a higher intracellular Li⁺ concentration was found in chromaffin than in SH-SY5Y cells. Anisotropic and isotropic motions were detected for extracellular Na⁺ in all cellular systems studied. Isotropic intracellular Na⁺ motions were observed in all types of cells, while anisotropic Na⁺ motions in the intracellular compartment were only detected in RBCs. ²³Na triple quantum signal efficiency for intracellular Na⁺ was SH-SY5Y > chromaffin > RBCs, while the reverse order was observed for the extracellular ions. ²³Na double quantum signal efficiency for intracellular Na⁺ was non-zero only in RBCs, and for extracellular Na⁺ the order RBCs > chromaffin > SH-SY5Y cells was observed. Li⁺ loading generally decreased intracellular Na⁺ isotropic movements in the cells, except for astrocytes incubated with a low Li⁺ concentration and increased anisotropic intracellular Na⁺ movements in RBCs. Li⁺ effects on the extracellular signals were more complex, reflecting Li⁺/Na⁺ competition for isotropic and anisotropic binding sites at the extracellular surface of cell membranes and also at the surface of the gel used for cell immobilisation. These results are relevant and contribute to the interpretation of the in vivo pharmacokinetics and sites of Li⁺ action.     

Hypo-osmotic stimulation of active Na+ transport in frog muscle: Apparent upregulation of Na+ pumps

Summary The purpose of this work was to determine if hypotonicity, in addition to the stimulation of active Na⁺ transport (Venosa, R.A., 1978,Biochim. Biophys. Acta 510:378–383), promoted changes in (i) active K⁺ influx, (ii) passive Na^– and K⁺ fluxes, and (iii) the number of³H-ouabain binding sites.The results indicate that a reduction of external osmotic pressure () to one-half of its normal value (=0.5) produced the following effects: (i) an increase in active K⁺ influx on the order of 160%, (ii) a 20% reduction in Na⁺ influx and K⁺ permeability (P _K), and (iii) a 40% increase in the apparent density of ouabain binding sites. These data suggest that the hypotonic stimulation of the Na⁺ pump is not caused by an increased leak of either Na⁺ (inward) or K⁺ (outward). It is unlikely that the stimulation of active Na⁺ extrusion and the rise in the apparent number of pump sites produced by hypotonicity were due to a reduction of the intracellular ionic strength. It appears that, at least in part, the stimulation of active Na⁺ transport takes place whenever muscles are transferred from one medium to another of lower tonicity even if neither one was hypotonic (for instance =2 to =1 transfer). Comparison of the present results with those previously reported indicate that in addition to the number of pump sites, the cycling rate of the pump is increased by hypotonicity. Active Na⁺ and K⁺ fluxes were not significantly altered by hypertonicity (=2).     

Skeletal Muscle-specific Calpain Is an Intracellular Na+-dependent Protease

Because intracellular [Na⁺] is kept low by Na⁺/K⁺-ATPase, Na⁺ dependence is generally considered a property of extracellular enzymes. However, we found that p94/calpain 3, a skeletal-muscle-specific member of the Ca²⁺-activated intracellular “modulator proteases” that is responsible for a limb-girdle muscular dystrophy (“calpainopathy”), underwent Na⁺-dependent, but not Cs⁺-dependent, autolysis in the absence of Ca²⁺. Furthermore, Na⁺ and Ca²⁺ complementarily activated autolysis of p94 at physiological concentrations. By blocking Na⁺/K⁺-ATPase, we confirmed intracellular autolysis of p94 in cultured cells. This was further confirmed using inactive p94:C129S knock-in (p94CS-KI) mice as negative controls. Mutagenesis studies showed that much of the p94 molecule contributed to its Na⁺/Ca²⁺-dependent autolysis, which is consistent with the scattered location of calpainopathy-associated mutations, and that a conserved Ca²⁺-binding sequence in the protease acted as a Na⁺ sensor. Proteomic analyses using Cs⁺/Mg²⁺ and p94CS-KI mice as negative controls revealed that Na⁺ and Ca²⁺ direct p94 to proteolyze different substrates. We propose three roles for Na⁺ dependence of p94; 1) to increase sensitivity of p94 to changes in physiological [Ca²⁺], 2) to regulate substrate specificity of p94, and 3) to regulate contribution of p94 as a structural component in muscle cells. Finally, this is the first example of an intracellular Na⁺-dependent enzyme.     

