Correlation between Possession of a Respiration-Dependent Na Pump and Na Requirement for Growth of Marine Bacteria

The possession of a respiration-dependent primary sodium pump and the requirement of Na for growth were investigated in bacterial isolates from marine environments. The bacteria in which NADH oxidase specifically required Na for maximum activity were believed to possess a primary sodium pump. All bacteria that failed to grow without the addition of NaCl possessed a primary Na pump. All bacteria that had no primary Na pump grew without additional NaCl. The primary Na pump seems to be involved in the Na requirement of marine bacteria, and this can be regarded as a criterion for the definition of marine bacteria.     

Respiratory Na+ pump and Na+-dependent energetics inVibrio alginolyticus

The marine bacteriumVibrio alginolyticus was found to possess the respiratory Na⁺ pump that generates an electrochemical potential of Na⁺, which plays a central role in bioenergetics ofV. alginolyticus, as a direct result of respiration. Mutants defective in the Na⁺ pump revealed that one of the two kinds of NADH: quinone oxidoreductase requires Na⁺ for activity and functions as the Na⁺ pump. The Na⁺ pump composed of three subunits was purified and reconstituted into liposomes. Generation of membrane potential by the reconstituted proteoliposomes required Na⁺. The respiratory Na⁺ pump coupled to the NADH: quinone oxidoreductase was found in wide varieties of Gramnegative marine bacteria belonging to the generaAlcaligenes, Alteromonas, andVibrio, and showed a striking similarity in the mode of electron transfer and enzymic properties. Na⁺ extrusion seemed to be coupled to a dismutation reaction, which leads to the formation of quinol and quinone from semi-quinone radical.     

A primary respiratory Na+ pump of an anaerobic bacterium: the Na+-dependent NADH:quinone oxidoreductase of Klebsiella pneumoniae

Membranes of Klebsiella pneumoniae, grown anaerobically on citrate, contain a NADH oxidase activity that is activated specifically by Na⁺ or Li⁺ ions and effectively inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Cytochromes b and d were present in the membranes, and the steady state reduction level of cytochrome b increased on NaCl addition. Inverted bacterial membrane vesicles accumulated Na⁺ ions upon NADH oxidation. Na⁺ uptake was completely inhibited by monensin and by HQNO and slightly stimulated by carbonylcyanide-p-trifluoromethoxy phenylhydrazone (FCCP), thus indicating the operation of a primary Na⁺ pump. A Triton extract of the bacterial membranes did not catalyze NADH oxidation by O₂, but by ferricyanide or menadione in a Na⁺-independent manner. The Na⁺-dependent NADH oxidation by O₂ was restored by adding ubiquinone-1 in micromolar concentrations. After inhibition of the terminal oxidase with KCN, ubiquinol was formed from ubiquinone-1 and NADH. The reaction was stimulated about 6-fold by 10 mM NaCl and was severely inhibited by low amounts of HQNO. Superoxide radicals were formed during electron transfer from NADH to ubiquinone-1. These radicals disappeared by adding NaCl, but not with NaCl and HQNO. It is suggested that the superoxide radicals arise from semiquinone radicals which are formed by one electron reduction of quinone in a Na⁺-independent reaction sequence and then dismutate in a Na⁺ and HQNO sensitive reaction to quinone and quinol. The mechanism of the respiratory Na⁺ pump of K. pneumoniae appears to be quite similar to that of Vibrio alginolyticus.     

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⁺.     

The Na+ cycle of extreme alkalophiles: A secondary Na+/H+ antiporter and Na+/solute symporters

Extremely alkalophilic bacteria that grow optimally at pH 10.5 and above are generally aerobic bacilli that grow at mesophilic temperatures and moderate salt levels. The adaptations to alkalophily in these organisms may be distinguished from responses to combined challenges of high pH together with other stresses such as salinity or anaerobiosis. These alkalophiles all possess a simple and physiologically crucial Na⁺ cycle that accomplishes the key task of pH homeostasis. An electrogenic, secondary Na⁺/H⁺ antiporter is energized by the electrochemical proton gradient formed by the proton-pumping respiratory chain. The antiporter facilitates maintenance of a pH_in that is two or more pH units lower than pH_out at optimal pH values for growth. It also largely converts the initial electrochemical proton gradient formed by respiration into an electrochemical sodium gradient that energizes motility as well as a plethora of Na⁺/solute symporters. These symporters catalyze solute accumulation and, importantly, reentry of Na⁺. The extreme nonmarine alkalophiles exhibit no primary sodium pumping dependent upon either respiration or ATP. ATP synthesis is not part of their Na⁺ cycle. Rather, the specific details of oxidative phosphorylation in these organisms are an interesting analogue of the same process in mitochondria, and may utilize some common features to optimize energy transduction.     

