Cation-Sugar Cotransport in the Melibiose Transport System of Escherichia coli

The entry of Na⁺ or H⁺ into cells of Escherichia coli via the melibiose transport system was stimulated by the addition of certain galactosides. The principal cell used in these studies (W3133) was a lactose transport negative strain of E. coli possessing an inducible melibiose transport system. Such cells were grown in the presence of melibiose, washed, and incubated in the presence of 25 μM Na⁺. The addition of thiomethylgalactoside (TMG) resulted in a fall in Na⁺ concentration in the incubation medium. No TMG-stimulated Na⁺ movement was observed in uninduced cells. In an α-galactosidase negative derivative of W3133 (RA11) a sugar-stimulated Na⁺ uptake was observed in meliboise-induced cells on the addition of melibiose, thiodigalactoside, methyl-α-galactoside, methyl-β-galactoside, and galactose, but not lactose. It was inferred from these studies that the substrates of the melibiose system enter the cell on the melibiose carrier associated with the simultaneous entry of Na⁺ when this cation is present in the incubation medium.

Extracellular pH was measured in unbuffered suspensions of induced cells in order to study proton movement across the membrane of cells exposed to different galactosides. In the absence of external Na⁺ or Li⁺ the addition of melibiose or methyl-α-galactoside resulted in marked alkalinization of the external medium (consistent with H⁺-sugar cotransport). On the other hand TMG, thiodigalactoside, and methyl-β-galactoside gave no proton movement under these conditions. When Na⁺ was present, the addition of TMG or melibiose resulted in acidification of the medium. This observation is consistent with the view that the entry of Na⁺ with TMG or melibiose carries into the cell a positive charge (Na⁺) which provides the driving force for the diffusion of protons out of the cell. It is concluded that the melibiose carrier recognition of cations differs with different substrates.     

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.     

Cysteine substitutions for individual residues in helix VI of the melibiose carrier of Escherichia coli

The melibiose carrier of Escherichia coli is a cytoplasmic membrane protein that mediates the cotransport of galactosides with H⁺, Na⁺, or Li⁺. In this study we used cysteine-scanning mutagenesis to try to gain information about the position of transmembrane helix VI in the three-dimensional structure of the melibiose carrier. We constructed 23 individual cysteine substitutions in helix VI and an adjacent loop of the carrier. The resulting melibiose carriers retained 22–100% of their ability to transport melibiose. We tested the effect of the hydrophilic sulfhydryl reagent p-chloromercuri-benzenesulfonic acid (PCMBS) on the cysteine-substitution mutants and we found that there was no inhibition of melibiose transport in any of the mutants. We suggest that helix VI is imbedded in phospholipid and does not face the aqueous channel through which melibiose passes. Received: 6 March 2001/Revised: 14 May 2001     

Reduced Na+ Affinity Increases Turnover of Salmonella enterica Serovar Typhimurium MelB

The melibiose permease of Salmonella enterica serovar Typhimurium (MelB_St) catalyzes symport of melibiose with Na⁺, Li⁺, or H⁺. Bioinformatics and mutational analyses indicate that a conserved Gly117 (helix IV) is a component of the Na⁺-binding site. In this study, Gly117 was mutated to Ser, Asn, or Cys. All three mutations increase the maximum rate (V_max) for melibiose transport in Escherichia coli DW2 and greatly decrease Na⁺ affinity, indicating that intracellular release of Na⁺ is facilitated. Rapid melibiose transport, particularly by the G117N mutant, triggers osmotic lysis in the lag phase of growth. The findings support the previous conclusion that Gly117 plays an important role in cation binding and translocation. Furthermore, a spontaneous second-site mutation (P148L between loop_4-5 and helix V) in the G117C mutant prevents cell lysis. This mutation significantly decreases V_max with little effect on cosubstrate binding in G117C, G117S, and G117N mutants. Thus, the P148L mutation specifically inhibits transport velocity and thereby blocks the lethal effect of elevated melibiose transport in the Gly117 mutants.     

Bacterial transporters: Charge translocation and mechanism

A comparative review of the electrophysiological characterization of selected secondary active transporters from Escherichia coli is presented. In melibiose permease MelB and the Na⁺/proline carrier PutP pre-steady-state charge displacements can be assigned to an electrogenic conformational transition associated with the substrate release process. In both transporters cytoplasmic release of the sugar or the amino acid as well as release of the coupling cation are associated with a charge displacement. This suggests a common transport mechanism for both transporters. In the NhaA Na⁺/H⁺ exchanger charge translocation due to its steady-state transport activity is observed. A new model is proposed for pH regulation of NhaA that is based on coupled Na⁺ and H⁺ equilibrium binding.     

