Use of the pH sensitive fluorescence probe pyranine to monitor internal pH changes in Escherichia coli membrane vesicles

Measurements of the fluorescent properties of 8-hydroxy-1,3,6-pyrenetrisulfonate (pyranine) enclosed within the internal space of Escherichia coli membrane vesicles enable recordings and quantitative analysis of: (i) changes in intravesicular pH taking place during oxidation of electron donors by the membrane respiratory chain; (ii) transient alkalization of the internal aqueous space resulting from the creation of outwardly directed acetate diffusion gradients across the vesicular membrane. Quantitation of the fluorescence variations recorded during the creation of transmembrane acetate gradients shows a close correspondence between the measured shifts in internal pH value and those expected from the amplitude of the imposed acetate gradients.     

Formation of pH and potential gradients by the reconstituted Azotobacter vinelandii cytochrome bd respiratory protection oxidase.

To directly characterize the bioenergetic properties of the cytochrome bd terminating branch of the Azotobacter vinelandii electron transport chain, the purified cytochrome bd oxidase was reconstituted into a phospholipid environment consisting of phosphatidylethanolamine and phosphatidylglycerol (3:1). The average diameter of the proteoliposomes after extrusion through a polycarbonate membrane was 94 +/- 4 nm. Initiation of respiration upon the addition of 20 microM ubiquinone-1 to proteoliposomes loaded with the pH-sensitive dye pyranine resulted in an immediate alkalization of the vesicle lumen by an average pH change of 0.11 unit. This pH gradient was readily collapsed upon the addition of nigericin, carbonyl cyanide p-(tri-fluoromethoxy) phenyl-hydrazone, gramicidin, Triton X-100, or 2-heptyl-4-hydroxyquinoline N-oxide (HQNO). Proteoliposomal respiration initiated in the presence of the potentiometric membrane dye rhodamine 123 caused the generation of a transmembrane potential; the potential was collapsed upon the addition of either valinomycin or HQNO. The formation of both pH and potential gradients during turnover demonstrates that the A. vinelandii cytochrome bd oxidase is coupled to energy conservation in vivo.     

Effects of transmembrane potential and pH gradient on the cytochrome c-promoted fusion of mitochondrial mimetic membranes

The present study investigated the effects of ΔΨ and ΔpH (pH gradient) on the interaction of cytochrome c with a mitochondrial mimetic membrane composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cardiolipin (CL) leading to vesicle fusion. ΔpH generated by lowered bulk pH (pH_out) of PCPECL liposomes, with an internal pH (pH_in) of 8.0, favored vesicle fusion with a titration sigmoidal profile (pK _a?~?6.9). Conversely, ΔpH generated by enhanced pH_in of PCPECL at a pH_out of 6.0 favored the fusion of vesicles with a linear profile. We did not observe a significant amount of liposome fusion when ΔpH was generated by lowered pH_in at a pH_out of 8.0. At bulk acidic pH, ΔΨ generated by Na⁺ gradient also favored cyt c-promoted vesicle fusion. At acidic and alkaline pH_out, the presence of ΔpH and ΔΨ did not affect cytochrome c binding affinity measured by pyrene quenching. Therefore, cytochrome c-mediated PC/PE/CL vesicle fusion is dependent of ionization of the protein site L (acidic pH) and the presence of transmembrane potential. The effect of transmembrane potential is probably related to the generation of defects on the lipid bilayer. These results are consistent with previous reports showing that cytochrome c release prior to the dissipation of the ΔΨ_M blocks inner mitochondrial membrane fusion during apoptosis.     

Transport of Catecholamines by Native and Reconstituted Rat Heart Synaptic Vesicles

Highly purified “heavy” synaptic vesicles were isolated from rat heart by differential centrifugation. Because of the high intravesicular concentrations of proteins, catecholamine, and ATP, resealed vesicle ghosts were prepared and used to study the detailed kinetics of catecholamine transport. ATP stimulated the uptake of /-norepinephrine and was saturable with a K_m for l-norepinephrine at 3.3 μM and 1.8 mM for ATP. The ghosts also accumulated 5-hydroxytryptamine and l-epinephrine via an ATP-dependent mechanism. Uptake was stereospecific for the l-form. A functional catecholamine transporter could be solubilized by the detergent octyl-glucoside and incorporated into phospholipid vesicles, which, after detergent removal, generated proteoliposomes that accumulated l-norepinephrine. Reserpine- and l-propranolol-sensitive accumulation against a concentration gradient is achieved by artificially creating a pH gradient across the membrane, and lends further support to the idea that at least the initial phase of catecholamine transport is driven by the trans-membrane pH gradient created by the proton-translocating ATPase.     

