Transmembrane effects of intracellular chloride on the inhibitory potency of extracellular H2DIDS. Evidence for two conformations of the transport site of the human erythrocyte anion exchange protein

The ping-pong model for the red cell anion exchange system postulates that the transport protein band 3 can exist in two different conformations, one in which the transport site faces the cytoplasm (Ei) and another in which it faces the outside medium (Eo). This model predicts that an increase in intracellular chloride should increase the fraction of sites in the outward-facing, unloaded form (Eo). Since external H2DIDS is a competitive inhibitor of chloride exchange that does not cross the membrane, it must bind only to the Eo form. Thus, an increase in Eo should cause an increase in H2DIDS inhibition. When intracellular chloride was increased at constant extracellular chloride, the inhibitory potency of H2DIDS rose, as predicted by the ping-pong model. This increase was not due to the concomitant changes in intracellular pH or membrane potential. When the chloride gradient was reversed, the inhibitory potency of H2DIDS decreased, again in qualitative agreement with the ping-pong model. These data provide support for the ping-pong model and also demonstrate that chloride gradients can be used to change the orientation of the transport protein.     

Asymmetry of the red cell anion exchange system. Different mechanisms of reversible inhibition by N-(4-azido-2-nitrophenyl)-2- aminoethylsulfonate (NAP-taurine) at the inside and outside of the membrane

In the dark, the photoaffinity reagent, N-(4-azido-2-nitrophenyl)-2- aminoethylsulfonate (NAP-taurine), acts as a reversible inhibitor of red cell anion exchange when it is present either within the cell or in the external solution. A detailed analysis of the inhibition kinetics, however, reveals substantial differences in the responses to the probe at the two sides of the membrane. On the inside of the cell, NAP- taurine is a relatively low affinity inhibitor of chloride exchange (Ki = 370 microM). Both the effects of chloride on NAP-taurine inhibition and the affinity of NAP-taurine for the system as a substrate are consistent with the concept that internal NAP-taurine competes with chloride for the substrate site of the anion exchange system. External NAP-taurine, on the other hand, is a far more potent inhibitor of chloride exchange (Ki = 20 microM). It acts at a site of considerably lower affinity for chloride than the substrate site, probably the modifier site, at which halide anions are reported to cause a noncompetitive inhibition of chloride transport. NAP-taurine therefore seems to interact preferentially with either the substrate or modifier site of the transport system, depending on the side of the membrane at which it is present. It is suggested that the modifier site is accessible to NAP-taurine only from the outside whereas the transport site may be accessible from either side.     

Transmembrane effects of irreversible inhibitors of anion transport in red blood cells. Evidence for mobile transport sites

Experiments were designed to determine whether band 3, the anion transport protein of the red cell membrane, contains a mobile element that acts as a carrier to move the anions across a permeability barrier. The transport site-specific, nonpenetrating irreversible inhibitor 4,4'-diisothiocyano-2,2'-stilbene disulfonate (DIDS) was found to be effective only when applied extracellularly. It was used to sequester transport sites on the extracellular side of the membrane in intact cells. The membranes were then coverted into inside-out vesicles. The number of anion transport sites available on the cytoplasmic side of the vesicle membranes was then estimated by measuring the binding of N-(-4-azido-2-nitrophenyl)-2-aminoethyl-sulfonate (NAP-taurine), a photoreactive probe. Pretreatment with DIDS from the extracullular side substantially reduced the binding of NAP-taurine at the cytoplasmic side. Since NAP-taurine does not appear to penetrate into the intravesicular (normally extracellular) space, a transmembrane effect is apparently involved. About 70% of the DIDS-sensitive NAP-taurine binding sites are located in band 3, with the remainder largely in a lower molecular weight (band 4) region. A similar pattern of reduction in NAP-taurine binding is produced by high concentrations of Cl-, but this anion has little or no effect in vesicles from cells pretreated with DIDS. Thus the DIDS-modulated sites seem to be capable of binding either NAP-taurine or Cl. It is suggested that band 3 contains a mobile transport element that can be recruited to the extracellular surface by DIDS, thus becoming unavailable to NAP-taurine at the cytoplasmic face of the membrane. The results are consistent with a model of carrier-mediated transport in which the movement of the transport site is associated with a local conformational change in band 3 protein.     