Quantification of Na+,K+ pumps and their transport rate in skeletal muscle: Functional significance

During excitation, muscle cells gain Na⁺ and lose K⁺, leading to a rise in extracellular K⁺ ([K⁺]_o), depolarization, and loss of excitability. Recent studies support the idea that these events are important causes of muscle fatigue and that full use of the Na⁺,K⁺-ATPase (also known as the Na⁺,K⁺ pump) is often essential for adequate clearance of extracellular K⁺. As a result of their electrogenic action, Na⁺,K⁺ pumps also help reverse depolarization arising during excitation, hyperkalemia, and anoxia, or from cell damage resulting from exercise, rhabdomyolysis, or muscle diseases. The ability to evaluate Na⁺,K⁺-pump function and the capacity of the Na⁺,K⁺ pumps to fill these needs require quantification of the total content of Na⁺,K⁺ pumps in skeletal muscle. Inhibition of Na⁺,K⁺-pump activity, or a decrease in their content, reduces muscle contractility. Conversely, stimulation of the Na⁺,K⁺-pump transport rate or increasing the content of Na⁺,K⁺ pumps enhances muscle excitability and contractility. Measurements of [³H]ouabain binding to skeletal muscle in vivo or in vitro have enabled the reproducible quantification of the total content of Na⁺,K⁺ pumps in molar units in various animal species, and in both healthy people and individuals with various diseases. In contrast, measurements of 3-O-methylfluorescein phosphatase activity associated with the Na⁺,K⁺-ATPase may show inconsistent results. Measurements of Na⁺ and K⁺ fluxes in intact isolated muscles show that, after Na⁺ loading or intense excitation, all the Na⁺,K⁺ pumps are functional, allowing calculation of the maximum Na⁺,K⁺-pumping capacity, expressed in molar units/g muscle/min. The activity and content of Na⁺,K⁺ pumps are regulated by exercise, inactivity, K⁺ deficiency, fasting, age, and several hormones and pharmaceuticals. Studies on the α-subunit isoforms of the Na⁺,K⁺-ATPase have detected a relative increase in their number in response to exercise and the glucocorticoid dexamethasone but have not involved their quantification in molar units. Determination of ATPase activity in homogenates and plasma membranes obtained from muscle has shown ouabain-suppressible stimulatory effects of Na⁺ and K⁺.

