Counter-transport mediated by the lactose permease of Escherichia coli.

When the two main energy yielding pathways, respiration and the membrane ATPase of Escherichia coli are poisoned, the lactose permease is unable to accomplish accumulative transport of thiogalactosides, but the efflux of preloaded substrate can be coupled to a transiently uphill transport of exogenous substrate. This transient uphill transport, called overshoot has been reexamined with the possibility of an obligate H+ cotransport in mind. Overshoot can be diminished but not suppressed by a proton-conducting uncoupler, carbonyl cyanide m chlorophenylhydrazone, (CCCP) and by a liposoluble cation, triphenyl-methyl phosphonium (TPMP+). The effect of other factors, such as temperature, amount of permease and pH were also explored. The overshoot was found to decrease with increasing pH, until at pH 8 it became negligible. This is in sharp contrast with the relatively flat pH dependence of uphill and downhill transport in unpoisoned cells. CCCP and TPMP+ had no inhibitory effect on the overshoot at pH 6 and below.     

An estimation of the light-induced electrochemical potential difference of protons across the membrane of Halobacterium halobium

The light-dependent uptake of triphenylmethylphosphonium (TPMP⁺) and of 5,5-dimethyloxazolidine-2,4-dione (DMO) by starved purple cells of Halobacterium halobium was investigated. DMO uptake was used to calculate the pH difference (ΔpH) across the membrane, and TPMP⁺ was used as an index of the electrical potential difference, Δψ.Under most conditions, both in the light and in the dark, the cells are more alkaline than the medium. In the light at pH 6.6, ΔpH amounts to 0.6–0.8 pH unit. Its value can be increased to 1.5–2.0 by either incubating the cells with TPMP⁺ (10^?3 M) or at low external pH (5.5). — ΔpH can be lowered by uncoupler or by nigericin. The TPMP⁺ uptake by the cells indicates a large Δψ across the membrane, negative inside. It was estimated that in the light, at pH 6.6, Δψ might reach a value of about 100 mV and that consequently the electrical equivalent of the proton electrochemical potential difference, , amounts under these conditions to about 140 mV.The effects of different ionophores on the light-driven proton extrusion by the cells were in agreement with the effects of these compounds on — ΔpH.     

A novel type of energetics in a marine alkali-tolerant bacterium: Δμ-Na-driven motility and sodium cycle

Motility of a marine alkali-tolerant bacterium, Vibrio alginolyticus, can be observed in the presence of high concentrations of a protonophorous uncoupler, CCCP. Motility in the CCCP-containing media is completely inhibited by decrease in extracellular [Na⁺] or by monensin-induced increase in intracellular [Na⁺]. A mutant has been selected that grows only in media supplemented with a substrate such as acetate requiring no Δ$\text{μ}\text{-}$_Na to be transported into the cell. Motility of the mutant was found to be completely inhibited by CCCP. Cyanide, CCCP and vanadate added separately or in twos inhibit motility only partially. The three poisons added together completely paralyse the cells. In this inhibitor cocktail, arsenate can substitute for CCCP + vanadate; cyanide can be replaced by anaerobiosis. It is concluded that (i) Δ$\text{μ}\text{-}$_Na rather than Δ$\text{μ}\text{-}$_NH powers the flagellar motor of V. alginolyticus in the presence of CCCP, and (ii) in addition to the Na⁺-motive respiratory chain [Tokuda, H. and Unemoto, T. (1982) J. Biol. Chem. 257, 10007–10014] there is a vanadate and arsenate-sensitive oxygen-independent mechanism of Δ$\text{μ}$_Na generation, presumably an ion-motive ATPase. A suggestion is put forward that circulation of Na⁺ can replace that of H⁺ in V. alginolyticus, Δ$\text{μ}\text{-}$_Na being formed by the Na⁺-motive respiratory chain and utilized by Na⁺-solute symporters, the Na⁺-driven flagellar motor and maybe by a reverse ion-motive ATPase.     

Concentrative uptake of melphalan, a cancer chemotherapeutic agent which is transported by the leucine-preferring carrier system.

The transport of melphalan, $\text{L}$-phenylalamine mustard, proceeded uphill against a concentration gradient and resulted in a distribution ratio of approximately 10. Concentrative uptake was temperature sensitive and was inhibited by the metabolic inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and $\text{L}$-leucine, a natural substrate of the transport carriers. These results indicate that melphalan transport is an energy requiring process and that naturally occurring competitive substrates such as leucine markedly reduce concentrative uptake of the drug.     