Proton efflux associated with melibiose permease activity in Salmonella typhimurium.

Transport of thiomethyl-β-D-galactoside (TMG) via the melibiose permease system (TMG permease II) in $\text{Salmonella typhimurium}$ is known to be a sodium-dependent co-transport system. We have shown that this co-transport of sodium and TMG is associated with extrusion of protons from the cells. The rate and extent of proton extrusion during TMG uptake were measured in wild-type cells and mutants containing internal and extended deletions in the $\text{pts}$ locus. No differences between these various strains were noted.     

Glutamate transport driven by an electrochemical gradient of sodium ion in membrane vesicles of Escherichia coli B.

Membrane vesicles prepared from $\text{E. coli}$ B strain 29–78 require Na⁺ for the accumulation of glutamate. Respiratory-driven transport of glutamate but not lysine is sensitive to the ionophore monensin. An artificially-imposed sodium gradient and/or membrane potential drives glutamate uptake. These results suggest that glutamate is accumulated via a Na⁺/glutamate symport.     

Double dependence of organic acid active transport in proximal tubules of surviving frog kidney on sodium ions I. Influence of sodium ions in bath medium on the uptake and run out of fluorescein and uranin

With the aid of direct microfluorimetric determination of marker organic anions (fluorescein and uranin) accumulated in the proximal tubules the influence of Na⁺ in the bath medium on the active transport of these anions was studied. Kinetic analysis of the rate dependence of organic acid active transport into tubules on their concentration in the bath medium with a constant Na⁺ concentration permitted to define values of apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and $\text{V}$ for uranin and fluorescein transport in the medium with different Na⁺ content. It was shown that a decrease of Na⁺ concentration in the medium increases $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and lowers the $\text{V/K}{}_{\mathrm{<mtext>m</mtext>}}$ ratio with uncharged $\text{V}$. By varying the Na⁺ concentration in the medium with a constant concentration of the marker anion the and values for fluorescein and uranin transport were determined. A value for fluorescein in twice as much that for uranin. The $\text{1/K}{}_{\mathrm{<mtext>m</mtext>}}$ value for uranin transport is a linear function of Na⁺ concentration, while for fluorescein transport it is a quadratic one. Therefore it is concluded that two Na⁺ from the medium participate in active transfer of one fluorescein anion whereas only one Na⁺ from the medium is required for active transfer of one uranin anion. The run out of fluorescein from tubules preloaded with this acid is sharply reinforced by the Na⁺ omission from the medium. Thus, active transport of organic acids in proximal tubules of frog kidney is Na⁺-dependent, and Na⁺ from the medium is likely to participate directly in formation of a transport complex. When Na⁺ is absent in the medium a carrier fulfils a facilitated diffusion only.     

The basic asymmetry of Na+-dependent glycine transport in Ehrlich cells

Influx and efflux of glycine have been examined as a function of external and internal Na⁺ concentrations, respectively, when $\text{Δ}\text{μ}{}_{\mathrm{<mtext>Na</mtext>}}\text{= 0}$. With $\text{Δ}\text{μ}{}_{\mathrm{<mtext>Na</mtext>}}\text{= 0}$ it was found that at comparable external and cellular Na⁺ levels, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for efflux was larger by an order of magnitude than the value for influx and the $\text{V}$ for efflux was several times greater than the $\text{V}$ for influx. For both fluxes the major effect of Na⁺ was to decrease the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value. The observations are consistent with the conclusion that the Na⁺-dependent transport system is asymmetric per se. Influx and efflux of glycine were increased in a near linear manner by increasing the Na⁺ concentration from 13 to 100 mM, the half-time for glycine equilibration being a function of the Na⁺ concentration in absence of an electrochemical potential difference for Na⁺. In Na⁺-free media ($\text{[}\text{Na}{}^{\mathrm{+}}\text{] < 5}\text{mM}$) equilibration of glycine between cells and medium was not achieved after 60 min at 25°C. With $\text{Δ}\text{μ}{}_{\mathrm{<mtext>Na</mtext>}}\text{= 0}$, efflux (or uptake) of glycine was not affected by internal (or external) K⁺ between 20 and 120 mM suggesting that K⁺ plays no direct role in Na⁺-dependent transport of glycine in Ehrlich cells.     