N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine) as a photoaffinity probe for identifying membrane components containing the modifier site of the human red blood cell anion exchange system

Exposure of cells to intense light with the photoactivatable reagent, N- (4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine), present in the external medium results in irreversible inhibition of chloride or sulfate exchange. This irreversible inhibition seems to result from covalent reaction with the same sites to which NAP-taurine binds reversibly in the dark. As shown in the preceding paper, high chloride concentrations decrease the reversible inhibition by NAP-taurine in the dark, in a manner suggesting that NAP-taurine and chloride compete for the modifier site of the anion transport system. In a similar fashion, high chloride concentrations in the medium during exposure to light cause a decrease in both the irreversible binding of NAP-taurine to the membrane and the inhibition of chloride exchange. Most of the chloride- sensitive irreversibly bound NAP-taurine is found in the 95,000 dalton polypeptide known as band 3 and, after pronase treatment of intact cells, in the 65,000 dalton fragment of this protein produced by proteolytic cleavage. After chymotrypsin treatment of ghosts, the NAP- taurine is localized in the 17,000 dalton transmembrane portion of this fragment. Although the possible involvement of minor labeled proteins cannot be rigorously excluded, the modifier site labeled by external NAP-taurine appears, therefore, to be located in the same portion of the 95,000 dalton polypeptide as is the transport site.     

Use of niflumic acid to determine the nature of the asymmetry of the human erythrocyte anion exchange system

Niflumic acid is a noncompetitive inhibitor of chloride exchange, which binds to a site different from the transport or modifier sites. When the internal Cl- concentration is raised, at constant extracellular Cl- , the inhibitory potency of niflumic acid increases. This effect cannot be attributed to changes in membrane potential, but rather it suggests that niflumic acid binds to the anion exchange protein band 3 only when the transport site faces outward. When the chloride gradient is reversed, with Clo greater than Cli , the inhibitory potency of niflumic acid decreases greatly, which indicates that the affinity of niflumic acid for band 3 with the transport site facing inward is almost 50 times less than when the transport site faces outward. Experiments in which Cli = Clo show no significant change in the inhibition by niflumic acid when Cl- is lowered from 150 to 10 mM. These data suggest that the intrinsic dissociation constants for Cl- at the two sides of the membrane are nearly equal. Thus, the chloride- loaded transport sites have an asymmetric orientation like that of the unloaded transport sites, with approximately 15 times more sites facing the inside than the outside. The asymmetry reflects an approximately 1.5 kcal/mol free energy difference between the inward-facing and outward-facing chloride-loaded forms of band 3. High concentrations of chloride (with Cli = Clo), which partially saturate the modifier site, have no effect on niflumic acid inhibition, which indicates that chloride binds equally well to the modifier site regardless of the orientation of the transport site.     

Evidence that anion transport by band 3 proceeds via a ping-pong mechanism involving a single transport site. A 35 Cl NMR study

