Carrier-mediated transport systems of tetraethylammonium in rat renal brush-border and basolateral membrane vesicles

Transport of [3H]tetraethylammonium, an organic cation, has been studied in brush-border and basolateral membrane vesicles isolated from rat kidney cortex. Some characteristics of carrier-mediated transport for tetraethylammonium were demonstrated in brush-border and basolateral membrane vesicles; the uptake was saturable, was stimulated by the countertransport effect, and showed discontinuity in an Arrhenius plot. In brush-border membrane vesicles, the presence of an H+ gradient ( [H+]i greater than [H+]o) induced a marked stimulation of tetraethylammonium uptake against its concentration gradient (overshoot phenomenon), and this concentrative uptake was completely inhibited by HgCl2. In contrast, the uptake of tetraethylammonium by basolateral membrane vesicles was unaffected by an H+ gradient. Tetraethylammonium uptake by basolateral membrane vesicles was significantly stimulated by a valinomycin-induced inside-negative membrane potential, while no effect of membrane potential was observed in brush-border membrane vesicles. These results suggest that tetraethylammonium transport across brush-border membranes is driven by an H+ gradient via an electroneutral H+-tetraethylammonium antiport system, and that tetraethylammonium is transported across basolateral membranes via a carrier-mediated system and this process is stimulated by an inside-negative membrane potential.     

Characterization of the transport system for beta-lactam antibiotics and dipeptides in rat renal brush-border membrane vesicles by photoaffinity labeling

The uptake of the alpha-aminocephalosporin cephalexin into brush-border membrane vesicles from rat renal cortex was independent on an inward H+-gradient in contrast to the intestinal transport system. The transport system could be irreversibly inhibited by photoaffinity labeling. Two binding polypeptides for beta-lactam antibiotics and dipeptides with apparent molecular weights 130,000 and 95,000 were identified by photoaffinity labeling with [3H]benzylpenicillin and N-(4-azido[3,5-3H]benzoyl) derivatives of cephalexin and glycyl-L-proline. The uptake of cephalexin and the labeling of the respective binding proteins was inhibited by beta-lactam antibiotics and dipeptides as with intestinal brush-border membranes. These data indicate that the transport systems for beta-lactam antibiotics and dipeptides in the brush-border membrane from rat kidney and small intestine are similar but not identical.     

Affinity labelling of the folate-binding protein in pig intestine.

A specific transport system for folate and a high-affinity folate-binding protein have been identified in pig intestinal brush-border membranes. To determine if the binding protein plays a role in folic acid (PteGlu) uptake in to the cell, the inactivation of folate binding and transport by N-hydroxysuccinimide esters of folic acid (NHS-PteGlu) was compared. In addition, the number of brush-border proteins modified by the affinity reagent was assessed. Brush-border vesicles were incubated with various concentrations of NHS-PteGlu or NHS-methotrexate. Transport and binding of [3H]PteGlu by the vesicles were measured at 37 and 4 degrees C respectively by using the vacuum-filtration technique. NHS-methotrexate and NHS-PteGlu specifically inhibited PteGlu transport. Incubating the vesicles with 1 microM-NHS-PteGlu inactivated [3H]PteGlu transport by 60% and binding by 80%. Half-maximal inhibition of both transport and binding was observed at similar concentrations of the affinity reagent (0.05 and 0.07 microM-NHS-PteGlu respectively). Treating the vesicles with radiolabelled NHS-PteGlu followed by gel electrophoresis and autoradiography revealed a specifically labelled protein with an Mr of 56,000. These results indicate that the intestinal folate-binding and transport proteins are identical and that the function of the folate-binding protein is to transport folate into the cell.     