Determination of ΔpH in cholinergic synaptic vesicles: Its effect on storage and release of acetylcholine

The interior of purified cholinergic Torpedo vesicles is acidic, pH_in = 5.2 at external pH = 7.4. The internal pH changes linearily as a function of external pH yielding ΔpH = 2.0 and 2.5 at pH_out = 6.3 and 9.1 respectively. The proton translocator carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) + the ionophore valinomycin dissipate the proton gradient across the vesicular membrane and concurrently induce acetylcholine release from vesicles suspended in K⁺ buffer. The effect of FCCP + valinomycin is not sensitive to external pH values between 6.3 and 9.1 and is diminished at lower external pH. The possible role of intravesicular pH and of the proton electrochemical gradient in the storage of acetylcholine within cholinergic vesicles is discussed.     

Solubilization and reconsitution of renal brush border Na+−H+ exchanger

Summary In order to permit future characterization and possible isolation of the Na⁺–H⁺ exchanger from the apical membrane of proximal tubular cells, studies were performed to solubilize and reconstitute this transporter. Rabbit brush border membranes were prepared by a magnesium aggregation method, solubilized with the detergent octyl glucoside, and reconstituted into artificial phospholipid vesicles. In the presence of a pH gradient (pH_in 6.0, pH_out 8.0), the uptake of 1mm ²²Na⁺ into the proteoliposomes was five- to sevenfold higher than into liposomes. Amiloride (2mm) inhibited proton gradient-stimulated uptake of sodium by 50%. As compared to proton gradient conditions, the uptake of sodium was lower in the absence of a pH gradient but was significantly higher when the outside and inside pH was 6.0 than 8.0. TheK _a for sodium in reconstituted proteoliposomes studied under pH gradient conditions was 4mm. The uptake of sodium in proteoliposomes prepared from heat-denatured membrane proteins was significantly decreased. These studies demonstrate that proteoliposomes prepared from octyl glucoside-solubilized brush border membrane proteins and asolectin exhibit proton gradient-stimulated, amiloride-inhibitable, electroneutral uptake of sodium. The ability to solubilize and reconstitute the Na⁺–H⁺ exchanger from the apical membrane of the proximal tubule will be of value in isolating and characterizing this transporter.     

Effects of triorganotin-mediated anion-hydroxide exchange upon reconstituted cytochrome c oxidase proteoliposomes

Proteoliposomes containing cytochrome c oxidase and an internally trapped fluorescent pH probe (pyranine) were used to monitor respiration-dependent internal alkalinization and membrane potential formation. A maximum steady-state pH gradient of about 0.4 pH unit (vesicle interior alkaline) was obtained during active respiration in presence of reducing substrates and cytochrome c. This pH gradient was abolished by the triorganotin compounds tripropyl-, tributyl-, and triphenyl-tin chloride. At the same time, the membrane potential, measured by carbocyanine dye uptake, was slightly increased in value. Valinomycin, which abolishes the membrane potential, restores the value of delta pH at low trialkyltin concentrations. The organotin compounds acted as electroneutral ionophores which exchanged intravesicular OH- ions with external SCN-, I-, and CI- ions, but not NO3- or SO4(2-) ions. Abolition of delta pH is accompanied by an increase in respiration rate, but full respiratory stimulation only occurs when both delta psi and delta pH are abolished by addition of both triorganotin and valinomycin. The triorganotin-valinomycin combination leads to active KCl accumulation by the respiring proteoliposome, and it is necessary to postulate an electrically neutral KCl efflux process to explain the continued steady respiration of the proteoliposomes in the presence of this ionophore combination.     