A novel mutant of the lac transport system of Escherichia coli.

A new type of lactose permease mutant, called lacY^f, does not actively transport the usual substrates; but it does facilitate the entry of β-galactosides into Escherichia coli K-12. The kinetics of facilitated entry, as assayed by hydrolysis of o-nitrophenyl-β-d-galactopyranoside by intact cells are identical to those observed with wild-type permease. However, the mutant permease activity is not affected by SH reagents or the substrate analog β-d-galactosyl-1-thio-β-d-galactopyranoside which strongly inhibit wild-type activity. Furthermore, the kinetics of formation of permease in the mutant following addition of inducer and the kinetics subsequent to removal of inducer differ strikingly from those observed in wild-type strains. The results are consistent with a block in the maturation of permease in the mutant resulting in the accumulation of a large amount of permease precursor. Studies of the $\text{lacY}{}^{\mathrm{+}}\text{lacY}{}^{\mathrm{f}}$ heterogenotes provide evidence for a subunit structure for the lactose permease.     

Phosphate transport in Neurospora: Kinetic characterization of a constitutive,low-affinity transport system

Log-phase cells of Neurospora crassa, grown in standard minimal medium, possess an energy-dependent transport system for inorganic phosphate, with a $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ (at pH 5.8) of 0.123 mM and a J_max of 1.64 mmoles/l cell water per min. Like the PO₄^3? transport system in yeast, the Neurospora system is stimulated by high intracellular K⁺. In addition, it is inhibited by high extracellular salt concentrations, an effect which may be related to the known depolarization of the Neurospora plasma membrane at high salt concentrations.The most striking property of the system is its strong dependence upon the extracellular pH. From pH 4.0 to pH 7.3, the J_max remains essentially constant but the $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ increases nearly 400-fold, from 0.01 to 3.62 mM. The increase cannot be accounted for by a single system with a preference for H₂PO₄^? (which would show only a 3-fold increase in apparent $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ over this pH range) nor by two systems with different affinities and pH optima (which would display nonlinear double-reciprocal plots at intermediate pH values). It can be explained, however, by a model in which OH^? or H⁺ is assumed to act as a modifier of the transport system, altering its affinity for substrate.     

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

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

Analysis of two chloride requirements for sodium-dependent amino acid and glucose transport by intestinal brush-border membrane vesicles of fish

Intestinal brush border vesicles of a Mediterranean sea fish (Dicentrarchus labrax) were prepared using the Ca²⁺-sedimentation method. The transport of glucose, glycine and 2-aminoisobutyric acid is energized by an Na⁺ gradient ($\text{out > in}$). In addition, amino acid uptake requires Cl^? in the extravesicular medium (2-aminoisobutyric acid more than glycine). This Na⁺- and Cl^?-dependent uptake is electrogenic, since it can be stimulated by negative charges inside the vesicles. The specific Cl^? requirement of glycine and 2-aminoisobutyric acid transport is markedly influenced by pH, a change from 6.5 to 8.4 reducing the role played by Cl^?. In the presence of Cl^?, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 2-aminoisobutyric acid uptake is reduced and its $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ is enhanced. Cl^? affects also a non-saturable Na⁺-dependent component of this amino acid uptake. Amino acid transport is also increased by intravesicular Cl^? (2-aminoisobutyric acid less than glycine). This effect is more concerned with glucose uptake, which can be then multiplied by 2.3. A concentration gradient ($\text{in > out}$) as well as the presence of Na⁺ in the incubation medium seems to enter into this requirement. This intravesicular Cl^? effect is not influenced by pH between 6.5 and 8.4.     

Internalization and degradation of a low affinity insulin ([LeuB24]insulin) by rat adipocytes

In Halobacterium halobium tactic responses towards light and chemoeffectors are accompanied by changes in the methylation level of methyl-accepting chemotaxis proteins (MCP). Taxis towards green light absorbed by the bacteriorhodopsin proton pump appears to be governed by Δ$\text{μ}$_H⁺-sensing. The addition of CCCP, an uncoupler, prevented the increase of MCP methylation in response to green light illumination, but had no effect on CH₃-incorporation followed by the addition of the attractants glucose, leucine and histidine. Similarly, CCCP did not change MCP demethylation in response to blue light illumination, a repelling stimuli.The sensitivity to an uncoupler of methylation linked to Δ$\text{μ}$_H⁺-mediated green light taxis is to be expected, while the independence of demethylation caused by the blue light of CCCP is an indication that in the latter case a specific photoreceptor governs phototaxis. Informed processing from the blue light receptor to MCP does not involve a change in the membrane potential.     

