Gerhard Burckhardt

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 ([Ki+] > [Ko+]) 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+ render Na+-dependent L-glutamic acid transport sensitive to changes in the transmembrane electrical potential difference.

Cloning of a Human Renal p–Aminohippurate Transporter, hROAT1

For several decades, p-aminohippurate (PAH) has served as a model substrate to study the renal excretion of amphiphilic organic anions [1]. PAH is freely filtered in the glomeruli and also efficiently secreted in the proximal tubules. In all species investigated so far, the uptake of PAH across the basolateral membrane of proximal tubule cells occurs by exchange with intracellular α-ketoglutarate through a PAH/ α-ketoglutarate antiporter [1–3]. The dicarboxylate α-ketoglutarate is recycled by a Na +-coupled transport mechanism and the Na +-ions are pumped out of the cell by the Na +, K+-ATPase. Overall, the uptake of one PAH molecule requires the expenditure of one ATP molecule. Detailed investigations on substrate specificity revealed that the PAH transporter in the basolateral membrane of rat proximal tubule cells accepts a large variety of amphiphilic anions, uncharged compounds and even some organic cations [4]. The specificities of three basolateral organic anion transporters (PAH/ α-ketoglutarate antiporter, Na +-dicarboxylate symporter, sulfate/anion antiporter) and of the organic cation transporter localized in the same membrane partially overlap, ensuring excretion of most water-soluble xenobiotics with the urine. The proteins mediating PAH/ α-ketoglutarate antiport at the basolateral membrane of rat [5, 6] and winter flounder [7] proximal tubule cells have recently been identified by an expression cloning strategy using Xenopus laevisoocytes. The cloned renal organic anion transporters, termed OAT1/ROAT1 for rat and fROAT for flounder, are between 551 and 562 amino acids long with 12 putative transmembrane domains. Rat and flounder ROATs show an amino acid identity of 47% and are related to the organic cation transporters, OCT1 and OCT2 [8]. Following expression of cRNA derived from rat OAT1/ROAT1 and flounder fROAT in X. laevisoocytes, a probenecid-inhibitable, saturable uptake of PAH could be demonstrated [5–7]. PAH uptake was cis-inhibited by α-ketoglutarate and glutarate in the bath, and trans-stimulated by these dicarboxylates loaded previously into the oocytes, indicating that OAT1/ROAT1 and fROAT most likely represent the PAH/ α-ketoglutarate antiporter of the basolateral membrane of proximal tubule cells. In line with this assumption is the broad substrate specificity: besides PAH and α-ketoglutarate, OAT1 transported labeled methotrexate, cAMP, cGMP, PGE 2, and urate [5]. Direct experimental evidence is lacking for the presence of a PAH/α-ketoglutarate antiporter in the basolateral membrane of human renal proximal tubule cells. However, given the evolutionary conservation of PAH/ α-ketoglutarate antiport in the basolateral membrane, we assumed that a human homologue to OAT1/ROAT1 and fROAT exists. Here we report the PCR cloning of hROAT1 1, which is highly homologous to rat OAT1/ROAT1. Basic Renal Research

Sodium-dependent dicarboxylate transport in rat renal basolateral membrane vesicles.

Dicarboxylate transport in basolateral membrane vesicles prepared from rat kidney cortex was studied using3H-methylsuccinate as a substrate. A sodium gradient (out > in) simulated methylsuccinate uptake and led to a transient overshoot. Lithium inhibited methylsuccinate uptake in the presence of sodium. The dependence of methylsuccinate uptake on sodium concentration indicated the interaction of more than one sodium ion with the transporter. Half-maximal stimulation was observed at 24 mmol/l sodium. Sodium-driven methylsuccinate uptake was electrogenic carrying a net positive charge. The basolateral dicarboxylate transport system exhibited an optimum at pH 7.0–7.5. In contrast, the sodium-dependent dicarboxylate transport system of brush border membranes depended much less on pH and had no optimum in the tested range. Cis-inhibition studies showed a preference of the system for dicarboxylates in the trans-configuration (fumarate) over cis-dicarboxylates (maleate). Citrate was accepted but oxalate andl-glutamate were not. DIDS exhibited a small inhibition. Among the monocarboxylates, gluconate and pyruvate inhibited methylsuccinate uptake whereas probenecid and p-aminohippurate (1 mmol/l) were without effect. The data indicate the presence of a sodium-dependent transport system in the basolateral membrane which accepts tricarboxylic acid cycle intermediates. This system is most likely not identical to the transport system responsible for organic anion secretion.