Growth responses of blue-green algae to sodium chloride concentration

Summary General characteristics of blue-green algal halotolerance were studied by growth experiments and selected analyses. Variation in NaCl concentration was used to mimic salinity. Marine isolates were more halotolerant (8–10% NaCl) than non-marine isolates (2% NaCl). The Na⁺ requirement for growth was saturated at 1 mg NaCl/l for non-marine isolates and 100mg NaCl/l for marine isolates. Intracellular Na⁺ values were affected by washing; however, bound-K⁺ values for both marine and fresh-water blue-green algae were fairly constant, 1–3 g/mg cells. A specific Na⁺ function was implied by the retention after washing of ²²Na⁺ (0.1 g/mg cells) by Agmenellum quadruplicatum (PR-6), a marine coccoid blue-green alga.High concentrations of NaCl apparently inhibit growth more by ionic (Na⁺) stress than by osmotic stress. Changes in light, temperature, pH, or composition of the basal medium failed to alleviate this stress.In contrast to marine bacteria, cells of PR-6 grown in Medium ASP-2+90 g NaCl/l did not undergo lysis when suspended in distilled water. However, viability of cells grown in Medium ASP-2+90 g NaCl/l decreased rapidly compared to cells grown in Medium ASP-2+18 g NaCl/l.Cells of PR-6 grown in ASP-2+90 g NaCl/l were larger than normal, formed chains (3–16 cells), and appeared bleached. Analyses of such cells revealed an overall decrease in fatty acids, hydrocarbons, and pigment levels. Electron micrographs showed that NaCl stressed cells were little altered in morphology.The photosynthesis of PR-6 cells was immediately depressed when the cells were transferred from 18 g NaCl/l to 70 g NaCl/l medium. When held in the latter for several hours the rate recovered and approached the initial photosynthetic rate maintained before NaCl-shock. This phenomenon was never seen with non-marine isolates. The explanation may lie in the ability of the cell to adjust to sudden Na⁺ increase via an ion (Na⁺) pump, for example, adenosine triphosphatase (ATPase). Subsequent assays suggested more ATPase activity in a marine isolated than in a nonmarine isolate. The ATPase was not, however, ouabain sensitive.It is suggested that marine blue-green algal isolates are characteristically more halotolerant, perhaps by selection, than fresh-water forms. This difference may be due in part to inherent capacity of the cell to extrude Na⁺. Alternatively, in freshwater forms rhe Na⁺ functional sites may be more Na⁺ sensitive than in marine forms.     

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.     

Na+ and K+ transport in Nitrosomonas europaea and Nitrobacter agilis

Potassium-depleted cells of Nitrosomonas europaea and Nitrobacter agilis were prepared by diethanolamine treatment and contained less than 5 mM intracellular K⁺. The addition of K⁺ to K⁺-depleted cells of N. europaea and N. agilis resulted in a depolarization of membrane potential (ΔΨ) by about 5 and 10 mV, respectively. This depolarization was, however, compensated by an equivalent increase in transmembrane pH gradient (ΔpH), so that the total proton-motive force (Δp) remained constant, indicating that K⁺ transport was electrogenic in both bacteria. Using ²²Na⁺-loaded cells, it is shown that both bacteria lack a respiration-dependent Na⁺ pump; however, antiporters for Na⁺/H⁺, K⁺/Na⁺ and K⁺/H⁺ were detected. Of these, at least the K⁺/Na⁺ antiporter required an electrochemical gradient for its operation. It is also shown that the unprotonated form of NH₄⁺ is transported into these bacteria by a simple diffusion mechanism.     

Inward-directed current generated by the Na+,K+ pump in Na+- and K+-free medium

In Na⁺- and K⁺-free solution, an inward-directed current can be detected in Xenopus oocytes, which is inhibited by cardic glycosides and activated by ATP. Therefore, it is assumed to be generated by the Na⁺, K⁺ pump. At negative membrane potentials, the pump current increases with more negative potentials and with increasing [H⁺] in the external medium. This current is not observed when Mg²⁺ instead of Ba²⁺ is the only divalent cation present in the bath medium, and it does not depend on whether Na⁺ or K⁺ is present internally. At 5 to 10 mM Na⁺ externally, maximum pump-generated current is obtained while no current can be detected in presence of physiological [Na⁺]. It is suggested that in low-Na⁺ and K⁺-free medium the Na⁺, K⁺ pump molecule can either form a conductive pathway that is permeable to Ba²⁺ or protons or operate in its conventional transport mode accepting Ba²⁺ as a K⁺ congener. A reversed pump mode or an electrogenic uncoupled Na⁺-efflux mode is excluded.     

