Isolation of basolateral and brush-border membranes from the rabbit kidney cortex: Vesicle integrity and membrane sidedness of the basolateral fraction

A rapid and reproducible method has been developed for the simultaneous isolation of basolateral and brush-border membranes from the rabbit renal cortex. The basolateral membrane preparation was enriched 25-fold in (Na⁺ + K⁺)-ATPase and the brush-border membrane fraction was enriched 12-fold in alkaline phosphatase, whereas the amount of cross-contamination was low. Contamination of these preparations by mitochondria and lysosomes was minimal as indicated by the low specific activities of enzyme markers, i.e., succinate dehydrogenase and acid phosphatase. The basolateral fraction consisted of 35–50% sealed vesicles, as demonstrated by detergent (sodium dodecyl sulfate) activation of (Na⁺ + K⁺)-ATPase activity and [³H]ouabain binding. The sidedness of the basolateral membranes was estimated from the latency of ouabain-sensitive (Na⁺ + K⁺)-ATPase activity assayed in the presence of gramicidin, which renders the vesicles permeable to Na⁺ and K⁺. These studies suggest that nearly 90% of the vesicles are in a right-side-out orientation.     

Sulfate transport in brush border membrane vesicles prepared from human placental syncytiotrophoblast

Isolated brush-border membrane vesicles prepared from human placenta are known to transport amino acids via a Na⁺-dependent mechanism akin to that found in gut and kidney vesicle preparations. We studied sulfate transport in placental vesicles and failed to identify any Na⁺-dependent uptake mechanism. Rather, uptake is a non-electrogenic process that is trans-stimulated by outwardly directed anion flux which is independent of cation. If anion exchange is tightly coupled $\text{in}\text{vivo}$, the net transfer of sulfate from mother to the growing fetus may be driven by the continuous flux of bicarbonate in the opposite direction.     

Identification of the renal Na+/H+ exchanger with N,N′-dicyclohexylcarbodiimide (DCCD) and amiloride analogues

Summary Dicyclohexylcarbodiimide (DCCD) and the 5-ethylisopropyl-6-bromo-derivative of amiloride (Br-EIPA) have been used as affinity and photoaffinity labels of the Na⁺/H⁺ exchanger in rat renal brush-border membranes. Intravesicular acidification by the Na^–/H⁺ exchanger was irreversibly inhibited after incubation of vesicles for 30 min with DCCD. The substrate of the antiporter, Na⁺, and the competitive inhibitor, amiloride, protected from irreversible inhibition. The Na⁺-dependent transport systems for sulfate, dicarboxylates, and neutral, acidic, and basic amino acids were inhibited by DCCD, but not protected by amiloride. An irreversible inhibition of Na⁺/H⁺ exchange was also observed when brush-border membrane vesicles were irradiated in the presence of Br-EIPA. Na⁺ and Li⁺ protected. [¹⁴C]-DCCD was mostly incorporated into three brush-border membrane polypeptides with apparent molecular weights of 88,000, 65,000 and 51,000. Na⁺ did not protect but rather enhanced labeling. In contrast, amiloride effectively decreased the labeling of the 65,000 molecular weight polypeptide. In basolateral membrane vesicles one band was highly labeled by [¹⁴C]-DCCD that was identified as the -subunit of the Na⁺, K⁺-ATPase. [¹⁴C]-Br-EIPA was mainly incorporated into a brushborder membrane polypeptide with apparent molecular weight of 65,000. Na⁺ decreased the labeling of this protein. Similar to the Na⁺/H⁺ exchanger this Na⁺-protectable band was absent in basolateral membrane vesicles. We conclude that a membrane protein with an apparent molecular weight of 65,000 is involved in rat renal Na⁺/H⁺ exchange.     