Introduction: Transport and content of Na⁺ and K⁺ in skeletal muscle

The Na⁺,K⁺-ATPase (also known as the Na⁺,K⁺ pump) is the major translator of metabolic energy in the form of ATP to electrical and chemical gradients for the two most common ions in the body. These gradients enable the generation of action potentials, which are essential for muscle cell function. Evaluation of the physiological and clinical significance of the Na⁺,K⁺ pumps requires measuring the transmembrane fluxes of Na⁺ and K⁺ in intact muscles or cultured muscle cells. The simplest approach involves incubating intact muscles isolated from small animals in temperature-controlled and oxygenated buffers with electrolyte and glucose concentration comparable to that normally present in blood plasma. Initial studies used cut hemi- or quarter-diaphragm muscles from rats, mice, or guinea pigs for incubation because these muscles were considered thin enough to allow adequate oxygenation under these conditions (Gemmill, 1940). However, such preparations have numerous cut muscle ends, allowing large passive movements of Na⁺ and K⁺ and free access of Ca²⁺ to the cell interior. This unavoidably boosts the energy required for active transport of Na⁺, K⁺, and Ca²⁺, and leads to impaired cell survival. Thus, in cut muscles, the components of O₂ consumption and ⁴²K uptake attributable to the Na⁺,K⁺ pump (i.e., the fraction suppressible by the cardiac glycoside ouabain, which binds to and inhibits the Na⁺,K⁺-ATPase) have been severely overestimated. (The ouabain-suppressible components of O₂ consumption or ⁴²K uptake are measured in isolated muscles incubated without or with ouabain and calculated as the difference.) Such overestimation led to the assumption that in skeletal muscle, the Na⁺,K⁺ pumps mediate a large fraction of total energy turnover, suggesting that a major part of the thermogenic action of thyroid hormone is caused by an increased rate of active Na⁺,K⁺ transport (Asano et al., 1976). In contrast, in intact resting muscle preparations, only 2–10% of the total energy turnover is used for active Na⁺,K⁺ transport (Creese, 1968; for details, see Clausen et al., 1991). Even during maximum contractile work in human muscles, only a small fraction (2%) (Medbø and Sejersted, 1990) of total energy release is used for the Na⁺,K⁺ pumps. Thus, in skeletal muscle, the thermogenic action of the Na⁺,K⁺ pumps is modest.For the analysis of Na⁺,K⁺ transport in skeletal muscle, isolated intact limb muscles are used, primarily mammalian soleus, extensor digitorum longus (EDL), extensor digitorum brevis, or epitrochlearis muscles. These preparations can survive during incubation for many hours and can undergo repeated excitation. More recently, the isolated rat sternohyoid muscle, which also offers thin dimensions, cellular integrity, and simple handling, has been introduced (Mu et al., 2011).Measurement of muscle Na⁺ and K⁺ content requires extraction. In the past, this was done by digesting the muscle preparation in nitric acid, a sometimes risky procedure. More recently, this approach has been superseded by homogenization of the tissue in 0.3 M trichloroacetic acid, followed by centrifugation to sediment the proteins (Kohn and Clausen, 1971). The clear supernatant may then be diluted for flame photometric determination of Na⁺ and K⁺ or counting of the isotopes ²²Na or ⁴²K. Because ⁴²K has a short half-life (12.5 h) and is of limited availability, the K⁺ analogue ⁸⁶Rb is often used as a tracer for K⁺. For the quantification of Na⁺,K⁺ pump–mediated (i.e., ouabain-suppressible) transport of K⁺, ⁸⁶Rb gives the same results as ⁴²K (Clausen et al., 1987; Dørup and Clausen, 1994). For other flux measurements, however, the results obtained with ⁸⁶Rb differ appreciably from those obtained with ⁴²K. For example, the fractional loss of ⁸⁶Rb from intact resting rat soleus muscles is only 45% of that measured using ⁴²K. Moreover, two agents shown to stimulate the Na⁺,K⁺ pumps in isolated rat soleus muscle, salbutamol (Clausen and Flatman, 1977) and rat calcitonin gene–related peptide (CGRP) (Andersen and Clausen, 1993), both induce a highly significant stimulation of ⁸⁶Rb efflux from the same muscle (Dørup and Clausen, 1994). In contrast, these same agents induced a rapid but transient (20-min duration) inhibition of the fractional loss of ⁴²K, indicating that a large fraction of the ⁴²K lost from the cells is reaccumulated as a result of stimulation of the Na⁺,K⁺ pumps (Andersen and Clausen, 1993; Dørup and Clausen, 1994). Finally, in skeletal muscle, bumetanide, an inhibitor of the NaK2Cl cotransporter, produces no inhibition of ⁴²K uptake but clear-cut inhibition of ⁸⁶Rb uptake (see ​and22 in Dørup and Clausen, 1994). Thus, results obtained with ⁸⁶Rb must be verified with ⁴²K or flame photometric measurements of changes in intracellular Na⁺ and K⁺ content. The activity of the Na⁺,K⁺ pump depends on ATP supplied by glycolysis or oxidative phosphorylation. Excitability of isolated rat soleus or EDL muscles can be maintained in the presence of the electron transport inhibitor cyanide or during anoxia (Murphy and Clausen, 2007; Fredsted et al., 2012), whereas contractions are markedly suppressed by 2-deoxyglucose, which interferes with the production of glycolytic ATP. Thus, in keeping with the studies of Glitsch (2001), these data suggest that glycolytic ATP is a primary source of energy for the Na⁺,K⁺ pumps (see also Okamoto et al., 2001). More focused studies showed that Na⁺,K⁺-pump function is not adequately supported when cytoplasmic ATP is at the normal resting concentration of ∼8 mM, but that the addition of as little as 1 mM phosphoenol pyruvate produces a marked increase in Na⁺,K⁺-pump function that is supported by endogenous pyruvate kinase bound within the t-tubular triad (Dutka and Lamb, 2007). In anoxic rat EDL muscles, contractility could be restored by stimulating the Na⁺,K⁺ pumps with the β₂ agonists salbutamol or terbutaline, effects that were abolished by ouabain or 2-deoxyglucose (Fredsted et al., 2012). Glycolytic ATP furnishes energy for contractile activity under the critical condition of anoxia. Because the Na⁺,K⁺ pumps are essential for the maintenance of excitability and contractions, it would not be surprising if they were also kept going on glycolytic ATP.