Specificity of d-glucose transport by the apical membrane of Nereis diversicolor epidermis

(1) The specificity of d-[6-³H]glucose influx by a Na⁺-dependent and phlorizin-sensitive transport system in the apical epidermal membrane of the polychaete worm, Nereis diversicolor, was investigated in vivo. (2) The inhibitory effect of eleven d-glucose analogues on d-[6-³H]glucose influx from a 5 μM external concentration was recorded. The inhibitors (each tested at 5, 50, 500 and 5000 μM) were selected to illuminate the configurational requirements for interaction with the d-glucose transport system. (3) The following compounds were found to be significant inhibitors: methyl α-d-glucoside, methyl β-d-glucoside, d-galactose, $\text{3-O-}\text{methyl-}\text{d}\text{-glucose}$, 2-deoxy-d-glucose, d-xylose, myo-inositol, β-d-fructose; the effect was graded according to inhibitor concentration. l-Glucose also inhibited d-glucose influx but to the same extent at all four concentrations tested, suggesting transport site heterogeneity. d-Mannose and l-arabinose did not inhibit influx. (4) The most potent inhibitor, methyl-α-d-glucoside, was itself a substrate, and its transport was inhibited by phlorizin and d-glucose, as well as by substitution of Na⁺ in the incubation medium with Li⁺ or choline⁺. (5) We conclude that the specificity of the Na⁺-dependent d-glucose transporter in the apical epidermal membrane of Nereis is similar to that in the apical membrane of vertebrate small intestinal and proximal tubular epithelium, and in the tapeworm integument.     

Regulation of canine renal vesicle Pi transport by growth hormone and parathyroid hormone

Renal phosphate (P_i) reabsorption is increased by growth hormone (GH) and decreased by parathyroid hormone (PTH). Na⁺-stimulated P_i transport across the brush border membrane of the proximal tubule is the initial step in the process of P_i reabsorption. To determine whether changes in P_i reabsorption induced by GH or PTH are accompanied by changes in brush border membrane Na⁺-gradient-stimulated P_i transport, we examined the effect of in vivo GH and PTH administration and thyroparathyroidectomy on P_i transport by isolated brush border membrane vesicles prepared from canine kidney. In experiments in which the effect of PTH administration was examined, the same animal provided the control kidney (before PTH administration) and the experimental kidney (after PTH administration). The Na⁺-gradient P_i overshoot in vesicles isolated from normal, GH-treated and thyroparathyroidectomized dogs was increased after in vivo PTH administration. GH administration and thyroparathyroidectomy increased the height of the overshoot compared to normal. PTH administration decreased the apparent $\text{V}$ value by 44% in vesicles from normal animals. The apparent $\text{V}$ value was increased, compared to normal, by GH (34%) and thyroparathyroidectomy (57%). PTH administration decreased the apparent $\text{V}$ in both the latter groups. GH administration to thyroparathyroidectomized dogs further increased the apparent $\text{V}$. Changes in the apparent $\text{V}$ paralleled changes in P_i reabsorption in vivo induced by experimental manipulations. We conclude that changes in renal P_i reabsorption induced by GH were like those induced by PTH, accompanied by changes in the Na⁺-stimulated P_i transport system in the renal brush border membrane, and that the effect of PTH on vesicular P_i transport in GH-treated dogs did not differ from the effect on vesicles from normal animals.     

Reconstitution into liposomes of glucose active transport from the rabbit renal proximal tubule. Characteristics of the system

This paper describes the characteristics of Na⁺-dependent d-glucose transport into liposomes made from soybean phospholipids into which have been reconstituted detergent-solubilized components from the rabbit renal proximal tubular brush border membrane. Conditions for optimal and quantitative reconstitution of glucose carriers are defined. Na⁺-dependent d-glucose uptake occurs via a saturable system with a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 0.125–0.135 mM, is responsive to the volume of the internal liposomal space, and shows ‘overshoot’ as seen in natural membranes. The rate of Na⁺-dependent d-glucose uptake and the magnitude of the ‘overshoot’ are proportional to the concentration of protein used in reconstitution.     