Effect of hyperglycemia on d-glucose transport across the brush-border and basolateral membrane of rat small intestine

Experimental hyperglycemia leads to an increase in the capacity of the rat small intestine to absorb glucose. This effect occurs within hours from the onset of hyperglycemia and is thought to involve an induction of glucose transport in the brush-border and/or basolateral membrane of the intestinal epithelium. We devised a protocol for the simultaneous preparation of brush-border vesicles and basolateral vesicles from rat small intestine to determine the locus for the inductioof glucose transporter in hyperglycemic rats. A 6 h period of intravenous infusion with a 30% glucose solution had no effect on the initial rate of glucose uptake across jejunal or ileal brush-border vesicles when measured in the absence of a Na⁺ gradient, suggesting that enhanced glucose uptake is not dependent on an increase in the number of Na⁺-dependent secondary active glucose transporters in the brush-border. Hyperglycemia did not effect the rate of glucose uptake across ileal basolateral vesicles but did cause a 78% increase in the initial rate of carrier-mediated d-glucose uptake across jejunal basolateral vesicles. The induction of glucose transport in the jejunal basolateral membrane was characterized by a rapid rate of glucose equilibration across the vesicles ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 46}\text{s}$ sorbitol infused controls, 18 s hyperglycemia) and a 75% increase in the V_max for carrier-mediated glucose uptake with no significant change in K_t. When the rats were pretreated with cycloheximide prior to intravenous infusion, the initial rate of d-glucose uptake dropped to 13% of that seen in jejunal basolateral vesicles prepared from untreated rats. These results suggest a rapid turnover rate for the Na⁺-independent glucose transporter in the basolateral membrane of the enterocyte. An increase in the number of functioning glucose transporters in the basolateral membrane may play an important role in the short-term induction of glucose absorption by the jejunum of the hyperglycemic animal.     

In vitro ethanol effects on the transport properties of isolated renal brush-border membrane vesicles

Summary Thein vitro effect of ethanol on membrane structure and transport properties was studied in isolated renal brush border membrane vesicles.³¹P-NMR studies showed a dose-dependent increase in the quantity of an isotropic, possibly inverted-micellar component of the renal brush-border membrane as a result of treatment with ethanol. Such structures have been shown to be instrumental in the translocation of material across membrane bilayers. A²³Na-NMR study of Na⁺ exchange in artificial phosphatidylcholine liposomes indicated that ethanol (0.1%) was capable of rending the otherwise inert vesicles permeable to sodium, supporting the idea that ethanol may exert its action via a direct effect on the structure of the phospholipid bilayer. In the isolated renal brush-border membrane vesicles, like in the artificial liposomes, amiloride-insensitive pathways of Na⁺ transport were shown to be markedly activated by ethanol. These results were consistent with the inhibitory effect ethanol had on Na⁺ gradient-dependent transport systems such as the Na⁺ gradient-dependentd-glucose transport and Na⁺/H⁺ exchange. In conclusion, our results indicate that ethanol exerts its effect on the renal brush-border membrane by causing a structural change in the phospholipid bilayer which activates sodium intake. The inhibitory effect of ethanol on glucose uptake and Na⁺/H⁺ exchange is secondary, as a result of the dissipation of the energy-producing Na⁺ gradient.     

Distinct transport activity of tetraethylammonium from l-carnitine in rat renal brush-border membranes

We investigated the contribution of the Na⁺/l-carnitine cotransporter in the transport of tetraethylammonium (TEA) by rat renal brush-border membrane vesicles. The transient uphill transport of l-carnitine was observed in the presence of a Na⁺ gradient. The uptake of l-carnitine was of high affinity (K_m=21 μM) and pH dependent. Various compounds such as TEA, cephaloridine, and p-chloromercuribenzene sulfonate (PCMBS) had potent inhibitory effects for l-carnitine uptake. Therefore, we confirmed the Na⁺/l-carnitine cotransport activity in rat renal brush-border membranes. Levofloxacin and PCMBS showed different inhibitory effects for TEA and l-carnitine uptake. The presence of an outward H⁺ gradient induced a marked stimulation of TEA uptake, whereas it induced no stimulation of l-carnitine uptake. Furthermore, unlabeled TEA preloaded in the vesicles markedly enhanced [¹⁴C]TEA uptake, but unlabeled l-carnitine did not stimulate [¹⁴C]TEA uptake. These results suggest that transport of TEA across brush-border membranes is independent of the Na⁺/l-carnitine cotransport activity, and organic cation secretion across brush-border membranes is predominantly mediated by the H⁺/organic cation antiporter.     