Analysis of the pH dependence of folate binding and transport by rat kidney brush border membrane vesicles

Folate reabsorption by the mammalian kidney occurs following a tight binding reaction with the renal brush border membrane. Previous studies have shown that transport of folic acid (PteGlu) by rat kidney brush border membrane vesicles occurs maximally at pH 5.6 via a saturable system that is associated with a binding component. The present studies have shown that the pH dependency of transport was due to the development of the transmembrane pH gradient (7.3 in/5.6 out), not to the acidic pH per se. The pH gradient-mediated transport was stimulated by an inwardly directed ionic gradient, either of NaCl or choline chloride. These gradients also stimulated the membrane binding of PteGlu suggesting that NaCl and choline chloride may have increased PteGlu transport by altering binding to the brush border membrane. Renal brush border membrane vesicular transport of PteGlu was not affected by induction of a relatively positive intravesicular space. Transport was inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, an anion exchange inhibitor. The results suggest that rat kidney brush border membrane transport of PteGlu is initiated by association with a specific membrane protein, followed by transfer of folate across the membrane. The overall activity is influenced by a transmembrane pH gradient.     

Evidence for an organic cation-proton antiport system in brush-border membranes isolated from the human term placenta

Uptake of guanidine, an endogenous organic cation, into brush-border membrane vesicles isolated from human term placentas was investigated. Initial uptake rates were manyfold greater in the presence of an outward-directed H+ gradient ([pH]o greater than [pH]i) than in the absence of a H+ gradient ([pH]o = [pH]i). Guanidine was transiently accumulated inside the vesicles against a concentration gradient in the presence of the H+ gradient. The H+ gradient-dependent stimulation of guanidine uptake was not due to a H+-diffusion potential because an ionophore (valinomycin or carbonylcyanide p-trifluoromethoxyphenylhydrazone)-induced inside-negative membrane potential failed to stimulate the uptake. In addition, uphill transport of guanidine could be demonstrated even in voltage-clamped membrane vesicles. The H+ gradient-dependent uptake of guanidine was inhibited by many exogenous as well as endogenous organic cations (cis-inhibition) but not by cationic amino acids. The presence of unlabeled guanidine inside the vesicles stimulated the uptake of labeled guanidine (trans-stimulation). These data provide evidence for the presence of an organic cation-proton antiporter in human placental brush-border membranes. Kinetic analysis of guanidine uptake demonstrated that the uptake occurred via two saturable, carrier-mediated transport systems, one being a high affinity, low capacity type and the other a low affinity, high capacity type. Studies on the effects of various cations on the organic cation-proton antiporter and the Na+-H+ exchanger revealed that these two transport systems are distinct.     

Characteristics of tetraethylammonium transport in rabbit renal plasma-membrane vesicles.

Transport of [14C]tetraethylammonium (TEA), an organic cation, was studied in brush-border (BBMV) and basolateral (BLMV) membrane vesicles isolated from rabbit kidney cortex. In BBMV, the presence of an outwardly directed H+ gradient induced a marked stimulation of TEA uptake against its concentration gradient (overshoot phenomenon), whereas a valinomycin-induced inside-negative potential had no effect on TEA uptake. In BLMV, TEA uptake was significantly stimulated by the presence of an outwardly directed H+ gradient and by an inside-negative potential, but the effect of H+ gradient was absent when the vesicles were chemically 'voltage clamped'. In BBMV, internal H+ stimulated TEA uptake in a non-competitive manner by binding at a site with apparent pKa of 6.87. External H+ inhibited TEA uptake through a direct interaction with the putative H+/organic-cation exchanger at a site with apparent pKa of 6.78. Changing external pH while maintaining the pH gradient constant produced a result similar to that obtained by changing external pH alone. Increasing external H+ showed a mixed-type inhibition of TEA uptake. These results suggest that in the rabbit TEA transport across the basolateral membranes is driven by an inside-negative potential and that transport across the brush-border membrane is driven by a H+ gradient via an electroneutral H+/TEA antiport system.     