Structure and vectorial properties of proteoliposomes containing cytochrome oxidase in the submitochondrial orientation

Cytochrome oxidase proteoliposomes were prepared from bovine heart oxidase. Size distributions determined by quasi-elastic light scattering (QELS) showed that there was a small population of large vesicles (120-200-nm diameter) and a large population of small vesicles (50-100-nm diameter). Trapping cytochrome c inside the proteoliposomes did not significantly alter this size distribution. Separation of the vesicles by gel filtration, however, revealed that the cytochrome c/cytochrome a ratio is higher in the larger vesicles. Internally trapped cytochrome c can be reduced by the membrane-permeable reductants 2,3,5,6-tetramethyl-p-phenylenediamine (DAD) or N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). Respiration on internal cytochrome c generated a membrane potential of 53 mV (positive inside) and a pH gradient of 0.2 (acid inside) as monitored by the optical probes oxonol V and pyranine, respectively. But the true magnitude of these gradients in individual proteoliposomes is complicated by vesicle heterogeneity. The membrane potential increased biphasically with increasing concentration of reductant. Ionophore sensitivity was higher for the "low Km" phase, and respiration became increasingly uncoupled as the reductant concentration was increased. These findings are consistent with a kinetic heterogeneity such that vesicles respiring at lower reductant concentrations generate a higher proton motive force than those with a larger Km. The steady-state internal acidification induced by turnover of the internally facing enzyme is probably maintained by both cytochrome oxidase proton translocation and a TMPD+/H+ antiport present in these vesicles [Cooper, C. E., & Nicholls, P. (1987) FEBS. Lett. 223, 155-160].     

Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.     

Transport by reconstituted lactose carrier from parental and mutant strains ofEscherichia coli

Summary The lactose transport carrier from parental (X71/F'W3747) and mutant cells (54/F'5441) was reconstituted into proteoliposomes. Transport by the counterflow assay showed slightly greater activity in proteoliposomes prepared from extracts of the mutant membranes compared with that for the parental cell. The mutant carrier showed a threefold lowerK _m but similarV _max compared to the parent. On the other hand proteoliposomes from the mutant showed a defect in protonmotive force-driven accumulation, compared with the parent. With a pH gradient (inside alkaline) plus a membrane potential (inside negative) the parental proteoliposomes accumulated lactose 25-fold over the medium concentration while the mutant proteoliposomes accumulated sixfold. In a series of experiments proteoliposomes were exposed to proteolytic enzymes. Chrymotrypsin treatment resulted in 30% inhibition of counterflow activity for the reconstituted carrier from both parent and mutant. Papain produced 84% inhibition of transport by the reconstituted parental carrier but only 41% of that of the mutant. Trypsin and carboxypeptidase Y treatment had no effect on counterflow activity of either parent or mutant. Exposure of purified lactose carrier in proteoliposomes to carboxypeptidase Y resulted in the release of alanine and valine, the two C-terminal amino acids predicted from the DNA sequence.     

ATP-induced ΔpH formation in chloroplast ATP synthase proteoliposomes

Summary A procedure to reconstitute CF₀CF₁ proteoliposomes by gel filtration through a Sephadex-column pre-equilibrated with valinomycin and potassium is described. Proteoliposomes reconstituted by this procedure catalyze an ATP-induced pH of 2.5 to 3.5 units. pH was measured with either 9-aminoacridine or with the pH indicator pyranine trapped inside the proteoliposomes. CF₀CF₁ proteoliposomes prepared by conventional techniques catalyzed an ATP-induced formation, but were unable to catalyze an ATP-induced pH even in the presence of valinomycin.The ATP-induced pH was sensitive to uncouplers and energy transfer inhibitors and was increased at low temperatures. It is suggested that ATP-induced pH was observed in these proteoliposomes due to the efficient removal of intravesicular ammonium introduced with the CF₀CF₁ preparation. The ammonium acted as an internal buffer, and thus prevented an observable pH formation.     

Solubilization and reconstitution of the oat root vacuolar h/ca exchanger

Calcium is sequestered into vacuoles of oat (Avena sativa L.) root cells via a H⁺/Ca²⁺ antiporter, and vesicles derived from the vacuolar membrane (tonoplast) catalyze an uptake of calcium which is dependent on protons (pH gradient [ΔpH] dependent). The first step toward purification and identification of the H⁺/Ca²⁺ antiporter is to solubilize and reconstitute the transport activity in liposomes. The vacuolar H⁺/Ca²⁺ antiporter was solubilized with octylglucoside in the presence of soybean phospholipids and glycerol. After centrifugation, the soluble proteins were reconstituted into liposomes by detergent dilution. A ΔpH (acid inside) was generated in the proteoliposomes with an NH₄Cl gradient (NH₄⁺_in » NH₄⁺_out) as determined by methylamine uptake. Fundamental properties of ΔpH dependent calcium uptake such as the K_m for calcium (~15 micromolar) and the sensitivity to inhibitors such as N,N′-dicyclohexylcarbodiimide, ruthenium red, and lanthanum, were similar to those found in membrane vesicles, indicating that the H⁺/Ca²⁺ antiporter has been reconstituted in active form.     

Pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate) as a probe of internal aqueous hydrogen ion concentration in phospholipid vesicles

The fluorescence intensity (at 510 nm) of the hydrophilic pyrene analogue 8-hydroxy-1,3,6-pyrenetrisulfonate (pyranine) is strongly dependent upon the degree of ionization of the 8-hydroxyl group (pKa = 7.2) and hence upon the medium pH, over the range pH 6--10. Because of its polyanionic character, pyranine does not bind significantly to phospholipid vesicles having a net anionic surface charge. As a result, it is possible to form vesicles in the presence of pyranine which, after removal of external probe by gel filtration, contain pyranine entrapped within the internal aqueous compartment. Once entrapped, pyranine does not readily leak out of the vesicles. Because the fluorescence properties of entrapped pyranine resemble closely the properties of bulk pyranine solution with respect to pH sensitivity, pyranine can be used as a reliable reporter of aqueous pH changes within anionic vesicles. When HCl is rapidly added to a suspension of unilamellar soybean phospholipid (asolectin) vesicles preincubated at alkaline pH, a biphasic decrease in the pH of the vesicle inner aqueous compartment is observed. An initial, very rapid and electrically uncompensated H+ influx (t 1/2 less than 1 s) results in the generation of a transmembrane electric potential opposing further H+ influx. This leads to the development of a much slower (t 1/2 approximately equal to 5 min), valinomycin-sensitive, proton--counterion exchange which continues until the proton concentration gradient is eliminated. Similar results were obtained in asolectin vesicles prepared by detergent dilution, in sonicated egg phosphatidylcholine vesicles, and in multilamellar asolectin liposomes. The rather high permeability of soybean lipid membranes to H+ is surprising in view of the widespread use of these lipids for the reconstitution of membrane proteins which are thought to generate or utilize H+ ion gradients in energy transduction reactions.     

Apparent inhibition of Na+/H+ exchange by amiloride and harmaline in acridine orange studies

Amiloride and harmaline were tested as inhibitors of proton movements in brush-border membrane vesicles from rat kidney cortex. Transmembrane pH differences were visualized using acridine orange. Fluorescence quenching due to Na⁺ gradient-driven intravesicular acidification was inhibited by amiloride and harmaline. However, a similar inhibition was observed for the Na⁺ gradient-driven electrogenic proton movements in the presence of gramicidin. Moreover, amiloride and harmaline decreased the fluorescence signal of electrogenic proton movements driven by a K⁺ gradient in the presence of valinomycin. The degree of inhibition of intravesicular acidification by both drugs was concentration dependent. Half-maximal inhibition (I₅₀) of Na⁺/H⁺ exchange and K⁺ gradient-driven proton movements occurred at 0.21 and 0.6 amiloride, respectively. The I₅₀ for harmaline was 0.21 mM in both cases. Amiloride also decreased the initial quenching of acridine orange fluorescence due to a preset pH gradient without affecting the rate of dissipation of the pH gradient. This effect was independent of the buffer capacity. In contrast, harmaline seemed to dissipate pH gradient in the same way as a permeant buffer. Amiloride and harmaline led to a concentration-dependent fluorescence decrease even in aqueous solution. The results suggest an interaction of amiloride and harmaline with acridine orange which overlaps a possible specific inhibition of Na⁺/H⁺ exchange by these drugs.     

Determination of the rate of rapid pH equilibration across isolated sarcoplasmic reticulum membranes

The rate of the transmembrane pH equilibration in the isolated vesicles of sarcoplasmic reticulum of skeletal muscle after extravesicular pH jump was determined for the first time. A highly water-soluble pH sensitive fluorescent dye, 8-hydroxy-1,3,6-pyrenetrisulphonic acid (pyranine), was used as intravesicular pH indicator in the stopped-flow fluorophotometry. The pH of the medium was controlled with 20 mM HEPES-K or PIPES-K. The fluorescence intensity changed monophasically as much as expected from its pH dependency for imposed delta pH. The half time for initial pH of 7.53 or 6.83 was about 6 msec. The H+/OH- permeability was 11 cm/sec. The results suggested that each vesicle contained large numbers of H+/OH- channels.     