Sodium uptake mechanisms in brush-border membrane vesicles prepared from rabbit renal cortex

Amiloride-sensitive and amiloride-insensitive components of ²²Na⁺ uptake were examined in brush-border membrane vesicles prepared from rabbit renal cortex. Both components could be stimulated by interior-negative electrical potentials, demonstrating a sodium conductance pathway and an effect of electrical potential on the initial rate of Na⁺/H⁺ exchange.     

A simple method for the isolation of basolateral plasma membrane vesicles from rat kidney cortex Enzyme activities and some properties of glucose transport

A procedure for preparing basolateral membrane vesicles from rat renal cortex was developed by differential centrifugation and Percoll density gradient centrifugation, and the uptake of d-[³H]glucose into these vesicles was studied by a rapid filtration technique. (Na⁺ + K⁺)-ATPase, the marker enzyme for basolateral membranes, was enriched 22-fold compared with that found in the homogenate. The rate of d-glucose uptake was almost unaffected by Na⁺ gradient (no overshoot).     

Relation of ATPases in rat renal brush-border membranes to ATP-driven H+ secretion

Summary In the presence of inhibitors for mitochondrial H⁺-ATPase, (Na⁺+K⁺)- and Ca²⁺-ATPases, and alkaline phosphatase, sealed brush-border membrane vesicles hydrolyse externally added ATP demonstrating the existence of ATPases at the outside of the membrane (ecto-ATPases). These ATPases accept several nucleotides, are stimulated by Ca²⁺ and Mg²⁺, and are inhibited by N,N-dicyclohexylcarbodiimide (DCCD), but not by N-ethylmaleimide (NEM). They occur in both brushborder and basolateral membranes. Opening of brush-border membrane vesicles with Triton X-100 exposes ATPases located at the inside (cytosolic side) of the membrane. These detergent-exposed ATPases prefer ATP, are activated by Mg²⁺ and Mn²⁺, but not by Ca²⁺, and are inhibited by DCCD as well as by NEM. They are present in brush-border, but not in basolateral membranes. As measured by an intravesicularly trapped pH indicator, ATP-loaded brush-border membrane vesicles extrude protons by a DCCD- and NEM-sensitive pump. ATP-driven H⁺ secretion is electrogenic and requires either exit of a permeant anion (Cl^–) or entry of a cation, e.g., Na⁺ via electrogenic Na⁺/d-glucose and Na⁺/l-phenylalanine uptake. In the presence of Na⁺, ATP-driven H⁺ efflux is stimulated by blocking the Na⁺/H⁺ exchanger with amiloride. These data prove the coexistence of Na⁺-coupled substrate transporters, Na⁺/H⁺ exchanger, and an ATP-driven H⁺ pump in brush-border membrane vesicles. Similar location and inhibitor sensitivity reveal the identity of ATP-driven H⁺ pumps with (a part of) the DCCD- and NEM-sensitive ATPases at the cytosolic side of the brush-border membrane.     