Na+-ATPases of halotolerant microalgae

Na⁺ homeostasis in the cytoplasm is a common property of all organisms, irrespective of their taxonomic position. Low Na⁺ concentrations in the cytoplasm of living cells are maintained by specialized Na⁺-transporting molecular machines operating in the cell membranes. In eukaryotic cells, Na⁺-transporting ATPases of P-type play the important role in keeping the Na⁺ homeostasis. This review summarizes the authors’ investigations demonstrating the operation of the Na⁺-transporting P-type ATPases in the plasma membrane of green marine microalgae. Experiments described here provided the first evidence for the existence of the primary Na⁺-pump in plasma membranes of organisms attributed to the plant kingdom. The significance of the Na⁺-ATPases in halotolerant microalgae Dunaliella maritima and Tetraselmis viridis inhabiting saline environments is discussed.     

Na+ uptake and extrusion in the cyanobacterium SynechocystisPCC6714 in response to hypersaline treatment. Evidence for transient changes in plasmalemma Na+ permeability

The kinetics of uptake and loss of Na⁺ have been studied using radioisotopic tracer techniques in cells of the cyanobacterium Synechocystis PCC6714 exposed to hyperosmotic stress. Cells transferred from a fresh-water-based medium to NaCl at 300–1000 mmol·dm⁻³ showed net Na⁺ uptake during the first 2 min following transfer, with the intracellular Na⁺ level at 2 min increasing as a direct function of the external NaCl concentration. Further incubation of cells in low-level hypersaline media (350–500 mmol · dm⁻³ NaCl) led to a marked reduction in cell Na⁺ within 20 min, indicating an efficient active Na⁺ extrusion system. In contrast, cells maintained in more extreme hypersaline media showed little (750 mmol · dm⁻³ NaCl) or no (1000 mmol · dm⁻³ NaCl) net Na⁺ extrusion following upshock. Cells grown in a saline medium (with NaCl at 500 mmol · dm⁻³ showed a greatly reduced net Na⁺ uptake after 2 min in media containing higher levels of NaCl. However, net Na⁺ uptake was also observed when these cells were downshocked to media containing 50–200 mmol · dm⁻³ NaCl. This is the first demonstration of downshock-induced Na⁺ accumulation in a microbial cell. Time-courses for Na⁺ extrusion in cells downshocked from 500 mmol · dm⁻³ to 100 mmol · dm⁻³ NaCl were similar to those for cells upshocked from freshwater to 500 mmol · dm⁻³ NaCl, requiring approximately 30 min to reach their lowest values. Net Na⁺ extrusion in upshocked cells was found to be markedly sensitive to the external K⁺ concentration, with limited net Na⁺ extrusion in the absence of external K⁺ and maximal reductions in cell Na⁺ in media containing K⁺ at 1–10 mmol · dm⁻³. Temperature was also shown to affect uptake and loss of cell Na⁺ during upshock: cells maintained at 5°C showed no capacity for net Na⁺ extrusion, while higher temperatures (up to 35°C) led to a progressive reduction in the amount of cell Na⁺ at 2 min following upshock and also in the rate of net Na⁺ extrusion after this time.     

Role of Tonoplast Proton Pumps and Na+/H+ Antiport System in Salt Tolerance of Populus euphratica Oliv.

Populus euphratica has been used as a plant model to study resistance against salt and osmotic stresses, with recent studies having characterized the tonoplast and the plasma membrane ATPases, and two Na⁺/H⁺ antiporters, homologs of the Arabidopsis tonoplast AtNHX1, were published in databases. In the present work we show that P. euphratica suspension-cultured cells are highly tolerant to high salinity, being able to grow with up to 150 mM NaCl in the culture medium without substantial modification of the final population size when compared to the control cells in the absence of salt. At a salt concentration of 300 mM, cells were unable to grow but remained highly viable up to 17 days after subculture. The addition of a 1-M-NaCl pulse to unadapted cells did not promote a significant loss in cell viability within 48 h. In tonoplast vesicles purified from cells cultivated in the absence of salt and from salt-stressed cells, vacuolar H⁺-pyrophosphatase (V-H⁺-PPase) seemed to be the primary tonoplast proton pump; however, there appears to be a decrease in V-H⁺-PPase activity with exposure to NaCl, in contrast to the sodium-induced increase in the activity of vacuolar H⁺-ATPase (V-H⁺-ATPase). Despite reports that in P. euphratica there is no significant difference in the concentration of Na⁺ in the different cell compartments under NaCl stress, in the present study, confocal and epifluorescence microscopic observations using a Na⁺-sensitive probe showed that suspension-cultured cells subject to a salt pulse accumulated Na⁺ in the vacuole when compared with control cells. Concordantly, a tonoplast Na⁺/H⁺ exchange system is described whose activity is upregulated by salt and, indirectly, by a salt-mediated increase of V-H⁺-ATPase activity.     