Effect of lithium ion on melibiose transport inEscherichia coli

Summary Both Li⁺ and Na⁺ stimulated the uptake of thiomethylgalactoside by the melibiose transport system ofEscherichia coli. On the other hand, Li⁺ inhibited the growth of cells on melibiose as a sole source of carbon. This inhibition was specific for melibiose, and Li⁺ had no effect on growth of cells on glucose, galactose, lactose, or glycerol. The effect of the cation on melibiose transport was investigated in a mutant which cannot utilize glucose. After entry into this cell, melibiose is cleaved into glucose and galactose by -galactosidase, and the resulting glucose is excreted. Since the entry step was found to be rate-limiting, glucose production could be taken as a measure of melibiose transport. Li⁺ inhibited the transport of melibiose, but not the induction of the melibiose operon nor the activity of -galactosidase. Li⁺ was found to inhibit the entry ofp-nitrophenyl--d-galactoside, but notp-nitrophenyl--d-galactoside entry. Thus, the cation specificity for the melibiose membrane carrier varies with different transport substrates.     

Effect of N-ethylmaleimide on leucine transport in chang liver cells. I. Stimulatory effect on Na+-independent transport at low concentrations of leucine

Pretreatment of Chang liver cells with $\text{N-}\text{ethylmaleimide}$ (0.5 or 1 mM) stimulated Na⁺-independent uptake of leucine at low concentrations (?1 mM). The stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on the uptake of leucine measured in Na⁺-replete medium was completely blocked by the addition of $\text{b-2-}\text{aminobicyclo}\text{[2,2,1]}\text{heptane}\text{-2-}\text{carboxylate}$ (5 mM), which shows that the L system participates in the stimulation. The Na⁺-dependent uptake of glycine was depressed by $\text{N-}\text{ethylmaleimide}$ pretreatment. The stimulation of the Na⁺-independent component of leucine uptake continued for at least 30 min after $\text{N-}\text{ethylmaleimide}$ treatment, while the inhibition of glycine uptake was progressive with time and the Na⁺-dependent uptake of leucine became depressed later, after the treatment. It has been demonstrated that treatment of cells with $\text{N-}\text{ethylmaleimide}$ is capable of increasing the Na⁺-independent influx of leucine and at the same time slightly decreasing the efflux of it. These results suggest that $\text{N-}\text{ethylmaleimide}$ attacks the Na⁺-independent system of amino acid transport at the reactive SH groups(s) of relevant protein(s) in favor of specific activation of that system in this cell.     

Thallium inhibition of ouabain-sensitive sodium transport and of the (Na++K+)-ATPase in human erythrocytes

The influence of Tl⁺ on Na⁺ transport and on the ATPase activity in human erythrocytes was studied. 0.1–1.0 mM Tl⁺ added to a K⁺-free medium inhibited the ouabain-sensitive self-exchange of Na⁺ and activated both the ouabain-sensitive ²²Na outward transport and the transport related ATPase. 5–10 mM external Tl⁺ caused inhibition of the ouabain-sensitive ²²Na efflux as well as the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{Tl}{}^{\mathrm{+}}$)-ATPase. Competition between the internal Na⁺ and rapidly penetrating thallous ions at the inner Na⁺-specific binding sites of the erythrocyte membrane could account for the inhibitory effect of Tl⁺. An increase of the internal Na⁺ concentration in erythrocytes or in ghosts protected the system against the inhibitory effect of high concentration of Tl⁺. A protective effect of Na⁺ was also demonstrated on the ($\text{Na}{}^{\mathrm{+}}\text{+ Tl}{}^{\mathrm{+}}$)-ATPase of fragmented erythrocyte membranes studied at various Na⁺ and Tl⁺ concentrations.     

Double dependence of organic acid active transport in proximal tubules of surviving frog kidney on sodium ions II. Relationship between counter-flows of fluorescein and sodium ion across cell layer