Amino acid stimulation of ATP cleavage by two Ehrlich cell membrane preparations in the presence of ouabain

Two membrane fractions prepared from the Ehrlich ascites-tumor cell show non-identical stimulatory responses to certain amino acids in their Mg²⁺-dependent activity to cleave ATP, despite the presence of ouabain and the absence of Na⁺ or K⁺. The first of these, previously described, shows little ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase activity, and is characteristically stimulated by the presence of certain diamino acids with low $\text{pK}{}_2$, and at pH values suggesting that the cationic forms of these amino acids are effective. The evidence indicates that these effects are not obtained through occupation of the kinetically discernible receptor site serving characteristically for the uphill transport of these amino acids into the Ehrlich cell. The second membrane preparation was purified with the goal of concentrating the ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase activity. It also is stimulated by the model diamino acid, 4-amino-1-methylpiperidine-4-carboxylic acid, and several ordinary amino acids. The diamino acids were most effective at pH values where the neutral zwitterionic forms might be responsible. Among the optically active amino acids tested, the effects of ornithine and leucine were substantially stronger for the l than for d isomers. The list of stimulatory amino acids again corresponds poorly to any single transport system, although the possibility was not excluded that stimulation might occur for both preparations by occupation of a membrane site which ordinarily is kinetically silent in the transport sequence. The high sensitivity to deoxycholate and to dicyclohexylcarbodiimide of the hydrolytic activity produced by the presence of l-ornithine and 4-amino-1-methyl-piperidine-4-carboxylic acid suggests that the stimulatory effect is not merely a general intensification of the background Mg⁺-dependent hydrolytic activity.     

Lactose transport in Escherichia coli cells Dependence of kinetic parameters on the transmembrane electrical potential difference

We determine the kinetic parameters $\text{V}$ and $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ of lactose transport in Escherichia coli cells as a function of the electrical potential difference (Δψ) at pH 7.3 and $\text{Δ}\text{pH}\text{= 0}$. We report that transport occurs simultaneously via two components: a component which exhibits a high $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ (larger than 10 mM) and whose contribution is independent of Δψ, a component which exhibits a low $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ independent of Δψ (0.5 mM) but whose $\text{V}$ increases drastically with increasing Δψ. We associate these components of lactose transport with facilitated diffusion and active transport, respectively. We analyze the dependence upon Δψ of $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ and $\text{V}$ of the active transport component in terms of a mathematical kinetic model developed by Geck and Heinz (Geck, P. and Heinz, E. (1976) Biochim. Biophys. Acta 443, 49–63). We show that within the framework of this model, the analysis of our data indicates that active transport of lactose takes place with a H⁺/lactose stoichiometry greater than 1, and that the lac carrier in the absence of bound solutes (lactose and proton(s)) is electrically neutral. On the other hand, our data relative to facilitated diffusion tend to indicate that lactose transport via this mechanism is accompanied by a H⁺/lactose stoichiometry smaller than that of active transport. We discuss various implications which result from the existence of H⁺/lactose stoichiometry different for active transport and facilitated diffusion.     

Effect of membrane potential and internal pH on active sodium-potassium transport and on ATP content in high-potassium sheep erythrocytes

Ouabain-sensitive Na⁺ and K⁺ fluxes and ATP content were determined in high potassium sheep erythrocytes at different values of membrane potential and internal pH. Membrane potential was adjusted by suspending erythrocytes in media containing different concentrations of MgCl₂ and sucrose. Concomitantly either the external pH was changed sufficiently to maintain a constant internal pH or the external pH was kept constant with a resultant change of internal pH. The erythrocytes were preincubated before the flux experiment started in a medium which produced increased ATP content in order to avoid substrate limitation of the pump. p] It was found that an increased cellular pH reduced the rates of active transport of Na⁺ and K⁺ without significantly altering the ratio of pumped $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$. This reduction was not due to limitation in the supply of ATP although ATP content decreased when internal pH increased. Changes of membrane potential in the range between ?10 and +60 mV at constant internal pH did not affect the rates of active transport of Na⁺ or K⁺.     

Na+-dependent methyl β-thiogalactoside transport in Salmonella typhimurium

We have studied the role of sodium ions in methyl β-thiogalactoside (TMG) transport via the melibiose permease (TMG II) in SalmonellaTMG uptake via TMG Il in anaerobic, starved and metabolically poisoned cells is dependent on an inward-directed Na⁺ gradient.Cells which have been partially depleted of endogenous substrates show H⁺ extrusion upon sodium-stimulated TMG influx.Measurements of the electrochemical H⁺ gradient in cells, starved in different ways for endogenous substrates, suggest that this proton extrusion is probably not linked to the actual translocation mechanism but is the result of metabolism induced by TMG plus Na⁺ uptake.     