Band 3 catalyzes the one-for-one exchange of monovalent anions across the red cell membrane. At least two anion binding sites have been postulated to exist on the transport unit: 1) a transport site that has been observed by saturation kinetics and by 35 Cl NMR studies of chloride binding, and 2) a 35Cl NMR-invisible inhibitory site that has been proposed to explain the inhibition of anion exchange at large anion concentrations. A number of independent studies have indicated that the transport site is alternately exposed to different sides of the membrane during the transport cycle. Yet the role, if any, of the postulated inhibitory site in the transport cycle is not known. Here it is shown that: 1) when the [Cl-], [Br-], or pH is varied, the band 3 transport sites on both sides of the membrane behave like a homogeneous population of simple anion binding sites in 35Cl NMR experiments, and 2) when the [Cl-] is varied, the outward-facing transport site behaves like a simple anion binding site. These results indicate that the postulated inhibitory site has no effect on chloride binding to the transport site. Instead, the results are quantitatively consistent with the ping-pong model (Gunn, R. B., and Fr?lich, O. (1979) J. Gen. Physiol. 74, 351-374), which states that the transport site is the only site involved in the transport cycle. Expressions are derived for the macroscopically observed characteristics of a ping-pong transporter: these characteristics are shown to be weighted averages of the microscopic properties of the inward- and outward-facing conformations of the transport site. In addition to supporting the simplicity of the transport mechanism, the high pH titration curve for chloride binding to the transport site provides insight into the structure of the site. The macroscopically observed pKA = 11.1 +/- 0.1 in the leaky ghost system indicates that an arginine must provide the essential positive charge in the inward- or outward-facing conformation of the transport site, or in both conformations.     

Identification of the Cl− transport site of human red blood cells by a kinetic analysis of the inhibitory effects of a chemical probe

H₂DIDS, the dihydro analog of DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid) can interact covalently with membrane sites, resulting in an irreversible inhibition of anion exchange. At low temperatures (0°C) and for relatively short times, however, its interaction is largely reversible, so that a kinetic analysis of the nature of its inhibitory effect on Cl^? self exchange can be performed. The effects of variations in the chloride concentration on the inhibitory potency of H₂DIDS are consistent with the concept that Cl^? and H₂DIDS compete for the transport site of the anion exchange system. The value of K_i for H₂DIDS is 0.046 μM, indicating that H₂DIDS has a higher affinity for the transport system than any other inhibitor so far examined. If, as seems probable, the covalent labelling of H₂DIDS occurs at the same site as the reversible binding, H₂DIDS can be used as a covalent label for the transport site. The specific localization of H₂DIDS in the band-3 protein thus indicates that this protein participates directly in anion exchange.     

Effects of membrane potential on electrically silent transport. Potential-independent translocation and asymmetric potential-dependent substrate binding to the red blood cell anion exchange protein

Tracer anion exchange flux measurements have been carried out in human red blood cells with the membrane potential clamped at various values with gramicidin. The goal of the study was to determine the effect of membrane potential on the anion translocation and binding events in the catalytic cycle for exchange. The conditions were arranged such that most of the transporters were recruited into the same configuration (inward-facing or outward-facing, depending on the direction of the Cl- gradient). We found that the membrane potential has no detectable effect on the anion translocation event, measured as 36Cl(-)-Cl- or 36Cl(-)-HCO3- exchange. The lack of effect of potential is in agreement with previous studies on red cells and is different from the behavior of the mouse erythroid band 3 gene expressed in frog oocytes (Grygorczyk, R., W. Schwarz, and H. Passow. 1987. J. Membr. Biol. 99:127-136). A negative potential decreases the potency of extracellular SO4= as an inhibitor of either Cl- or HCO3- influx. Because of the potential-dependent inhibition by SO4=, conditions could be found in which a negative intracellular potential actually accelerates 36Cl- influx. This effect is observed only in media containing multivalent anions. The simplest interpretation of the effect is that the negative potential lowers the inhibitory potency of the multivalent anion by lowering its local concentration near the transport site. The magnitude of the effect is consistent with the idea that the anions move through 10-15% of the transmembrane potential between the extracellular medium and the outward-facing transport site. In contrast to its effect on extracellular substrate binding, there is no detectable effect of membrane potential on the competition between intracellular Cl- and SO4= for transport sites. The lack of effect of potential on intracellular substrate binding suggests that the access pathway leading to the inward-facing transport site is of lower electrical resistance than that leading to the extracellular substrate site.     