Hydrogel-assisted functional reconstitution of human P-glycoprotein (ABCB1) in giant liposomes

This paper describes the formation of giant proteoliposomes containing P-glycoprotein (P-gp) from a solution of small proteoliposomes that had been deposited and partially dried on a film of agarose. This preparation method generated a significant fraction of giant proteoliposomes that were free of internalized vesicles, making it possible to determine the accessible liposome volume. Measuring the intensity of the fluorescent substrate rhodamine 123 (Rho123) inside and outside these giant proteoliposomes determined the concentration of transported substrates of P-gp. Fitting a kinetic model to the fluorescence data revealed the rate of passive diffusion as well as active transport by reconstituted P-gp in the membrane. This approach determined estimates for the membrane permeability coefficient (P_s) of passive diffusion and rate constants of active transport (k_T) by P-gp as a result of different experimental conditions. The P_s value for Rho123 was larger in membranes containing P-gp under all assay conditions than in membranes without P-gp indicating increased leakiness in the presence of reconstituted transmembrane proteins. For P-gp liposomes, the k_T value was significantly higher in the presence of ATP than in its absence or in the presence of ATP and the competitive inhibitor verapamil. This difference in k_T values verified that P-gp was functionally active after reconstitution and quantified the rate of active transport. Lastly, patch clamp experiments on giant proteoliposomes showed ion channel activity consistent with a chloride ion channel protein that co-purified with P-gp. Together, these results demonstrate several advantages of using giant rather than small proteoliposomes to characterize transport properties of transport proteins and ion channels.     

Proton Transport Activity of the Purified Vacuolar H-ATPase from Oats : Direct Stimulation by Cl

To determine whether the detergent-solubilized and purified vacuolar H⁺-ATPase from plants was active in H⁺ transport, we reconstituted the purified vacuolar ATPase from oat roots (Avena sativa var Lang). Triton-solubilized ATPase activity was purified by gel filtration and ion exchange chromatography. Incorporation of the vacuolar ATPase into liposomes formed from Escherichia coli phospholipids was accomplished by removing Triton X-100 with SM-2 Bio-beads. ATP hydrolysis activity of the reconstituted ATPase was stimulated twofold by gramicidin, suggesting that the enzyme was incorporated into sealed proteoliposomes. Acidification of K⁺-loaded proteoliposomes, monitored by the quenching of acridine orange fluorescence, was stimulated by valinomycin. Because the presence of K⁺ and valinomycin dissipates a transmembrane electrical potential, the results indicate that ATP-dependent H⁺ pumping was electrogenic. Both H⁺ pumping and ATP hydrolysis activity of reconstituted preparations were completely inhibited by <50 nanomolar bafilomycin A₁, a specific vacuolar type ATPase inhibitor. The reconstituted H⁺ pump was also inhibited by N,N′-dicyclohexylcarbodiimide or NO₃⁻ but not by azide or vanadate. Chloride stimulated both ATP hydrolysis by the purified ATPase and H⁺ pumping by the reconstituted ATPase in the presence of K⁺ and valinomycin. Hence, our results support the idea that the vacuolar H⁺-pumping ATPase from oat, unlike some animal vacuolar ATPases, could be regulated directly by cytoplasmic Cl⁻ concentration. The purified and reconstituted H⁺-ATPase was composed of 10 polypeptides of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kilodaltons. These results demonstrate conclusively that the purified vacuolar ATPase is a functional electrogenic H⁺ pump and that a set of 10 polypeptides is sufficient for coupled ATP hydrolysis and H⁺ translocation.     

Functional Reconstitution of an ATP-Driven Ca-Transport System from the Plasma Membrane of Commelina communis L

The protein(s) that constitute(s) the ATP-driven Ca²⁺-translocator of plasma membrane enriched vesicles obtained by aqueous two-phase partitioning from leaves of Commelina communis L. has/have been solubilized and reincorporated into tightly sealed liposomes. The reconstituted Ca²⁺-transport system was studied using ATP-driven ⁴⁵Ca²⁺ import into the proteoliposomes as a measure of activity. The detergent, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate proved to be the most suitable and was used at 10 millimolar concentration, i.e. just above its critical micellar concentration. The presence of additional phospholipid (2 milligrams phosphatidylcholine per milliliter) and ATP (5 millimolar) improved the solubilization and/or reconstitution. The characteristics of the reconstituted system were similar to those of the plasma membrane-bound activity, including the apparent K_m for Ca²⁺ (5.2 micromolar), inhibition by relatively high levels of vanadate (IC₅₀ = 500 micromolar) and lacking response to added calmodulin. The reconstituted transport system was very strongly inhibited by erythrosine B (IC₅₀ = 0.01 micromolar) and had a low apparent K_m for ATP (11.4 micromolar). As in the plasma membrane vesicles, the protonophore carbonylcyanide m-chlorophenyl hydrazone did not affect Ca²⁺-transport detectably in the reconstituted system. However, low levels of the Ca²⁺-ionophore A 23187 instantaneously discharged 90% of the Ca²⁺ associated with the vesicles, proving that it had been accumulated in the intravesicular volume in soluble, freely exchangeable form. Ca²⁺-transport in the reconstituted system was thus primary active, through a Ca²⁺-translocating ATPase. The system reported here may serve as a valuable tool for purifying the Ca²⁺-ATPase and for studying structural and functional aspects of the purified enzyme.     