Factors affecting the transport of β-amino acids in rat renal brush-border membrane vesicles. The role of external chloride

The effect of a variety of ions and other solutes on the accumulation of the β-amino acid, taurine, was examined in rat renal brush-border membrane vesicles. Initial taurine uptake (15 and 30 s) is sodium-dependent with a typical overshoot. This Na⁺ effect was confirmed by exchange diffusion and gramicidin inhibition of taurine uptake. External K⁺ or Li⁺ do not increase taurine accumulation more than Na⁺-free mannitol, except that the combination of external K⁺ and Na¹ in the presence of nigericin enhances uptake. Of all anions tested, including more permeant (SCN⁻ and NO₃⁻) or less permeant (SO₄²⁻), chloride supported taurine accumulation to a significantly greater degree. Preloading vesicles with choline chloride reduced taurine uptake, suggesting that external Cl⁻ stimulates uptake. Since this choline effect could be related to volume change, due to the slow diffusion of choline into vesicles, brush-border membrane vesicles were pre-incubated with LiCl, LiNO₃ and LiSO₄. Internal LiCl, regardless of the final Na⁺ anion mixture, reduced initial rate (15 and 60 s) and peak (360 s) taurine uptake. Internal LiNO₃ or LiSO₄ with external NaCl resulted in similar or higher values of uptake at 15, 60 and 360 s, indicating a role for external Cl⁻ in taurine uptake in addition to Na⁺ effect. Although uptake by vesicles is greatest at pH 8.0 and inhibited at acidic pH values (pH less than 7.0), an externally directed H⁺ gradient does not influence uptake. Similarly, amiloride, an inhibitor of the Na⁺/H⁺ antiporter, had no influence on taurine accumulation over a wide variety of concentrations or at low Na⁺ concentrations. Taurine uptake is blocked only by other β-amino acids and in a competitive fashion. d-glucose and p-aminohippurate at high concentrations (> 10⁻³ M) reduce taurine uptake, possibly by competing for sodium ions, although gramicidin added in the presence of d-glucose inhibits taurine uptake even further. These studies more clearly define the nature of the renal β-amino acid transport system in brush-border vesicles and indicate a role for external Cl in this uptake system.     

Taurine transport by brush border membrane vesicles isolated from the flounder kidney

Summary Taurine transport was investigated in brush border membrane vesicles isolated from renal tubules of the winter flounder (Pseudopleuronectes americanus). Taurine uptake by the vesicles was greater in the presence of NaCl as compared to uptake in KCl. The Na⁺-dependent taurine transport was electrogenic and demonstrated tracer replacement and inhibition by -alanine and HgCl₂, indicating the presence of Na⁺-dependent, carrier-mediated taurine transport. In contrast to Na⁺-dependent taurine transport across the basolateral membrane, there was not a specific Cl^– dependency for transport in the brush border membrane. No evidence was obtained for Na⁺-independent carrier-mediated taurine transport. The possible involvement of the brush border Na⁺-dependent transport system in the net secretion of taurine from blood to tubular lumen in vivo (Schrock et al. 1982) is discussed.     

Sodium gradient- and sodium plus potassium gradient-dependentl-glutamate uptake in renal basolateral membrane vesicles

Summary A membrane preparation enriched in the basolateral segment of the plasma membrane was isolated from the rat renal cortex by a procedure that included separation of particulates on a self-generating Percoll gradient. The uptake ofl-glutamate by the basolateral membrane vesicles was studied. A Na⁺ gradient ([Na⁺] _o >[Na⁺] _i ) stimulated the uptake ofl-glutamate and provided the driving force for the uphill transport of the acidic amino acid, suggesting a Na⁺-l-glutamate cotransport system in the basolateral membrane. A K⁺ gradient ([K⁺] _i >[K⁺] _o ) increased the uptake additionally. This effect was specific for K⁺ (Rb⁺). The action of the K⁺ gradient in enhancing the uptake ofl-glutamate had an absolute requirement for Na⁺. In the presence of Na⁺, but in the absence of a Na⁺ gradient. i.e., [Na⁺] _o =[Na⁺] _i , the K⁺ gradient also energized the concentrative uptake ofl-glutamate. This effect of the K⁺ gradient was not attributable to an alteration in membrane potential. The finding of a concentrative uptake system forl-glutamate energized by both Na⁺ ([Na⁺] _o >[Na⁺] _i and K⁺ ([K⁺] _i >[K⁺] _o ) gradients in the basolateral membrane, combined with previous reports of an ion gradient-dependent uphill transport system for this amino acid in the brush border membrane, suggests a mechanism by whichl-glutamate is accumulated intracellularly in the renal proximal tubule to extraordinarily high concentrations.     