With the aid of a direct microfluorimetric method a dependence of organic onion (fluorescein) transport into proximal tubules of surviving frog kidney on Na⁺-flow in the opposite direction was studied. It was shown that the complete removal of Na⁺ from the tubules lumen resulted in inhibition of fluorescein transport of about 30%. After a specific inhibitor of sodium channels, amiloride (10^-3M) having been introduced into lumen of the tubules, the fluorescein transport was inhibited to the same extent. Amiloride affects only when Na⁺ is present in the tubular lumen. S present in the tubular lumen. Strophantin K (5 · 10^?5 M), a specific inhibitor of (Na⁺, K⁺)-ATPase, reduced fluorescein transport about twice. Substances increasing the 3′,5′-AMP level in cells (theophylline, NaF) and exogenous 3′,5′-AMP inhibited fluorescein transport while substance that decreased the 3′,5′-AMP level intracellularly (carbachol) stimulated it. For realization of these effects Na⁺ should be present in proximal tubules lumen.Thus, the various effects on the Na⁺ flow from lumen of the tubules to medium at the level of both the basal and apical membranes alter the rate of organic acid active transport from medium to lumen as a result of changes in the maximum rate of transport ($\text{V}$) with unchanged $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. It is suggested that the system of Na⁺ extrusion from proximal tubules produces peritubular membrane-side (near the membrane) gradient of Na⁺ concentration which may be higher than the summary Na⁺ gradient between the medium and the cytoplasm. The magnitude of this gradient affects the maximal rate value of Na⁺-dependent organic acid transport. So, there is a double dependence of the active transport system on Na⁺, and the stages where Na⁺ is needed are: (1) the formation of a carrier-substrate-Na⁺ complex and (2) the production of substantial membrane-side Na⁺ gradient at the expense of Na⁺ extrusion from the tubules.     

The uptake of (3H)dopamine by homogenates of rat corpus striatum: effects of cations

The uptake of [³H]dopamine was studied with a synaptosomal preparation of the corpus striatum. The accumulation of dopamine was found to be temperature-dependent and very rapid, but linear over time for at least 5 min. at 37°C with characteristics of saturable kinetics. The optimum concentrations for Na⁺ and K⁺ were 150–160 m$\text{M}$ and 2.5–4.8 m$\text{M}$, respectively, while uptake was progressively inhibited at concentrations of K⁺ greater than 5 m$\text{M}$. Rubidium was capable of substituting for potassium whereas cesium was a much less effective replacement. The uptake of DA was blocked by the antibiotics, valinomycin and gramicidin-D which bind K⁺ or both Na⁺ and K⁺, respectively, and thereby might interfere with the transport of cations across neuronal membranes. Similarly, ouabain which blocks the active transport of Na⁺ markedly antagonized the accumulation of DA into striatal homogenates. In contrast, tetrodotoxin which does not prevent the active transport of Na⁺, had no effect. Uptake appeared not to require Ca⁺⁺ and it was not inhibited by increasing total osmolarity to 400 mos$\text{M}$. In general, the cationic requirements for DA-uptake in striatal tissue and its responses to several inhibition of ionic transport, do not appear to be greatly different from those reported for NE with synaptosomes prepared from whole brain.     

The effects of removal of sodium ions from the mucosal solution on sugar absorption by rabbit ileum

Net absorption and accumulation of d-galactose, β-methyl d-glucose and low concentrations of $\text{3-O-}\text{methyl}\text{-}\text{d}\text{-}\text{glucose}$ by sheets of rabbit ileum are observed even when Na⁺ in the mucosal solution is replaced by choline. This indicates that active sugar transport can occur in the direction opposite to the brush-border Na⁺ gradient.     

Interactions between Na+-dependent uptake of d-glucose,phosphate and l-alanine in rat renal brush border membrane vesicles

d-Glucose decreases phosphate reabsorption in rat proximal tubule. It is also postulated that some amino acids interact with phosphate reabsorption. To investigate the mechanism of these interactions, phosphate, d-glucose and l-alanine transport kinetics were measured in brush border membrane vesicles isolated from superficial rat kidney cortex by the calcium precipitation technique. At pH 7.4, Na⁺-dependent phosphate transport was inhibited in the presence of either d-glucose (39 mM) or l-alanine (2.4 mM). In this model, with d-glucose or with l-alanine the $\text{V}$ value of the phosphate uptake was decreased, whereas the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for the phosphate uptake was not affected. However, some inhibition of phosphate transport was observed in the presence of l-glucose, d-alanine or d-glucose after phlorizin preincubation. A 30% Na⁺-dependent l-alanine (0.1 mM) transport inhibition was observed in the presence of 5 mM phosphate. d-Glucose (1 mM) was also inhibited by 20% when 5 mM phosphate was added to incubation medium. According to several authors, in our model, d-glucose decreased the l-alanine transport and vice versa. Moreover, when the membrane potential was abolished, a clear inhibition of d-glucose by l-alanine persisted. These multiple interactions could be explained by the accelerated dissipation of the Na⁺ gradient insofar as the rate of the Na⁺ uptake was increased with d-glucose, l-alanine or phosphate and since the absence of variations in membrane potential did not suppress these inhibitions.     