Titration of transport and modifier sites in the red cell anion transport system

This work demonstrates the existence of titratable transport and modifier sites in the anion transport system of human red cells. Effects of alkaline extracellular pH on chloride exchange were studied up to pH 13 at 0 degrees C. The studies revealed two sets of reversible titratable groups. One set, having a pK of or approximately 11, appeared to be identical with the inhibitory halide-binding modifier site. Deprotonation of this site stimulated anion transport. The apparent dissociation constants of chloride and iodide at this modifier site were 0.3 and 0.06 M, respectively, and it was confirmed that the organic sulfonate NAP-taurine inhibits anion transport reversibly by a high-affinity interaction with halide-binding modifier sites at the extracellular side of the membrane. Other groups, with apparent pK of or approximately 12 at chloride concentrations above 0.1 M, were named as "transport sites" because transport function depended totally on their protonation. The apparent pK decreased when extracellular halide concentrations was lowered below 0.1 M. It was dependent of the intracellular chloride concentration, and was equally sensitive to extracellular pH of 13, was fully reversible. Hydroxyl ions were not transported to an appreciable extent by the anion exchange system. The pK values of both sets of groups make it likely that they are both arginyl residues, functioning as anion recognition sites similar to the role of functionally essential arginyl residues observed with numerous enzymes.     

Relationship of net chloride flow across the human erythrocyte membrane to the anion exchange mechanism

The parallel effects of the anion transport inhibitor DIDS (4,4'- diisothiocyanostilbene-2,2'-disulfonate) on net chloride flow and on chloride exchange suggest that a major portion of net chloride flow takes place through the anion exchange system. The "slippage" model postulates that the rate of net anion flow is determined by the movement of the unloaded anion transport site across the membrane. Both the halide selectivity of net anion flow and the dependence of net chloride flux on chloride concentration over the range of 75 to 300 mM are inconsistent with the slippage model. Models in which the divalent form of the anion exchange carrier or water pores mediate net anion flow are also inconsistent with the data. The observations that net chloride flux increases with chloride concentration and that the DIDS- sensitive component tends to saturate suggest a model in which net anion flow involves "transit" of anions through the diffusion barriers in series with the transport site, without any change in transport site conformation such as normally occurs during the anion exchange process. This model is successful in predicting that the anion exchange inhibitor NAP-taurine, which binds to the modifier site and inhibits the conformational change, has less effect on net chloride flow than on chloride exchange.     

Anion transport in red blood cells. III. Sites and sidedness of inhibition by high-affinity reversible binding probes

Studies of binding of the reversible inhibitor DNDS (for abbreviations, see Nomenclature) and red blood cell membranes revealed 8.6 +/- 0.7 x 10(5) high-affinity binding sites per cell (KD = 0.8 +/- 0.4 muM). Under conditions of "mutual depletion," inhibition studies of anion exchange revealed 8.0 +/- 0.7 x 10(5) DNDS inhibitory sites per cell (KD = 0.87 +/- 0.04 muM). Binding and kinetics studies with DNDS indicate that there are 0.8 -- 0.9 x 10(6) functional anion transport sites per blood cell. The transport of DNDS displayed high temperature and concentration dependencies, chemical specificity, susceptibility to inhibition by DIDS, and differences between egress and ingress properties. Under conditions of no DNDS penetration (e.g., 0 degrees C), inhibition of anion exchange by DNDS showed marked sidedness from the outside inhibitions and were demonstrable at micromolar concentrations, whereas from the inside no inhibition occurred even at millimolar concentrations. The asymmetry of DNDS transport properties and the sidedness of binding and inhibition suggest that anion transport sites have a very low affinity for or are inaccessible to DNDS at the inner membrane face. The site of DNDS permeation, although susceptible to DIDS, is apparently not the site of anion exchange.     

Pre-steady state transport by erythrocyte band 3 protein: uphill countertransport induced by the impermeant inhibitor H2DIDS.