Enzymatic removal of alkaline phosphatase from renal brush-border membranes. Effect on phosphate transport and on phosphate binding

Brush-border membrane vesicles prepared from rabbit kidney cortex were incubated at 37 degrees C for 30 min with phosphatidylinositol-specific phospholipase C. This maneuver resulted in a release of approx. 85% of the brush-border membrane-linked enzyme alkaline phosphatase as determined by its enzymatic activity. Transport of inorganic [32P]phosphate (100 microM) by the PI-specific phospholipase C-treated brush-border membrane vesicles was measured at 20-22 degrees C in the presence of an inwardly directed 100 mM Na+ gradient. Neither initial uptake rates, as estimated from 10-s uptake values (103.5 +/- 6.8%, n = 7 experiments), nor equilibrium uptake values, measured after 2 h (102 +/- 3.4%) were different from controls (100%). Control and PI-specific phospholipase C-treated brush-border membrane vesicles were extracted with chloroform/methanol to obtain a proteolipid fraction which has been shown to bind Pi with high affinity and specificity (Kessler, R.J., Vaughn, D.A. and Fanestil, D.D. (1982) J. Biol. Chem. 257, 14311-14317). Phosphate binding (at 10 microM Pi) by the extracted proteolipid was measured. No significant difference in binding was observed between the two types of preparations: 31.0 +/- 9.37 in controls and 29.8 +/- 8.3 nmol/mg protein in the proteolipid extracted from PI-specific phospholipase C-treated brush-border membrane vesicles. It appears therefore that alkaline phosphatase activity is essential neither for Pi transport by brush-border membrane vesicles nor for Pi binding by proteolipid extracted from brush-border membrane. These results dissociate alkaline phosphatase activity, but not brush-border membrane vesicle transport of phosphate, from phosphate binding by proteolipid.     

pH-sensitive transport of Fe2+ across purified brush-border membrane from mouse intestine

Recent studies of Fe2+ uptake by mouse proximal intestine brush-border membrane vesicles revealed low-affinity, NaCl-sensitive and high-affinity, NaCl-insensitive, components of uptake (Simpson, R.J. and Peters, T.J. (1985) Biochim. Biophys. Acta 814, 381-388). In this study, the former component is demonstrated to show a strong pH dependence with an optimum of pH 6.8-6.9. Studies at pH 6.5, where the low affinity component is inhibited by more than 25-fold compared with pH 7.2, suggest that the pH-sensitive component represents transport across the brush-border membrane followed by intravesicular binding. Cholate extracts of brush-border membrane vesicles contain pH- and NaCl-sensitive Fe2+ binding moieties which may be involved in the transfer of Fe2+ across the intestinal brush-border membrane and subsequent binding inside the vesicles. Fe2+ uptake by brush-border membrane vesicles from the duodenum of hypoxic mice is higher than uptake by vesicles from control-fed animals, suggesting the existence of a regulable brush-border membrane Fe2+ carrier.     

Na(+)-dependent fructose transport via rNaGLT1 in rat kidney

We found a system of Na(+)-dependent uptake of fructose by rat renal brush-border membrane vesicles. It consisted of two saturable components, and was thought to involve at least two transporters. rNaGLT1, a novel glucose transporter in rat kidney, showed fructose uptake as well as alpha-methyl-D-glucopyranoside uptake by transfected HEK293 cells. The features of the lower affinity type of fructose transporter in the brush-border membranes, such as affinity and substrate recognition, were very comparable with those of rNaGLT1-transfected HEK293 cells. These results indicated that rNaGLT1 is a primary fructose transporter in rat renal brush-border membranes.     

Phosphate transport into brush-border membrane vesicles isolated from rat small intestine.

Uptake of Pi into brush-border membrane vesicles isolated from rat small intestine was investigated by a rapid filtration technique. The following results were obtained. 1. At pH 7.4 in the presence of a NaCl gradient across the membrane (sodium concentration in the medium higher than sodium concentration in the vesicles), phosphate was taken up by a saturable transport system, which was competitively inhibited by arsenate. Phosphate entered the same osmotically reactive space as D-glucose, which indicates that transport into the vesicles rather than binding to the membranes was determined. 2. The amount of phosphate taken up initially was increased about fourfold by lowering the pH from 7.4 to 6.0.3. When Na+ was replaced by K+, Rb+ or Cs+, the initial rate of uptake decreased at pH 7.4 but was not altered at pH 6.0.4. Experiments with different anions (SCN-,Cl-, SO42-) and with ionophores (valinomycin, monactin) showed that at pH 7.4 phosphate transport in the presence of a Na+ gradient is almost independent of the electrical potential across the vesicle membrane, whereas at pH 6.0 phosphate transport involves the transfer of negative charge. It is concluded that intestinal brush-border membranes contain a Na+/phosphate co-transport system, which catalyses under physiological conditions an electroneutral entry of Pi and Na+ into the intestinal epithelial cell. In contrast with the kidney, probably univalent phosphate and one Na+ ion instead of bivalent phosphate and two Na+ ions are transported together.     