Light-driven sodium transport in sub-bacterial particles of Halobacterium halobium

Light-induced Na⁺ efflux was observed in sub-bacterial particles of Halobacterium halobium loaded and suspended in 4 M NaCl solution. The Na⁺ efflux was not ATP driven, since ATPase inhibitors were without effect or even enhanced efflux at low light intensity. Uncouplers, on the other hand, inhibited Na⁺ efflux, the inhibition being complete at low light intensity. The Na⁺ efflux was accompanied by proton influx. Both processes were dependent on light intensity, unaffected or enhanced by ATPase inhibitors and similarly affected by uncouplers. Proton influx was not observed in particles loaded with 4 M KCl instead of 4 M NaCl. Na⁺ transport in the dark could be induced by artificial formation of a pH difference across the membrane; changing the sign of the pH difference reversed the direction of the Na⁺ transport. Proton influx in the dark followed the artificial formation of a sodium gradient ($\text{[Na}{}^{\mathrm{+}}\text{]}{}_{\mathrm{in}}\text{> [Na}{}^{\mathrm{+}}\text{]}{}_{\mathrm{out}}$). These results may be explained by a Na⁺/H⁺ antiport mechanism. The fluxes of Na⁺ and H⁺ were of comparable magnitude, but the initial rate of Cl^? efflux in the same experiment was one-third of the initial rate of Na⁺ efflux. Consequently Cl^? is not regarded as a participant in the Na⁺ efflux mechanism.     

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 pre-loaded 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 $\text{m}$ 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.     

Effects of cholera toxin on cellular and paracellular sodium fluxes in rabbit ileum

The diarrhea observed in patients which cholera is known to be related to secretion of water and electrolytes into the intestinal lumen. However, the exact mechanisms involved in these secretory processes have remained unclear. Although it is clear that purified toxin acts on epithelial cell metabolism, its activity on Na⁺ transport across intestinal mucosa is equivocal: reported either to prevent net Na⁺ absorption or to cause net secretion of Na⁺ from serosa to mucosa. Since total transmural Na⁺ fluxes across “leaky” epithelia involve very significant movement via a paracellular shunt pathway, we studied the effects of cholera toxin on the cellular and paracellular pathways of Na⁺ movement. Unidirectional Na⁺ fluxes were examined as functions of applied potential in control tissues and in tissues from the same animal treated with purified cholera toxin. Treatment of rabbit ileum in vitro with toxin stimulated the cellular component of serosa-to-mucosa Na⁺ flux (from $\text{2.41 ± 0.49 μ}\text{equiv./h}$ per cm² under control conditions to $\text{4.71 ± 0.43 μ}\text{equiv./h}$ per cm² after treatment with toxin, $\text{P < 0.01}$). The effect of cholera toxin on Na⁺ movement through the cells from mucosa to serosa appeared to be insignificant. Finally, a marked decrease in the Na⁺ permeability ($\text{P < 0.01}$) and no detectable significant changes in transference number for Na⁺ of the paracellular shunt pathway were observed following treatment with cholera toxin. These results provide direct evidence for the hypothesis that purified cholera toxin stimulates active sodium secretion but has minimal effect on sodium absorption.     

Effect of N-ethylmaleimide on leucine transport in the chang liver cell. II. Effect on the kinetics of Na+-independent transport

The Na⁺-independent leucine transport system is resolved into two components by their different affinity ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ about 44 μM and 8.0 mM) for leucine in the Chang liver cell. Treatment of the cells with $\text{N-}\text{ethylmaleimide}$ (1 mM) specifically stimulates the high-affinity component of the Na⁺-independent system by greatly increasing its $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ value, whereas the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ value of the low-affinity component is markedly lowered. The stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on leucine transport is reduced by prior treatment of the cells with 2,4-dinitrophenol, but this phenomenon seems to be irrelevant to the ATP-depleting action of the uncoupler. The treatment with 2,4-dinitrophenol has been found not to be inhibitory on the subsequent Na⁺-independent leucine uptake itself. Treatment with dibucaine, a phospholipid-interacting drug, also reduces to varying degrees (depending on its concentration) the stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on the subsequent leucine uptake, although pretreatment with dibucaine can stimulate the Na⁺-independent leucine uptake itself. We conclude that the stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on leucine transport is not correlated with the energy level of cell, but involves the perturbation of the membrane bilayer structures.