Pre-steady state Cl- efflux experiments have been performed to test directly the idea that the transport inhibitor H2DIDS (4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate) binds preferentially to the outward-facing state of the transporter. Cells were equilibrated with a medium consisting of 150 mM sodium phosphate, pH 6.2, N2 atmosphere, and 80-250 microM 36Cl-. Addition of H2DIDS (10-fold molar excess compared with band 3) induces a transient efflux of Cl-, as expected if H2DIDS binds more tightly to outward-facing than to inward-facing states. The size of the H2DIDS-induced efflux depends on the Cl- concentration and is about 700,000 ions per cell at the highest concentrations tested. The size of the transient efflux is larger than would be expected if the catalytic cycle for anion exchange involved one pair of exchanging anions per band 3 dimer. These results are completely consistent with a ping-pong mechanism of anion exchange in which the catalytic cycle consists of one pair of exchanging anions per subunit of the band 3 dimer.     

Cytosolic pH regulation in osteoblasts. Interaction of Na+ and H+ with the extracellular and intracellular faces of the Na+/H+ exchanger

The interaction of Na and H ions with the extracellular and intracellular sites of the Na+/H+ exchanger of the osteosarcoma cell line UMR-106 was investigated. Na ions interact with a single, saturable extracellular transport site. H+ and amiloride appear to compete with Na+ for binding to this site. The apparent affinity for extracellular Na+ (Nao+) and amiloride was independent of intracellular H+ (Hi+), Nai+, or an outwardly directed H+ gradient. The interaction of H+ with the intracellular face of the exchanger had a sigmoidal characteristic with a Hill coefficient of approximately 2. The apparent affinity for Hi+ was independent of Nao+ between 25 and 140 mM. The apparent affinity for Hi+, but not the number of intracellular sites, increased with the increase in the outwardly directed H+ gradient across the membrane. Nai+/Ho+ exchange (reverse mode) is an electroneutral process with a Na+/H+ stoichiometry of 1. The dependence of Nai+/Ho+ exchange on Nai+ was sigmoidal, with a Hill coefficient of 2.16. Nai+ competes with Hi+ for binding to at least the transport site. The apparent affinity for Nai+ decreased with the increase in the outwardly directed H+ gradient. High Ho+ inhibited exchange activity in the reverse mode. We conclude that intracellular Na+ and H+ can activate the exchanger. The exchanger has two separate and asymmetric extracellular and intracellular transport sites. The relative apparent affinities of the internal transport site for Na+ and H+ are determined by the direction and magnitude of the H+ gradient across the membrane. Kinetic characterization of the exchanger suggests that Na+/H+ exchange is compatible with a simultaneous transport model, although a ping-pong transport model could not be excluded.     

The kinetic equation for the chloride transport cycle of band 3. A 35Cl and 37Cl NMR study

The nature of a transmembrane transport process depends largely on the identity of the reaction that is rate-limiting in the transport cycle. The one-for-one exchange of two chloride ions across the red cell membrane by band 3 can be decomposed into two component reactions: 1) the binding and dissociation of chloride at the transport site, and 2) the translocation of bound chloride across the membrane. The present work utilizes 35 Cl NMR and 37 Cl NMR to set lower limits on the rates of chloride binding and dissociation at the saturated inward- and outward-facing band 3 transport sites (greater than or equal to 10(5) events site-1 s-1 in all cases). At both 0-3 and 37 degrees C, the NMR data specify that chloride binding and dissociation at the saturated transport sites are not rate-limiting, indicating that translocation of bound chloride across the membrane is the slowest step in the overall transport cycle. Using these results, it is now possible to describe many features of the kinetic equation for the ping-pong transport cycle of band 3. This transport cycle can be decomposed into two half-reactions associated with the transport of two chloride ions in opposite directions across the membrane, where each half-reaction is composed of sequential binding, translocation, and dissociation events. One half-reaction contains the rate-limiting translocation event that controls the turnover of the transport cycle; in this half-reaction, translocation must be slower than binding and dissociation. The other half-reaction contains the non-rate-limiting translocation event that in principle could be faster than binding or dissociation. However, when the following sufficient (but not necessary) condition is satisfied, both translocation events are slower than binding and dissociation: if the non-rate-limiting translocation rate is within a factor of 10(2) (0-3 degrees C) or 2 (37 degrees C) of the overall turnover rate, then translocation is rate-limiting in each saturated half-reaction. Thus, even though chloride appears to migrate through a channel that leads from the transport site to solution, the results support a picture in which the binding, dissociation, and channel migration events are rapid compared to the translocation of bound chloride across the membrane. In this case, chloride binding to the transport site can be described by a simple dissociation constant (KD = kappa OFF/kappa ON) rather than by a Michaelis-Menten constant (KM = (kappa OFF + kappa TRANSLOCATION)/KAPPA ON).     