Table 1.

Acute stimulation of the Na⁺,K⁺ pumps in skeletal muscle

Study on the dehydrating effect of the red cell Na+/K+-pump in nystatin-treated cells with varying Na+ and water contents

Using the antibiotic Nystatin, we have developed a systematic method for the preparation of red blood cells with independently selected levels of intracellular Na⁺ concentrations and water content. Such cells provided an experimental model to study the effect of Na⁺/K⁺ pump stimulation on red cell water content. Even in initially dehydrated cells, stimulation of the Na⁺/K⁺ pump by elevated intracellular Na⁺ caused subsequent further loss of cell water. Cell water loss was reflected in decreased monovalent cation content per unit mass of hemoglobin and by a shift in the density distribution of the cell populations to higher densities on discontinuous Stractan gradients. We conclude that the 3 Na_out⁺ : 2 K_in⁺ stoichiometry of the Na⁺/K⁺ pump results in a net desalting effect with increased pump activity. Under the conditions of these experiments, the cell appears to have no effective mechanism to compensate for a net loss of ions and water.     

Na+ Sensitivity of ROMK1 K+ Channel: Role of the Na+/H+ Antiporter

To examine the extracellular Na⁺ sensitivity of a renal inwardly rectifying K⁺ channel, we performed electrophysiological experiments on Xenopus oocytes or a human kidney cell line, HEK293, in which we had expressed the cloned renal K⁺ channel, ROMK1 (Kir1.1). When extracellular Na⁺ was removed, the whole-cell ROMK1 currents were markedly suppressed in both the oocytes and HEK293 cells. Single-channel ROMK1 activities recorded in the cell-attached patch on the oocyte were not affected by removal of Na⁺ from the pipette solution. However, macro-patch ROMK1 currents recorded on the oocyte were significantly suppressed by Na⁺ removal from the bath solution. A blocker of Na⁺/H⁺ antiporters, amiloride, largely inhibited the Na⁺ removal-induced suppression of whole-cell ROMK1 currents in the oocytes. The pH-insensitive K80M mutant of ROMK1 was much less sensitive to Na⁺ removal. Na⁺ removal was found to induce a significant decrease in intracellular pH in the oocytes using H⁺-selective microelectrodes. Coexpression of ROMK1 with NHE3, which is a Na⁺/H⁺ antiporter isoform of the kidney apical membrane, conferred increased sensitivity of ROMK1 channels to extracellular Na⁺ in both the oocytes and HEK293 cells. Thus, it is concluded that the ROMK1 channel is regulated indirectly by extracellular Na⁺, and that the interaction between NHE transporter and ROMK1 channel appears to be involved in the mechanism of Na⁺ sensitivity of ROMK1 channel via regulating intracellular pH. Received: 13 April 1999/Revised: 15 July 1999     

Na+,K+-ATPase Na+ Affinity in Rat Skeletal Muscle Fiber Types

Previous studies in expression systems have found different ion activation of the Na⁺/K⁺-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used to quantify Na⁺,K⁺-ATPase activity, and the Na⁺ affinity of Na⁺,K⁺-ATPase was studied in total membranes from rat muscle and purified membranes from muscle with different fiber types. The Na⁺ affinity was higher (K _m lower) in oxidative muscle compared with glycolytic muscle and in purified membranes from oxidative muscle compared with glycolytic muscle. Na⁺,K⁺-ATPase isoform analysis implied that heterodimers containing the β₁ isoform have a higher Na⁺ affinity than heterodimers containing the β₂ isoform. Immunoprecipitation experiments demonstrated that dimers with α₁ are responsible for approximately 36% of the total Na,K-ATPase activity. Selective inhibition of the α₂ isoform with ouabain suggested that heterodimers containing the α₁ isoform have a higher Na⁺ affinity than heterodimers containing the α₂ isoform. The estimated K _m values for Na⁺ are 4.0, 5.5, 7.5 and 13 mM for α₁β₁, α₂β₁, α₁β₂ and α₂β₂, respectively. The affinity differences and isoform distributions imply that the degree of activation of Na⁺,K⁺-ATPase at physiological Na⁺ concentrations differs between muscles (oxidative and glycolytic) and between subcellular membrane domains with different isoform compositions. These differences may have consequences for ion balance across the muscle membrane.     

Na+,K+-ATPase enzyme units in skeletal muscle from lean and obese mice.