Effective use of renal cortical slices in transport and metabolic studies

The uptake and metabolism of two water-soluble vitamins were measured in rat renal cortical slices, isolated tubules, and vesicles of the brush-border and basolateral cell membranes to determine (a) whether it is possible to produce slices that have open tubules and, (b) whether slices and tubules metabolize vitamins similarly. Transport of ascorbic acid is sodium-dependent in slices and in brush-border vesicles but is sodium-independent in basolateral vesicles, suggesting that the brush-border membrane of slices is accessible to components of the bathing solution. Nicotinic acid was metabolized similarly (97–98%) in both slices and isolated tubules. Oxygen consumption by slices maintained in a closed chamber was constant as pO₂ decreased from 88% to 58%. Slices are concluded to be a suitable model for transport and metabolic studies providing that care is taken in their preparation and use.     

Coupling of Na<Superscript>+</Superscript>/α-ketoglutarate symport and PAH/α-ketoglutarate antiport in epithelial cells. Estimation of probability of the anion exchanger spontaneous re-orientation

In this work, dynamics was studied of uptake of p-aminohippurate by basolateral membrane vesicles isolated from rat kidney proximal tubules. The uphill PAH transport into the basolateral membrane vesicles was shown to occur in the presence of α-ketoglutarate and Na⁺-gradient. Based on mathematical model of symport and antiport cooperation, the mechanism of energy coupling of PAH transport via exchanger with Na⁺-dicarboxylate symport is discussed. Based on comparison of our own and literature data, the data analysis shows adequacy of the proposed mathematical model to describe the symport and antiport cooperation. This model has been shown to enable estimation of re-orientation probability of the empty anion exchanger (without substrate) from one membrane side to the other.     

Renal palmitate transport: possible sites for interaction with a plasma membrane fatty acid-binding protein

Summary In previous studies from this laboratory [14], a mediated transport system for long chain fatty acids was observed in rat renal basolateral membrane vesicles. Transport was measured in the absence of albumin and indicated the presence of a Na⁺ independent anion exchange mechanism. The present experiments were done to characterize renal transport of fatty acids derived from fatty acid-albumin complexes. ³H-palmitate uptake by brush border (BBMV) and basolateral membrane vesicles (BLMV) isolated from rat renal cortex was determined using a rapid filtration technique. All incubation media contained 100 µM ³H-palmitate complexed to 100 µM bovine serum albumin. Up to 65% of initially bound fatty acid-albumin complexes were displaceable by washing with solution containing 0.1% albumin. Total palmitate uptake was measured as the remaining non-displaceable radioactivity. In BBMV in low ionic strength (300 mM mannitol) or ionic buffers (100 mM mannitol + 100 mM NaCl or KCl), total palmitate uptake at 15 sec did not differ from equilibrium (60 min) values of 10–11 nmoles/mg protein. Uptake was primarily due to binding. A similar pattern was seen with BLMV in 300 mM mannitol buffer: In BLMV in 100 mM NaCl or KCl buffers, equilibrium uptake was 10-fold lower than at 15 sec. This suggests binding followed by cation-dependent translocation. If a putative FABP_PM is involved in transport only, its presence should be confined to BLMV.     