Cytosolic pH regulation in osteoblasts. Regulation of anion exchange by intracellular pH and Ca2+ ions

Measurements of cytosolic pH (pHi) 36Cl fluxes and free cytosolic Ca2+ concentration ([Ca2+]i) were performed in the clonal osteosarcoma cell line UMR-106 to characterize the kinetic properties of Cl-/HCO3- (OH-) exchange and its regulation by pHi and [Ca2+]i. Suspending cells in Cl(-)-free medium resulted in rapid cytosolic alkalinization from pHi 7.05 to approximately 7.42. Subsequently, the cytosol acidified to pHi 7.31. Extracellular HCO3- increased the rate and extent of cytosolic alkalinization and prevented the secondary acidification. Suspending alkalinized and Cl(-)-depleted cells in Cl(-)-containing solutions resulted in cytosolic acidification. All these pHi changes were inhibited by 4',4',-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) and H2DIDS, and were not affected by manipulation of the membrane potential. The pattern of extracellular Cl- dependency of the exchange process suggests that Cl- ions interact with a single saturable external site and HCO3- (OH-) complete with Cl- for binding to this site. The dependencies of both net anion exchange and Cl- self-exchange fluxes on pHi did not follow simple saturation kinetics. These findings suggest that the anion exchanger is regulated by intracellular HCO3- (OH-). A rise in [Ca2+]i, whether induced by stimulation of protein kinase C-activated Ca2+ channels, Ca2+ ionophore, or depolarization of the plasma membrane, resulted in cytosolic acidification with subsequent recovery from acidification. The Ca2+-activated acidification required the presence of Cl- in the medium, could be blocked by DIDS, and H2DIDS and was independent of the membrane potential. The subsequent recovery from acidification was absolutely dependent on the initial acidification, required the presence of Na+ in the medium, and was blocked by amiloride. Activation of protein kinase C without a change in [Ca2+]i did not alter pHi. Likewise, in H2DIDS-treated cells and in the absence of Cl-, an increase in [Ca2+]i did not activate the Na+/H+ exchanger in UMR-106 cells. These findings indicate that an increase in [Ca2+]i was sufficient to activate the Cl-/HCO3- exchanger, which results in the acidification of the cytosol. The accumulated H+ in the cytosol activated the Na+/H+ exchanger. Kinetic analysis of the anion exchange showed that at saturating intracellular OH-, a [Ca2+]i increase did not modify the properties of the extracellular site. A rise in [Ca2+]i increased the apparent affinity for intracellular OH- (or HCO3-) of both net anion and Cl- self exchange. These results indicate that [Ca2+]i modifies the interaction of intracellular OH- (or HCO3-) with the proposed regulatory site of the anion exchanger in UMR-106 cells.     