Effect of the preparation method on Na+-H+ exchange and ion permeabilities in rat renal brush-border membranes.

The delta pH-dependent quenching of Acridine orange was used to characterize Na+-H+ exchange and K+ and H+ conductances in brush-border membrane vesicles isolated by precipitation with either CaCl2 or MgCl2 from rat kidney cortex. A transmembrane pH difference of 2.5 units (inside acidic) was imposed and the initial rate of its dissipation was followed after injecting a puls of tetramethylammonium gluconate (control) or sodium or potassium gluconate. In membranes isolated by CaCl2, the Na+-H+ exchange was partially electroneutral (45% to 77% of the total exchange) and the rest was due to electrically coupled Na+ and H+ movements through conductive pathways in the membranes. In membranes prepared by MgCl2, the rate of total Na+-H+ exchange was about twice as high as that in membranes obtained by CaCl2 precipitation. However, total and electroneutral exchanges were equal indicating negligible electrically coupled Na+ and H+ movements in these membranes. K0.5 for Na+ in all preparations was in the same range, being in average 30 mM. Amiloride was a competitive inhibitor of Na+-H+ exchange in membranes obtained with both preparations; Ki values ranged between 0.1 and 0.58 mM. The rates of delta pH-dissipation with K+ gradients (+/- valinomycin) were by 50% to 150% higher in membranes prepared with CaCl2 than in membranes isolated with MgCl2 indicating much higher H+ and K+ conductances in membranes obtained with CaCl2. Therefore, the rate of Na+-H+ exchange as well as the conductances for various ions in the isolated brush-border membranes depend on membrane preparation.

The influence of pH on phosphate transport into rat renal brush border membrane vesicles

Sodium-dependent transport of inorganic phosphate into brush border membrane vesicles is strongly influenced by altering pH of the incubation medium (pHo). At constant total phosphate concentration an increase in pHo leads to an increase in the uptake of inorganic phosphate. Uptake of inorganic phosphate, however, is not affected by the intravesicular pH (pHi) or by transmembrane pH differences (pHo-pHi). If initial phosphate uptake is studied as a function of total phosphate concentration in the medium the half saturation concentration increases when pHo is raised from 6.3–6.9 but remains unaltered between pHo 6.9 and 7.8Vmax increases about 3-fold between pHo 6.3 and 6.9 and by a factor of about 1.6 between pHo 6.9 and 7.4. The pHo-dependence of phosphate uptake is diminished by increasing sodium concentrations.Altering transmembrane electrical potential difference by potassium + valinomycin-induced diffusion potentials or by anion replacement fails to demonstrate electrogenicity of sodium-phosphate cotransport. Experiments using a potential-sensitive fluorescent dye, however, indicate a vesicle inside positive electrical potential difference when inorganic phosphate is added. The phosphate-induced alterations in the electrical potential difference are sodium-dependent and more pronounced at low pHo values.Together with earlier observations there results suggest that translocation of inorganic phosphate across the proximal tubular brush border membrane is mediated by cotransport of 2 sodium ions with one either monovalent or divalent phosphate molecule according to its availability in the tubular fluid. The pH sensitivity of this transport system is rather due to alterations in the transport system itself than to pH-dependent alteration in the ratio of monovalent to divalent phosphate.

Evidence for a cytosolic inhibitor of epithelial chloride channels.