Phenylalanine uptake in isolated renal brush border vesicles

The uptake of l-phenylalanine into brush border microvilli vesicles and basolateral plasma membrane vesicles isolated from rat kidney cortex by differential centrifugation and free flow electrophoresis was investigated using filtration techniques.Brush border microvilli but not basolateral plasma membrane vesicles take up l-phenylalanine by an Na⁺-dependent, saturable transport system. The apparent affinity of the transport system for l-phenylalanine is 6.1 mM at 100 mM Na⁺ and for Na⁺ 13 mM at 1 mM l-phenylalanine. Reduction of the Na⁺ concentration reduces the apparent affinity of the transport system for l-phenylalanine but does not alter the maximum velocity.In the presence of an electrochemical potential difference for Na⁺ across the membrane (η_Na0 >η_Na1) the brush border microvilli accumulate transiently l-phenylalanine over the concentration in the incubation medium (overshoot phenomenon). This overshoot and the initial rate of uptake are markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potentials induced by the use of highly permeant anions, of valinomycin in the presence of an outwardly directed K⁺ gradient and of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence of an outward-directed proton gradient.These results indicate that the entry of l-phenylalanine across the brush border membrane into the proximal tubular epithelial cells involves cotransport with Na⁺ and is dependent on the concentration difference of the amino acid, on the concentration difference of Na⁺ and on the electrical potential difference. The exit of l-phenylalanine across the basolateral plasma membranes is Na⁺-independent and probably involves facilitated diffusion.     

Mechanisms of phosphate uptake into brush-border membrane vesicles from goat jejunum

This study concerns the uptake of inorganic phosphate into brush-border membrane vesicles prepared from jejunal tissues of either control or Ca-and/or P-depleted goats. The brush-border membrane vesicles showed a time-dependent accumulation of inorganic phosphate with a typical overshoot phenomenon in the presence of an inwardly directed Na⁺ gradient. The Na⁺-dependent inorganic phosphate uptake was completely inhibited by application of 5 mmol·l^-1 sodium arsenate. Half-maximal stimulation of inorganic phosphate uptake into brush-border membrane vesicles was found with Na⁺ concentrations in the order of 5 mmol·l^-1. Inorganic phosphate accumulation was not affected by a K⁺ diffusion potential (inside negative), suggesting an electroneutral transport process. Stoichiometry suggested an interaction of two or more Na ions with one inorganic phosphate ion at pH 7.4. Na⁺-dependent inorganic phosphate uptake into jejunal brush-border membrane vesicles from normal goats as a function of inorganic phosphate concentration showed typical Michaelis-Menten kinetic with V _max=0.42±0.08 nmol·mg^-1 protein per 15 s^-1 and K _m=0.03±0.01 mmol·l^-1 (n=4, x ±SEM). Long-term P depletion had no effect on these kinetic parameters. Increased plasma calcitriol concentrations in Ca-depleted goats, however, were associated with significant increases of V _max by 35–80%, irrespective of the level of P intake. In the presence of an inwardly directed Na⁺ gradient inorganic phosphate uptake was significantly stimulated by almost 60% when the external pH was decreased to 5.4 (pH_out/pH_in=5.4/7.4). The proton gradient had no effect on inorganic phosphate uptake in absence of Na⁺. In summary, in goats Na⁺ and calcitriol-dependent mechanisms are involved in inorganic phosphate transport into jejunal brush-border membrane vesicles which can be stimulated by protons.Abbreviations AP activity of alkaline phosphatase - BBMV brush-border membrane vesicles - EGTA ethyleneglycol-triacetic acid - n _app apparent Hill coefficient - P _i inorganic phosphate - PTH parathyroid hormone     

Na+-independent l-arginine transport in rabbit renal brush border membrane vesicles