pH homeostasis in promyelocytic leukemic HL60 cells

By measuring the membrane potential using the influx of the lipophilic cation tetraphenylphosphonium and intracellular pH using 2,7-biscarboxy-ethyl-5(6)-carboxyfluorescein and the distribution of the weak acid 5,5-dimethyl-2,4-oxazolidinedione, we have determined that intracellular pH is 0.9-1.1 pH units above electrochemical equilibrium in undifferentiated HL60 cells, indicating that these cells actively extrude proton equivalents. The Na/H exchanger is not the system responsible for keeping the pH above the electrochemical equilibrium, since adding inhibitors of this transport system (dimethylamiloride and ethylisopropylamiloride) or removing the extracellular sodium has no effect on intracellular pH. In contrast, the addition of the Cl/HCO3 exchange inhibitors H2 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) or pentachlorophenol (PCP) causes a drop in intracellular pH, and the removal of extracellular chloride in the presence of bicarbonate leads to a large intracellular alkalinization, which indicates a role for the anion exchanger in pH homeostasis in these cells. In addition, we find that the intracellular chloride concentration is about one order of magnitude above electrochemical equilibrium. We conclude that an H2DIDS and PCP inhibitable system, probably the Cl/HCO3 exchanger, is at least partially responsible for keeping intracellular pH above electrochemical equilibrium in HL60 cells under resting conditions. We also find no change in intracellular pH when cells differentiate along the granulocytic pathway (having been induced by the addition of dimethylsulfoxide or of retinoic acid), which indicates that changes in intracellular pH are not causally related to cell differentiation.     

The asymmetry of chloride transport at 38 degrees C in human red blood cell membranes

Band 3-mediated Cl- exchange in human red blood cells and resealed ghosts was measured at 38 degrees C by the continuous flow tube method. When external Cl- concentration, C(o), is varied with constant internal Cl- concentration, C(i), the flux fits a simple Michaelis-Menten saturation curve (MM fit), with K1/2o = 3.8 +/- 0.4 mM. When the Cl- concentration is varied simultaneously at both sides of the membrane in resealed ghosts (C(i) = C(o) = C(i = o)), the flux rises toward a flat maximum between 200 and 450 mM Cl-, and then decreases at very high C(i = o). An MM fit to the data with C(i = o) < 500 mM gives K1/2s of 106 +/- 13 mM; fits including modifier site inhibition (MS fit) give an over threefold higher K1/2s. Despite this uncertainty, the intrinsic asymmetry of unloaded transport sites, A (defined as E(o)/E(i) with C(i) = C(o), where E(i) is the fraction of unloaded inward-facing sites and E(o) is the fraction of unloaded outward-facing sites), calculated from K1/2s and K1/2o, ranges only from 0.046 to 0.107. A new method, which uses the initial slope of a plot of Cl- flux versus C(i = o), gives A values of 0.023 to 0.038. Flufenamic acid (FA) inhibits Cl- exchange by binding to an external site different from the transport site. At 38 degrees C, FA binds 24-36 times more tightly to E(o) than to E(i). Estimates of A from FA inhibitory potency range from 0.01 to 0.05. All methods, including bicarbonate data from the preceding paper, indicate that at 38 degrees C, like 0 degree C, far more band 3 molecules are in the E(i) than in the E(o) form. The agreement of various methods supports the ping-pong model for anion exchange, and demonstrates that the intrinsic asymmetry is very slightly, if at all, affected by temperature.     

Reaction mechanism and asymmetric orientation of the reconstituted chloroplast phosphate translocator.

Proteoliposomes loaded with varying levels of internal substrates were used in bisubstrate initial velocity studies to gain insight into the transport mechanism of the reconstituted chloroplast phosphate translocator. The kinetic response to trans substrates clearly indicated that the one-to-one exchange mediated by this translocator proceeds via a ping-pong type, and excluded a sequential type of reaction mechanism. It is also shown that reconstitution of the protein leads to an unidirectional orientation of the protein within the liposomes being orientated right-side-out with respect to chloroplasts. Different transport affinities were observed on either side of the membrane and only the outward-facing transport site of the translocator is able to bind inhibitors i.e. pyridoxal 5'-phosphate (PLP) and 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS).     