Triphenylmethylphosphonium uptake by pancreatic islet cells

Uptake of triphenylmethylphosphonium cation (TPMP⁺) was studied in pancreatic islet cells. Islets rich in β-cells were prepared from non-inbred ob/ob-mice and incubated with [³H]TPMP⁺ and l-[1-¹⁴C]glucose. Conjoined with the Nernst equation, the values for TPMP⁺ uptake in excess of the extracellular (l-glucose) space predicted membrane electric potentials far from those previously recorded with intracellular electrodes. Improved agreement with the electrode data was achieved by correcting for assumed voltage-independent binding of TPMP⁺; plausible correction terms were derived from the kinetics of TPMP⁺ efflux and from the uptake of [³H]TPMP⁺ in islets treated with non-radioactive TPMP⁺ at such a high concentration (50 μM) as to abolish the glucose oxidation. In whole islets the magnitude of the TPMP⁺-derived potentials decreased with increasing extracellular K⁺ in the range 5.9–130 mM, and was diminished by 20 mM d-glucose or 0.5 mM 2,4-dinitrophenol, but not by 20 mM 3-O-methyl-d-glucose, 20 mM d-mannoheptulose alone, or 10 μM chlorotetracycline. The effect of d-glucose was not observed in the presence of d-mannoheptulose and was diminished when 130 mM NaCl in the medium was replaced by sodium isethionate. The magnitude of TPMP⁺ uptake and the effects of K⁺ and dinitrophenol were reproduced with dispersed islet cells from ob/ob-mice and with whole islets of normal inbred NMRI-mice; the d-glucose effect was reproduced with NMRI-mouse islets. The results support our previous hypotheses that the depolarizing and insulin-releasing actions of d-glucose are in part mediated by electrodiffusion mechanisms involving K⁺ and Cl^?.     

Na+-driven Ca2+ transport in alkalophilic Bacillus

Ca²⁺ transport was studied in membrane vesicles of alkalophilic Bacillus. When Na⁺-loaded membrane vesicles were suspended in KHCO₃/KOH buffer (pH 10) containing Ca²⁺, rapid uptake of Ca²⁺ was observed. The apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for Ca²⁺ measured at pH 10 was about 7 μM, and the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value shifted to 24 μM when measured at pH 7.4. The efflux of Ca²⁺ was studied with Ca²⁺-loaded vesicles. Ca²⁺ was released when Ca²⁺-loaded vesicles were suspended in medium containing 0.4 M Na⁺.Ca²⁺ was also transported in membrane vesicles driven by an artificial pH gradient and by a membrane potential generated by K⁺-valinomycin in the presence of Na⁺.These results indicate the presence of Ca²⁺/Na⁺ and H⁺/Na⁺ antiporters in the alkalophilic Bacillus A-007.     

Active K+ transport in Mycoplasma mycoides var. Capri. Net and unidirectional K+ movements

Analysis of the cation composition of growing Mycoplasma mycoides var. Capri indicates that these organisms have a high intracellular K⁺ concentration ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{: 200–300}\text{mM}$) which greatly exceeds that of the growth medium, and a low Na⁺ concentration ($\text{Na}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}\text{: 20}\text{mM}$). Unlike $\text{Na}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}\text{, K}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}$ varies with cell aging.The K⁺ transport properties studied in washed organisms resuspended in buffered saline solution show that cells maintain a steady and large K⁺ concentration gradient across their membrane at the expense of metabolic energy mainly derived from glycolysis. In starved cells, $\text{K}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}$ decreases and is partially compensated by a gain in Na⁺. This substitution completely reverses when metabolic substrate is added (K⁺ reaccumulation process). Kinetic analysis of K⁺ movement in cells with steady K⁺ level shows that most of K⁺ influx is mediated by an autologous K⁺-K⁺ exchange mechanism. On the other hand, during K⁺ reaccumulation by K⁺-depleted cells, a different mechanism (a K⁺ uptake mechanism) with higher transport capacity and affinity drives the net K⁺ influx. Both mechanisms are energy-dependent.Ouabain and anoxia have no effect on K⁺ transport mechanisms; in contrast, both processes are completely blocked by dicyclohexylcarbodiimide, an inhibitor of the Mg²⁺-dependent ATPase activity.