It has been known for several years that the outwardly rectifying 30-pS chloride channel, the regulation of which has been reported to be defective in cystic fibrosis, can be activated by excision of a membrane patch from a cell. This suggested that the cytosol contains an inhibitory factor, which diffuses away after excision, thereby releasing the channel block. To test for such an inhibitor we have isolated cytosol from two epithelial cell lines, and in larger quantities from pig kidney cortex. Kidney cortex was chosen because published and unpublished evidence suggested that proximal tubular cells might also have a tonically suppressed Cl− conductance in the brush-border membrane, which is activated during isolation of membrane vesicles. The inhibitory effect of the cytosol preparations was assessed by: (a) measuring conductive Cl− fluxes on renal proximal tubular brush-border membrane vesicles preloaded with or without cytosol, and (b) recording single Cl− channel currents from excised membrane patches of nasal polyp epithelia and CFPAC-1 cells in the presence and absence of cytosol. All cytosol preparations tested were found to inhibit both conductive Cl− flux in membrane vesicles and single Cl− channels in patch-clamp experiments. In the latter case a type of flicker block was observed with a reduction of channel open probability. Stepwise dilution of the cytosol consistently reduced the inhibitory potency. Since the inhibition was preserved after boiling the cytosol for 10 min, we conclude that the inhibitor is a heat-stable substance. Whether it is identical with the postulated intracellular regulator that couples the defective function of the cystic fibrosis gene product to Cl− channel inhibition cannot be decided at present.

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

SummaryDicyclohexylcarbodiimide (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 α-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.

Single chloride channels in endosomal vesicle preparations from rat kidney cortex.

SummaryEndocytotic vesicles from rat kidney cortex, isolated by differential centrifugation and enriched on a Percoll gradient, contain both an electrogenic H+ translocation system and a conductive chloride pathway. Using the dehydration/rehydration method, we fused vesicles of enriched endosomal vesicle preparations and thereby made them accessible to the patch-clamp technique. In the fused vesicles, we observed Cl− channels with a single-channel conductance of 73±2 pS in symmetrical 140mm KCl solution (n=25). The current-voltage relationship was linear in the range of −60 to +80 mV, but channel kinetic properties dependended on the clamp potential. At positive potentials, two sublevels of conductance were discernible and the mean open time of the channel was 10–15 msec. At negative voltages, only one substate could be resolved and the mean open time decreased to 2–6 msec. Clamp voltages more negative than −50 mV caused reversible channel inactivation. The channel was selective for anions over cations. Ion substitution experiments revealed an anion permeability sequence of Cl−=Br−=I−>SO42−≈F−. Gluconate, methanesulfonate and cyclamate were impermeable. The anion channel blockers 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS, 1.0mm) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 0.1mm) totally inhibited channel activity. Comparisons with data obtained from radiolabeled Cl−-flux measurements and studies on the H+ pump activity in endocytotic vesicle suspensions suggest that the channel described here is involved in maintenance of electroneutrality during ATP-driven H+ uptake into the endosomes.

Relation of ATPases in Rat Renal Brush-Border Membranes to ATP-Driven H + Secretion

SummaryIn 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.

p-Aminohippurate/2-oxoglutarate exchange in bovine renal brush-border and basolateral membrane vesicles

The transport of the amphiphilic organic anion, P-aminohippurate (PAH), across the luminal (brush-border) and contraluminal (basolateral) membrane of renal proximal tubule cells was studied with membrane vesicles isolated from bovine kidney cortex. On the basis of the enrichment of specific activities of marker enzymes, leucine aminopeptidase and Na+/K+-ATPase, brush-border and basolateral membrane vesicles can be obtained from bovine kidneys in reasonably pure form. The uptake of [3H]PAH into both brush-border and basolateral membrane vesicles was trans-stimulated by intravesicular PAH and by 2-oxoglutarate. In the absence of Na+, [3H]PAH/2-oxoglutarate exchange was cis-inhibited by unlabelled 2-oxoglutarate in the medium. In the presence of an inward Na+ gradient, 10 μM 2-oxoglutarate, but no other Krebs cycle derivative, cis-stimulated [3H]PAH uptake, indicating that a Na3-coupled dicarboxylate transporter and PAH/2-oxoglutarate exchanger cooperate in both membranes to enhance [3H]PAH uptake. [3H]PAH uptake showed a non-saturable and a saturable component with similar apparent Km values in brush-border and basolateral membranes. Although one negatively charged PAH molecule exchanges with one doubly negatively charged 2-oxoglutarate molecule the exchange was electroneutral. Probenecid inhibited [3H]PAH/2-oxoglutarate exchange in brush-border and basolateral membrane vesicles with indistinguishable kinetics. We conclude that similar or identical PAH transporters are located in brush-border and basolateral membranes of bovine kidney proximal tubule cells. This arrangement seems species-specific since a Na+ gradient plus 2-oxoglutarate caused concentrative [3H]PAH uptake in brush-border membrane vesicles from bovine, but not from rat kidney.