Biotin uptake mechanisms in brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex

Biotin transport was studied using brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex. An inwardly directed Na+ gradient stimulated biotin uptake into brush-border membrane vesicles and a transient accumulation of the anion against its concentration gradient was observed. In contrast, uptake of biotin by basolateral membrane vesicles was found to be Na+-gradient insensitive. Generation of a negative intravesicular potential by valinomycin-induced K+ diffusion potentials or by the presence of Na+ salts of anions of different permeabilities enhanced biotin uptake by brush-border membrane vesicles, suggesting an electrogenic mechanism. The Na+ gradient-dependent uptake of biotin into brush-border membrane vesicles was saturable with an apparent Km of 28 microM. The Na+-dependent uptake of tracer biotin was significantly inhibited by 50 microM biotin, and thioctic acid but not by 50 microM L-lactate, D-glucose, or succinate. Finally, the existence in both types of membrane vesicles of a H+/biotin- cotransport system could not be demonstrated. These results are consistent with a model for biotin reabsorption in which the Na+/biotin- cotransporter in luminal membranes provides the driving force for uphill transport of this vitamin.     

Characterization of basolateral membrane Na/H antiport in rat jejunum

Na uptake studies were performed in order to examine the activity of a Na/H exchanger in basolateral membrane vesicles isolated from rat jejunum. Experiments were carried out under voltage-clamped conditions in order to avoid electrodiffusional ionic movements. 1 mM Na uptake was found to be enhanced by an outward proton gradient and its initial rate was further increased by the presence of monensin or nigericin. The pH gradient-driven Na uptake was inhibited by 2 mM amiloride and unaffected by 0.1 mM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. The initial rate of the proton gradient-induced Na uptake was saturable with respect to external Na, with a Km of 13.6 +/- 1.4 mM and a Vmax of 35.4 +/- 2.2 nmol/mg protein per min. Li competed with Na for the exchange process, whereas K, Rb, Cs, tetramethylammonium had no effect. We conclude that rat jejunal basolateral membrane contains a Na/H exchanger whose properties are similar to those of the antiporter identified in the brush-border membrane.     

A proton gradient, not a sodium gradient, is the driving force for active transport of lactate in rabbit intestinal brush-border membrane vesicles.

An inward-directed H+ gradient markedly stimulated lactate uptake in rabbit intestinal brush-border membrane vesicles, and uphill transport against a concentration gradient could be demonstrated under these conditions. Uptake of lactate was many-fold greater in the presence of a H+ gradient than in the presence of a Na+ gradient. Moreover, there was no evidence for uphill transport of lactate in the presence of a Na+ gradient. The H+-gradient-dependent stimulation of lactate uptake was not due to the effect of a H+-diffusion potential. The uptake process in the presence of a H+ gradient was saturable [Kt (concn. giving half-maximal transport) for lactate 12.7 +/- 4.5 mM] and was inhibited by many monocarboxylates. It is concluded that a H+ gradient, not a Na+ gradient, is the driving force for active transport of lactate in rabbit intestinal brush-border membrane vesicles.     