Insights into the mechanism of electron transfer and sodium translocation of the Na+-pumping NADH:quinone oxidoreductase

Na⁺-NQR is a unique energy-transducing complex, widely distributed among marine and pathogenic bacteria. It converts the energy from the oxidation of NADH and the reduction of quinone into an electrochemical Na⁺-gradient that can provide energy for the cell. Na⁺-NQR is not homologous to any other respiratory protein but is closely related to the RNF complex. In this review we propose that sodium pumping in Na⁺-NQR is coupled to the redox reactions by a novel mechanism, which operates at multiple sites, is indirect and mediated by conformational changes of the protein. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

The Basis of Higher Na+ Transport by Inner Medullary Collecting Duct Cells from Dahl Salt-Sensitive Rats: Implicating the Apical Membrane Na+ Channel

The present experiments were designed to examine the function of Na/K pumps from Dahl salt-sensitive (S) and salt-resistant (R) rats. Previous reports have suggested that there is a difference in primary sequence in the α₁ subunit, the major Na/K pump isoform in the kidney. This sequence difference might contribute to differences in NaCl excretion in these two strains which in turn could influence the systemic blood pressure. Using ``back-door' phosphorylation of pumps isolated from basolateral membranes of kidney cortex, we found no differences between S and R strains. We also examined the Na/K pumps from cultured inner medullary collecting duct (IMCD) cells. This approach takes advantage of the fact that monolayers cultured from S rats transport about twice as much Na⁺ as monolayers cultured from R rats. In cells whose apical membrane was made permeable with amphotericin B, comparison of the affinities for ouabain, Na⁺, and K⁺, respectively, showed only small or no differences between S and R monolayers. Ouabain binding showed no difference in the number of Na/K pumps on the basolateral membrane of cultured cells, despite a 2-fold difference in Na⁺ transport rates. The analysis of the steady-state Na⁺ transport indicates that Na/K pumps in IMCD monolayers from S rats operate at a higher fraction of their maximum capacity than do pumps in monolayers from R rats. The results, taken together, suggest that the major reason for the higher rate of Na⁺ transport in S monolayers is because of a primary increase in the conductive permeability of the apical membrane to Na⁺. They suggest that the epithelial Na⁺ channel is intrinsically different or differently regulated in S and R rats. Received: 6 May 1996/Revised: 16 October 1996     

Inhibitors of the Na+ K+-atpase

1. The major ionmotive ATPase, in animal cells, is the Na⁺, K⁺-ATPase or sodium pump.2. This membrane bound enzyme is responsible for the translocation of Na⁺ ions and K⁺ ions across the plasma membrane, an active transport mechanism that requires the expenditure of the metabolic energy stored within the ATP molecule.3. This ubiquitous enzyme controls directly or indirectly many essential cellular functions, such as, cell volume, free calcium concentration and membrane potential.4. It is, therefore, apparent that alterations in its regulation may play key roles in pathological processes.     

Immunocytochemical localization of Na+/K+-ATPase and V-H+-ATPase in the salivary glands of the cockroach,Periplaneta americana

The acinous salivary glands of the cockroach (Periplaneta americana) consist of four morphologically different cell types with different functions: the peripheral cells are thought to produce the fluid component of the primary saliva, the central cells secrete the proteinaceous components, the inner acinar duct cells stabilize the acini and secrete a cuticular, intima, whereas the distal duct cells modify the primary saliva via the transport of water and electrolytes. Because there is no direct information available on the distribution of ion transporting enzymes in the salivary glands, we have mapped the distribution of two key transport enzymes, the Na⁺/K⁺-ATPase (sodium pump) and a vacuolar-type H⁺-ATPase, by immunocytochemical techniques. In the peripheral cells, the Na⁺/K⁺-ATPase is localized to the highly infolded apical membrane surface. The distal duct cells show large numbers of sodium pumps localized to the basolateral part of their plasma membrane, whereas their highly folded apical membranes have a vacuolar-type H⁺-ATPase. Our immunocytochemical data are supported by conventional electron microscopy, which shows electrondense 10-nm particles (portasomes) on the cytoplasmic surface of the infoldings of the apical membranes of the distal duct cells. The apically localized Na⁺/K⁺-ATPase in the peripheral cells is probably directly involved in the formation of the Na⁺-rich primary saliva. The latter is modified by the distal duct cells by transport mechanisms energized by the proton motive force of the apically localized V-H⁺-ATPase.     