K+/H+ antiporter in alkaliphilic Bacillus sp. no. 66 (JCM 9763)

A K⁺/H⁺ antiport system was detected for the first time in right-side-out membrane vesicles prepared from alkaliphilic Bacillus sp. no. 66 (JCM 9763). An outwardly directed K⁺ gradient (intravesicular K⁺ concentration, K_in, 100 mM; extravesicular K⁺ concentration, K_out, 0.25 mM) stimulated uphill H⁺ influx into right-side-out vesicles and created the inside-acidic pH gradient (ΔpH). This H⁺ influx was pH-dependent and increased as the pH increased from 6.8 to 8.4. Addition of 100 μM quinine inhibited the H⁺ influx by 75%. This exchange process was electroneutral, and the H⁺ influx was not stimulated by the imposition of the membrane potential (interior negative). Addition of K⁺ at the point of maximum ΔpH caused a rapid K⁺-dependent H⁺ eflux consistent with the inward exchange of external K⁺ for internal H⁺ by a K⁺/H⁺ antiporter. Rb⁺ and Cs⁺ could replace K⁺ but Na⁺ and Li⁺ could not. The H⁺ efflux rate was a hyperbolic function of K⁺ and increased with increasing extravesicular pH (pH_out) from 7.5 to 8.5. These findings were consistent with the presence of K⁺/H⁺ antiport activity in these membrane vesicles. Received: March 20, 1997 / Accepted: May 22, 1997     

Ion pathways in renal brush border membranes

The absorbance change of the weak base dye probe, Acridine orange, was used to monitor alterations of pH gradients across renal brush border membrane vesicles. The presence of Na⁺/H⁺ or Li⁺/H⁺ exchange was demonstrated by diluting Na₂SO₄ or Li₂SO₄ loaded vesicles into Na⁺- or Li⁺-free solutions, which caused dye uptake. About 20% of the uptake was abolished by lipid permeable cations such as valinomycin-K⁺ or tetraphenylphosphonium, indicating perhaps the presence of a finite Na⁺ conductance smaller than electroneutral Na⁺/H⁺ exchange. The protonophore tetrachlorosalicylanilide raised the rate of dye uptake under these conditions, hence the presence of an Na⁺ conductance greater than the H⁺ conductance was suggested. K⁺ gradients also induced changes of pH, at about 10% of the Na⁺ or Li⁺ rate. Partial inhibition (21%) was seen with 0.1 mM amiloride indicating that K⁺ was a low affinity substrate for the Na⁺/H⁺ exchange. Acceleration both by tetrachlorosalicylanilide (2-fold) and valinomycin (4-fold) suggested the presence of 2 classes of vesicles, those with high and those with low K⁺ conductance. The larger magnitude of the valinomycin dependent signal suggested that 75% of the vesicles had a low K⁺ conductance. Inward Cl^? gradients also induced acidification, partially inhibited by the presence of tetraphenylphosphonium, and accelerated by tetrachlorosalicylanilide. Thus both a Cl^? conductance greater than the H⁺ conductance and a Cl^?/OH^? exchange were present. The rate of Na⁺/H⁺ exchange was amiloride sensitive with a pH optimum of 6.5 and an apparent K_m for Na⁺ or Li⁺ of about 10 mM and an E_A of 14.3 kcal per mol. A 61-fold Na₂SO₄ gradient resulted in a pH gradient of 1.64 units which increased to 1.8 with gramicidin. An equivalent NaCl gradient gave a much lower ΔpH even in the presence of gramicidin showing that the H⁺ and Cl^? pathways could alter the effects of the Na⁺/H⁺ exchange.