Sulphate-ion/sodium-ion co-transport by brush-border membrane vesicles isolated from rat kidney cortex

Uptake of SO(4) (2-) into brush-border membrane vesicles isolated from rat kindey cortex by a Ca(2+)-precipitation method was investigated by using a rapid-filtration technique. Uptake of SO(4) (2-) by the vesicles was osmotically sensitive and represented transport into an intra-vesicular space. Transport of SO(4) (2-) by brush-border membranes was stimulated in the presence of Na(+), compared with the presence of K(+) or other univalent cations. A typical ;overshoot' phenomenon was observed in the presence of an NaCl gradient (100mm-Na(+) outside/zero mm-Na(+) inside). Radioactive-SO(4) (2-) exchange was faster in the presence of Na(+) than in the presence of K(+). Addition of gramicidin-D, an ionophore for univalent cations, decreased the Na(+)-gradient-driven SO(4) (2-) uptake. SO(4) (2-) uptake was only saturable in the presence of Na(+). Counter-transport of Na(+)-dependent SO(4) (2-) transport was shown with MoO(4) (2-) and S(2)O(3) (2-), but not with PO(4) (2-). Changing the electrical potential difference across the vesicle membrane by establishing different diffusion potentials (anion replacement; K(+) gradient+/-valinomycin) was not able to alter Na(+)-dependent SO(4) (2-) uptake. The experiments indicate the presence of an electroneutral Na(+)/SO(4) (2-)-co-transport system in brush-border membrane vesicles isolated from rat kidney cortex.     

Effect of acidosis on glutamine transport by isolated rat renal brush-border and basolateral-membrane vesicles.

Glutamine uptake was examined in isolated renal brush-border and basolateral-membrane vesicles from control and acidotic rats. In brush-border vesicles from acidotic animals, there was a significant increase in the initial rate of glutamine uptake compared with that in controls. Lowering the pH of the medium increased the initial rate of glutamine uptake in brush-border vesicles from acidotic, but not from control, rats. In brush-border vesicles from both groups of animals, two saturable transport systems mediated glutamine uptake. There was a 2-fold increase in the Vmax. of the low-affinity high-capacity system in the brush-border vesicles from the acidotic animals compared with that from control animals, with no alteration in the other kinetic parameters. There was no difference in glutamine uptake by the two saturable transport systems in basolateral vesicles from control and acidotic animals. Lowering the incubation-medium pH increased the uptake of glutamine by basolateral vesicles from both control and acidotic rats to a similar extent. The data indicate that during acidosis there are alterations in glutamine transport by both the basolateral and brush-border membrane which could enhance its uptake by the renal-tubule cell for use in ammoniagenesis.     

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 microM) 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.     

The mechanism of uptake of retinol by plasma-membrane vesicles.

The mechanism of retinol uptake by human placental brush-border membrane vesicles was investigated using initial-velocity studies of [3H]retinol uptake from the [3H]retinol-RBP (retinol-binding protein) complex. The process was rapid and time- and temperature-dependent. The uptake was specifically reversed by the addition of native or apo-RBP, but not by serum albumin. By contrast, uptake of free [3H]retinol was temperature-independent, partially reversible and showed no requirement for a specific protein for reversibility. Treatment of membrane vesicles with p-chloromercuribenzenesulphonate (PCMBS), which inhibited 125I-RBP binding, also inhibited the uptake of retinol from RBP, but the uptake of free retinol was unaffected. Addition of PCMBS after the attainment of steady-state uptake equilibrium abolished the binding of RBP, but did not affect the retinol already taken up from RBP. The results suggest that binding of RBP to its specific receptor is obligatory for the subsequent delivery of retinol to the membrane. Since the studies were carried out on isolated membrane vesicles, endocytosis of RBP is most unlikely to be involved in the placental transport of retinol. A double-reciprocal plot of initial velocity versus [3H]retinol-RBP concentration gave an apparent Km of 116 +/- 13 nM. Transthyretin decreased the rate of uptake of [3H]retinol from RBP without substantially altering the steady-state uptake levels, suggesting that membranes take up retinol from uncomplexed RBP. High-pressure gel-filtration chromatography showed that [3H]retinol is largely transferred to a membrane component with an apparent molecular mass of 125 kDa.     