Effects of salt stress and exogenous Ca2+ on Na+ compartmentalization,ion pump activities of tonoplast and plasma membrane in Nitraria tangutorum Bobr. leaves

Nitraria tangutorum Bobr. is a typical halophyte with superior tolerance to salinity. However, little is known about its physiological adaptation mechanisms to the salt environment. In the present study, N. tangutorum seedlings were treated with different concentrations of NaCl (100, 200, 300 and 400 mmol L^?1) combined with five levels of Ca²⁺ (0, 5, 10, 15 and 20 mmol L^?1) to investigate the effects of salt stress and exogenous Ca²⁺ on Na⁺ compartmentalization and ion pump activities of tonoplast and plasma membrane (PM) in leaves. Na⁺ and Ca²⁺ treatments increased the fresh weight and dry weight of N. tangutorum seedlings. The absorption of Na⁺ in roots, stems and leaves was substantially increased with the increases of NaCl concentration, and Na⁺ was mainly accumulated in leaves. Exogenous Ca²⁺ reduced Na⁺ accumulation in roots but promoted Na⁺ accumulation in leaves. The absorption and transportation of Ca²⁺ in N. tangutorum seedlings were inhibited under NaCl treatments. Exogenous Ca²⁺ promoted Ca²⁺ accumulation in the plant. Na⁺ contents in apoplast and symplast of leaves were also significantly increased, and symplast was the main part of Na⁺ intracellular compartmentalization. The tonoplast H⁺-ATPase and H⁺-PPase activities were significantly promoted under salt stress (NaCl concentrations ≤300 mmol L^?1). PM H⁺-ATPase activities gradually increased under salt stress (NaCl concentrations ≤200 mmol L^?1) followed by decreases with NaCl concentration increasing. The tonoplast H⁺-ATPase, H⁺-PPase and PM H⁺-ATPase activities increased first with the increasing exogenous Ca²⁺ concentration, reached the maximums at 15 mmol L^?1 Ca²⁺, and then decreased. The tonoplast and PM Ca²⁺-ATPase activities showed increasing trends with the increases of NaCl and Ca²⁺ concentration. These results suggested that certain concentrations of exogenous Ca²⁺ effectively enhanced ion pump activities of tonoplast and PM as well as promoted the intracellular Na⁺ compartmentalization to improve the salt tolerance of N. tangutorum.     

Evidence for Na+/H+ antiport inMethanospirillum Hungatei

The growth and methane formation ofMethanospirillum hungatei were inhibited by an inhibitor of Na⁺/H⁺ antiport amiloride. After addition of NaCl or LiCl, when the cells had a lower intracellular pH and were deenergized, they extruded protons into the external medium. The acidification of the external medium was stimulated by protonophores and inhibited by amiloride. These findings suggest the existence of an Na⁺/H⁺ antiport in the cytoplasmic membrane ofM. hungatei and its role in the energetics of methanogenic bacteria.     

On the Concept of Resting Potential—Pumping Ratio of the Na+/K+ Pump and Concentration Ratios of Potassium Ions Outside and Inside the Cell to Sodium Ions Inside and Outside the Cell

In animal cells, the resting potential is established by the concentration gradients of sodium and potassium ions and the different permeabilities of the cell membrane to them. The large concentration gradients of sodium and potassium ions are maintained by the Na⁺/K⁺ pump. Under physiological conditions, the pump transports three sodium ions out of and two potassium ions into the cell per ATP hydrolyzed. However, unlike other primary or secondary active transporters, the Na⁺/K⁺ pump does not work at the equilibrium state, so the pumping ratio is not a thermodynamic property of the pump. In this article, I propose a dipole-charging model of the Na⁺/K⁺ pump to prove that the three Na⁺ to two K⁺ pumping ratio of the Na⁺/K⁺ pump is determined by the ratio of the ionic mobilities of potassium to sodium ions, which is to ensure the time constant τ and the τ-dependent processes, such as the normal working state of the Na⁺/K⁺ pump and the propagation of an action potential. Further, the concentration ratios of potassium ions outside and inside the cell to sodium ions inside and outside the cell are 0.3027 and 0.9788, respectively, and the sum of the potassium and sodium equilibrium potentials is ?30.3 mV. A comparative study on these constants is made for some marine, freshwater and terrestrial animals. These findings suggest that the pumping ratio of the Na⁺/K⁺ pump and the ion concentration ratios play a role in the evolution of animal cells.