Na⁺-independent l-arginine uptake was studied in rabbit renal brush border membrane vesicles. The finding that steady-state uptake of l-arginine decreased with increasing extravesicular osmolality and the demonstration of accelerative exchange diffusion after preincubation of vesicles with l-arginine, but not d-arginine, indicated that the uptake of l-arginine in brush border vesicles was reflective of carrier-mediated transport into an intravesicular space. Accelerative exchange diffusion of l-arginine was demonstrated in vesicles preincubated with l-lysine and l-ornithine, but not l-alanine or l-proline, suggesting the presence of a dibasic amino acid transporter in the renal brush border membrane. Partial saturation of initial rates of l-arginine transport was found with extravesicular [arginine] varied from 0.005 to 1.0 mM. l-Arginine uptake was inhibited by extravesicular dibasic amino acids unlike the Na⁺-independent uptake of l-alanine, l-glutamate, glycine or l-proline in the presence of extravesicular amino acids of similar structure. l-Arginine uptake was increased by the imposition of an H⁺ gradient (intravesicular pH<extravesicular pH) and H⁺ gradient stimulated uptake was further increased by FCCP. These findings demonstrate membrane-potential-sensitive, Na⁺-independent transport of l-arginine in brush border membrane vesicles which differs from Na⁺-independent uptake of neutral and acidic amino acids. Na⁺-independent dibasic amino acid transport in membrane vesicles is likely reflective of Na⁺-independent transport of dibasic amino acids across the renal brush border membrane.     

Electrogenic transport of 5-oxoproline in rabbit renal brush-border membrane vesicles Effect of intravesicular potassium

The Na⁺-dependent transport of 5-oxoproline into rabbit renal brush-border vesicles was stimulated by a K⁺ diffusion potential (interior-negative) induced by valinomycin. Na⁺ salts of two anions of different epithelial permeabilities also affected 5-oxoproline transport. These results show that the Na⁺-dependent 5-oxoproline transport in renal brush-border vesicles is an electrogenic process which results in a net transfer of positive charge. Maximum transport of 5-oxoproline occurred at an extravesicular pH of 6.0 to 8.0 and over that pH range, 5-oxoproline exists completely as an anion with a negative charge. The simplest stoichiometry consistent with this process is, therefore, the cotransport of one 5-oxoproline anion with two sodium ions. The presence of K⁺ inside the vesicles stimulated the Na⁺-dependent transport of 5-oxoproline. This stimulatory effect was specific for K⁺ and required the presence of Na⁺. The presence of Na⁺ gradient was not mandatory for the K⁺ action. The stimulation by the intravesicular K⁺ was seen in the presence as well as in the absence of a K⁺ gradient. Therefore, the increased influx of 5-oxoproline was not coupled to the simultaneous efflux of K⁺. The presence of K⁺ in the extravesicular medium alone did not affect the Na⁺-dependent transport of 5-oxoproline, showing that the site of K⁺ action was intravesicular. Glutamate did not interact with the Na⁺-dependent 5-oxoproline transport even in the presence of an outward K⁺ gradient.     

The effects of potassium and membrane potential on sodium-dependent glutamic acid uptake

The uptake of l-glutamic acid into brush-border membrane vesicles isolated from rat renal proximal tubules is Na⁺-dependent. In contrast to Na⁺-dependent uptake of d-glucose, pre-equilibration of the vesicles with K⁺ stimulates l-glutamic acid uptake. Imposition of a K⁺ gradient ($\text{[}\text{K}{}_{\mathrm{i}}{}^{\mathrm{+}}\text{] > [}\text{K}{}_{\mathrm{o}}{}^{\mathrm{+}}\text{]}$) further enhances Na⁺-dependent l-glutamic acid uptake, but leaves K⁺-dependent glucose transport unchanged. If K⁺ is present only at the outside of the vesicles, transport is inhibited. Intravesicular Rb⁺ and, to a lesser extent, Cs⁺ can replace intravesicular K⁺ to stimulate l-glutamic acid uptake. Changes in membrane potential incurred by the imposition of an H⁺-diffusion potential or anion replacement markedly affect Na⁺-dependent glutamic acid uptake only in the presence of K⁺. Experiments with a potential-sensitive cyanine dye also indicate that, in the presence of intravesicular K⁺ a charge movement is involved in Na⁺-dependent transport of l-glutamic acid.The data indicate that Na⁺-dependent l-glutamic acid transport can be additionally energized by a K⁺ gradient. Furthermore, intravesicular K⁺ renders Na⁺-dependent l-glutamic acid transport sensitive to changes in the transmembrane electrical potential difference.