Sulphate and phosphate transport in the renal proximal tubule

Experiments performed on microperfused proximal tubules and brush-border membrane vesicles revealed that inorganic phosphate is actively reabsorbed in the proximal tubule involving a 2 Na+-HPO2-4 or H2PO-4 co-transport step in the brush-border membrane and a sodium-independent exit step in the basolateral cell membrane. Na+-phosphate co-transport is competitively inhibited by arsenate. The transtubular transport regulation is mirrored by the brush-border transport step: it is inhibited by parathyroid hormone intracellularly mediated by cyclic AMP. Transepithelial inorganic phosphate (Pi) transport and Na+-dependent Pi transport across the brush-border membrane correlates inversely with the Pi content of the diet. Intraluminal acidification as well as intracellular alkalinization led to a reduction of transepithelial Pi transport. Data from brush-border membrane vesicles indicate that high luminal H+ concentrations reduce the affinity for Na+ of the Na+-phosphate co-transport system, and that this mechanism might be responsible for the pH dependence of phosphate reabsorption. Contraluminal influx of Pi from the interstitium into the cell could be partly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). It is not, however, changed when dicarboxylic acids are present or when the pH of the perfusate is reduced to pH 6. Sulphate is actively reabsorbed, involving electroneutral 2 Na+-SO2-4 co-transport through the brush-border membrane. This transport step is inhibited by thiosulphate and molybdate, but not by phosphate or tungstate. The transtubular active sulphate reabsorption is not pH dependent, but is diminished by the absence of bicarbonate. The transport of sulphate through the contraluminal cell side is inhibited by DIDS and diminished when the capillary perfusate contains no bicarbonate or chloride. The latter data indicate the presence of an anion exchange system in the contraluminal cell membrane like that in the erythrocyte membrane.     

High affinity binding of amiloride analogs at an internal site in renal microvillus membrane vesicles.

Amiloride analogs with hydrophobic substitutions on the 5-amino nitrogen atom are relatively high affinity inhibitors of the plasma membrane Na(+)-H+ exchanger. We demonstrated that a high affinity-binding site for [3H]5-(N-methyl-N-isobutyl)amiloride ([3H]MIA) (Kd = 6.3 nM, Bmax = 1.2 pmol/mg of protein) is present in microvillus membrane vesicles but not in basolateral membrane vesicles isolated from rabbit renal cortex, in accord with the known membrane localization of the Na(+)-H+ exchanger in this tissue. The rank order potency for inhibition of microvillus membrane [3H]MIA binding by amiloride analogs was: MIA (I50 approximately 10 nM) greater than amiloride (I50 approximately 200 nM) greater than benzamil (I50 approximately 1200 nM). This correlated with a qualitatively similar rank order potency for inhibition of Na(+)-H+ exchange: MIA (I50 approximately 4 microM) greater than amiloride (I50 approximately 15 microM) greater than benzamil (I50 approximately 100 microM), but did not correlate with the rank order potency for inhibition of the organic cation-H+ exchanger in microvillus membrane vesicles: MIA approximately benzamil (I50 approximately 0.5 microM) greater than amiloride (I50 approximately 10 microM). However, tetraphenylammonium, an inhibitor of organic cation-H+ exchange, inhibited the rate of [3H]MIA binding without an effect on equilibrium [3H]MIA binding; the dissociation of bound [3H]MIA was inhibited by preloading the membrane vesicles with tetraphenylammonium. These findings indicated that high affinity [3H]MIA binding to renal microvillus membrane vesicles takes place at an internal site to which access is rate-limited by the tetraphenylammonium-sensitive organic cation transporter. Equilibrium [3H]MIA binding was inhibited by H+ but was unaffected by concentrations of Na+ or Li+ that saturate the external transport site of the Na(+)-H+ exchanger. Binding of MIA to its high affinity binding site had no effect on the rate of Na(+)-H+ exchange. This study suggests that the renal Na(+)-H+ exchanger has a high affinity internal binding site for amiloride analogs that is distinct from the external amiloride inhibitory site.