[³H]-Ouabain binding to muscle preparations was utilized to estimate the number of Na⁺,K⁺-ATPase enzyme units in hindlimbs from 8 week old lean and obese mice. Specific [³H]-ouabain binding per mg particulate protein was 36% lower in obese mice; whereas, the affinity of the binding sites for ouabain was similar in obese and lean mice. Since obese mice had less muscle than lean mice, the number of Na⁺,K⁺-ATPase enzyme units in hindlimbs from obese mice was less than half the number observed in lean mice.     

Na+, K+-ATPase in HeLa cells after prolonged growth in low K+ or ouabain

Effects of long-term, subtotal inhibition of Na⁺-K⁺ transport, either by growth of cells in sublethal concentrations of ouabain or in low-K⁺ medium, are described for HeLa cells. After prolonged growth in 2 × 10^?8 M ouabain, the total number of ouabain molecules bound per cell increases by as much as a factor of three, mostly due to internalization of the drug. There is only about a 20% increase in ouabain-binding sites on the plasma membrane, representing amodest induction of Na⁺, K⁺-ATPase. In contrast, after long-term growth in low K⁺ there can be a twofold or greater increase in ouabain binding per cell, and in this case the additional sites are located in the plasma membrane. The increase is reversible. To assess the corresponding transport changes, we have separately estimated the contributions of increased intracellular [Na⁺] and of transport capacity (number of transport sites) to transport regulation. During both induction and reversal, short-term regulation is achieved primarily by changes in [Na⁺]_i. More slowly, long-term regulation is achieved by changes in the number of functional transporters in the plasma membrane as assessed by ouabain binding, V_max for transport, and specific phosphorylation. Parallel exposure of cryptic Na⁺, K⁺-ATPase activity with sodium dodecyl sulfate in the plasma membranes of both induced and control cells showed that the induction cannot be accounted for by an exposure of preexisting Na⁺, K⁺-ATPase in the plasma membrane. Analysis of the kinetics of reversal indicates that it may be due to a post-translational event.     

Generation of plasma membrane potential by the Na+-pump coupled to proton extrusion

Lettré cells maintain a plasma membrane potential near — 60mV, yet are scarcely depolarized by 80 mM Rb⁺ and are relatively impermeable to ⁸⁶Rb⁺. They are depolarized by ouabain without a concomitant change in intracellular cation content. Addition of K⁺ to cells suspended in a K⁺ free medium, or of Na⁺ to cells in a Na⁺ free medium, hyperpolarizes the cells. They contain electroneutral transport mechanisms for Na⁺, K⁺ and H⁺ which can function as Na⁺:K⁺ and Na⁺:H⁺ exchanges. It is concluded that plasma membrane potential of Lettré cells, in steady-state for Na⁺ and K⁺, is produced by an electrogenic Na⁺ pump sustained by electroneutral exchanges, and restricted by anion leakage.     

Na+-K+ pumps in the transverse tubular system of skeletal muscle fibers preferentially use ATP from glycolysis

The Na⁺-K⁺ pumps in the transverse tubular (T) system of a muscle fiber play a vital role keeping K⁺ concentration in the T-system sufficiently low during activity to prevent chronic depolarization and consequent loss of excitability. These Na⁺-K⁺ pumps are located in the triad junction, the key transduction zone controlling excitation-contraction (EC) coupling, a region rich in glycolytic enzymes and likely having high localized ATP usage and limited substrate diffusion. This study examined whether Na⁺-K⁺ pump function is dependent on ATP derived via the glycolytic pathway locally within the triad region. Single fibers from rat fast-twitch muscle were mechanically skinned, sealing off the T-system but retaining normal EC coupling. Intracellular composition was set by the bathing solution and action potentials (APs) triggered in the T-system, eliciting intracellular Ca²⁺ release and twitch and tetanic force responses. Conditions were selected such that increased Na⁺-K⁺ pump function could be detected from the consequent increase in T-system polarization and resultant faster rate of AP repriming. Na⁺-K⁺ pump function was not adequately supported by maintaining cytoplasmic ATP concentration at its normal resting level (8 mM), even with 10 or 40 mM creatine phosphate present. Addition of as little as 1 mM phospho(enol)pyruvate resulted in a marked increase in Na⁺-K⁺ pump function, supported by endogenous pyruvate kinase bound within the triad. These results demonstrate that the triad junction is a highly restricted microenvironment, where glycolytic resynthesis of ATP is critical to meet the high demand of the Na⁺-K⁺ pump and maintain muscle excitability. muscle fatigue; sodium-potassium-adenosinetriphosphatase; excitation-contraction coupling; T-system; excitability     