Binding of 125I-labelled albumin by isolated rat renal brush-border membrane vesicles. Evidence for uptake and internalization process

1. In the kidney, filtered proteins are rapidly reabsorbed by the proximal tubule via adsorptive endocytosis. This process starts with the protein binding to the luminal brush-border membrane. 2. The binding of 125I-labelled albumin to rat renal brush-border membrane vesicles and the effect of a low molecular weight protein lysozyme on that binding was assessed by the filtration method. 3. The Scatchard plot revealed a one-component binding-type curve with a dissociation constant Kd of 430.9 nM and 39.6 pmol/mg membrane protein for the number of binding sites. 4. Albumin binding was saturable and reversible, time and temperature dependent and the initial rate enhanced by increasing amounts of lysozyme. 5. The fact that association of albumin with the brush-border membrane vesicles was dependent upon the intravesicular space suggested a double process, binding of the ligand to the membrane surface and its internalization. These data suggest that albumin has a different binding site than that of a low-molecular weight protein lysozyme, with a constant affinity value near physiological loads. That specificity may confer selectivity upon the endocytic uptake process.     

Carrier-mediated transport system for choline and its related quaternary ammonium compounds on rat intestinal brush-border membrane.

The characteristics of the intestinal transport system for choline were investigated using isolated brush-border membrane vesicles from rat small intestine. In spite of the diminutive lipid solubility, the uptake of choline by membrane vesicles reflected smooth permeation into intravesicular space rather than the binding to the membrane surface. Physiological conditions, present in the intact intestine, such as an inward-directed Na+ or H+ gradient and inside negative membrane potentials, didn't directly involve in choline transport across the brush-border membrane. Moreover, an outward-directed H+ gradient had no significant effect on the time course of choline transport. However, in the absence of a driving-force, the initial uptake of choline exhibited a saturable manner. A kinetic analysis of the initial uptake rate gave an apparent Km of 159 microM. Furthermore, unlabeled choline caused both cis-inhibition and trans-stimulation for labeled choline transport, suggesting the existence of a carrier-mediated transport system for choline, classified as so-called 'facilitated diffusion'. Since tetramethylammonium, acetylcholine, and N1-methylnicotinamide caused both cis-inhibition and trans-stimulation, they appear to be accepted as the substrate of choline carrier. On the other hand, quaternary ammonium compounds (QACs) such as those which possessed hydrophobic parts in their molecules exhibited only cis-inhibition. They also inhibited Na(+)-dependent D-glucose transport, indicating that they influenced various carrier-mediated transport systems non-specifically due to interaction with the membrane. These findings strongly suggest that the choline transport system on the brush-border membrane of rat intestine recognizes only small molecular QACs as its substrate.     

Proton pathways in rat renal brush-border and basolateral membranes

The quenching of acridine orange fluorescence was used to monitor the formation and dissipation of pH gradients in brush-border and basolateral membrane vesicles isolated from rat kidney cortex. The fluorescence changes of acridine orange were shown to be sensitive exclusively to transmembrane delta pH and not to membrane potential difference. In brush-border membrane vesicles, an Na+ (Li+)-H+ exchange was confirmed. At physiological Na+ concentrations, 40-70% of Na+-H+ exchange was mediated by the electroneutral Na+-H+ antiporter; the remainder consisted of Na+ and H+ movements through parallel conductive pathways. Both modes of Na+-H+ exchange were saturable, with half-maximal rates at about 13 and 24 mM Na+, respectively. Besides a Na+ gradient, a K+ gradient was also able to produce an intravesicular acidification, demonstrating conductance pathways for H+ and K+ in brush-border membranes. Experiments with Cl- or SO2-4 gradients failed to demonstrate measurable Cl--OH- or SO2-4-OH- exchange by an electroneutral antiporter in brush-border membrane vesicles; only Cl- conductance was found. In basolateral membrane vesicles, neither Na+(Li+)-H+ exchange nor Na+ or K+ conductances were found. However, in the presence of valinomycin-induced K+ diffusion potential, H+ conductance of basolateral membranes was demonstrated, which was unaffected by ethoxzolamide and 4,4'-diisothiocyanostilbene-2,2-disulfonic acid. A Cl- conductance of the membranes was also found, but antiporter-mediated electroneutral Cl--OH- or SO2-4-OH- exchange could not be detected by the dye method. The restriction of the electroneutral Na+-H+ exchanger to the luminal membrane can explain net secretion of protons in the mammalian proximal tubule which leads to the reabsorption of bicarbonate.