Na+/Na+ exchange and Na+/H+ antiport in rabbit erythrocytes: Two distinct transport systems

Summary Rabbit erythrocytes are well known for possessing highly active Na⁺/Na⁺ and Na⁺/H⁺ countertransport systems. Since these two transport systems share many similar properties, the possibility exists that they represent different transport modes of a single transport molecule. Therefore, we evaluated this hypothesis by measuring Na⁺ transport through these exchangers in acid-loaded cells. In addition, selective inhibitors of these transport systems such as ethylisopropyl-amiloride (EIPA) and N-ethylmaleimide (NEM) were used. Na⁺/Na⁺ exchange activity, determined as the Na _o ⁺ -dependent²²Na efflux or Na _i ⁺ -induced²²Na entry was completely abolished by NEM. This inhibitor, however, did not affect the H _i ⁺ -induced Na⁺ entry sensitive to amiloride (Na⁺/H⁺ exchange activity). Similarly, EIPA, a strong inhibitor of the Na⁺/H⁺ exchanger, did not inhibit Na⁺/Na^– countertransport, suggesting the independent nature of both transport systems. The possibility that the NEM-sensitive Na⁺/Na⁺ exchanger could be involved in Na⁺/H⁺ countertransport was suggested by studies in which the net Na⁺ transport sensitive to NEM was determined. As expected, net Na⁺ transport through this transport system was zero at different [Na⁺] _i /[Na⁺] _o ratios when intracellular pH was 7.2. However, at pH _i =6.1, net Na⁺ influx occurred when [Na⁺] _i was lower than 39mm. Valinomycin, which at low [K⁺] _o was lower than 39mm. Valinomycin, which at low [K⁺] _o clamps the membrane potential close to the K⁺ equilibrium potential, did not affect the net NEM-sensitive Na⁺ entry but markedly stimulated, the EIPA-and NEM-resistant Na⁺ uptake. This suggest that the net Na⁺ entry through the NEM-sensitive pathway at low pH _i , is mediated by an electroneutral process possibly involving Na⁺/H⁺ exchange. In contrast, the EIPA-sensitive Na⁺/H⁺ exchanger is not involved in Na⁺/Na⁺ countertransport, because Na⁺ transport through this mechanism is not affected by an increase in cell Na^– from 0.4 to 39mm. Altogether, these findings indicate that both transport systems: the Na⁺/Na⁺ and Na⁺/H⁺ exchangers, are mediated by distinct transport proteins.     

Intracellular Na+ and K+ contents of Zygosaccharomyces rouxii mutants defective in salt tolerance

Two of five Zygosaccharomyces rouxii mutants defective in salt tolerance, 152S (sat1) and 1717S (SAT3), were inviable in a nutrient medium (YPD) containing more than 1% NaCl. These two mutant cells contained significantly higher amounts of Na⁺ (298 μmol and 285 μmol per g cells of 152S and 1717S, respectively) but lower amounts of K⁺ (242 μmol and 176 μmol per g cells of 152S and 1717S, respectively) than three other mutants, 41S (sat2-1 [98 μmol Na⁺ and 326 μmol K⁺/g cells]), 197S (sat2-2 [103μmol Na⁺ and 336 μmol K⁺/g cells]), 1611S (SAT4 [139 μmol Na⁺ and 294 μmol K⁺/g cells]), as well as a wild-type strain, AN39 (61 μmol Na⁺ and 349 μmol K⁺/g cells), when cultured in YPD medium containing 0.8% NaCl. A KCl supplement, optimally 0.6 M, added to the medium somewhat restored the NaCl-hypersensitivity of 152S and 1717S with a concomitant decrease of intracellular Na⁺. This finding suggests that the NaCl-hypersensitive mutations are due to a defect in the Na⁺-regulating mechanism. The other three mutants showed weak responses to KCl in high NaCl-YPD. These five salt sensitive mutants and the wild-type strain retained the same levels of intracellular glycerol and arabitol when transferred into NaCl (5%)-YPD from YDP medium. This suggests that polyol accumulation is not the only mechanism of salt tolerance in Z. rouxii.