Mechanism of the increase in cation permeability of human erythrocytes in low-chloride media. Involvement of the anion transport protein capnophorin

When human erythrocytes are suspended in low-Cl- media (with sucrose replacing Cl-), there is a large increase in both the net efflux and permeability of K+. A substantial portion (greater than 70% with Cl- less than 12.5 mM) of this K+ efflux is inhibited by the anion exchange inhibitor DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). This inhibition cannot be explained as an effect of DIDS on net Cl- permeability (Pcl) and membrane potential, but rather represents a direct effect on the K+ permeability. When cells are reacted with DIDS for different times, the inhibition of K+ efflux parallels that of Cl- exchange, which strongly indicates that the band 3 anion exchange protein (capnophorin) mediates the net K+ flux. Since a noncompetitive inhibitor of anion exchange, niflumic acid, has no effect on net K+ efflux, the net K+ flow does not seem to involve the band 3 conformational change that mediates anion exchange. The data suggest that in low-Cl- media, the anion selectivity of capnophorin decreases so that it can act as a very low-conductivity channel for cations. Na+ and Rb+, as well as K+, can utilize this pathway.     

Volume-induced increase of anion permeability in human lymphocytes

Peripheral blood mononuclear cells (PBM) readjust their volumes after swelling in hypotonic media. This regulatory volume decrease (RVD) is associated with a loss of cellular K+ and is thought to be promoted by an increased permeability to this ion. In contrast, no change in volume was observed when K+ permeability of PBM in isotonic media was increased to comparable or higher levels using valinomycin. Moreover, valinomycin-induced 86Rb+ loss in K+-free medium was considerably slower than in K+-rich medium. These results suggest that anion conductance limits net salt loss in isotonic media. Direct measurements of relative conductance confirmed that in volume-static cells, anion conductance is lower than that of K+. In volume-regulating cells depolarization occurred presumably as a result of increased anion conductance. Accordingly, the efflux of 36Cl from PBM was markedly increased by hypotonic stress. Since both membrane potential and intracellular 36Cl concentration are reduced in hypotonically swollen cells, the increased efflux is probably due to a change in Cl- permeability. Anions and cations seem to move independently through the volume-induced pathways: the initial rate of 86Rb uptake in swollen cells was not affected by replacement of external Cl- by SO=4; conversely, 36Cl fluxes were unaffected by substitution of K+ by Na+. The data indicate that anion conductance is rate-determining in salt and water loss from PBM. An increase in anion conductance is suggested to be the critical step of RVD of human PBM.     

Rapid electrogenic sulfate-chloride exchange mediated by chemically modified band 3 in human erythrocytes

One of the modes of action of the red blood cell anion transport protein is the electrically silent net exchange of 1 Cl- for 1 SO4= and 1 H+. Net SO4(=)-Cl- exchange is accelerated by low pH or by conversion of the side chain of glutamate 681 into an alcohol by treatment of intact cells with Woodward's reagent K (WRK) and BH4-. The studies described here were performed to characterize the electrical properties of net SO4(=)-Cl- exchange in cells modified with WRK/BH4-. The SO4= conductance measured in 100 mM SO4= medium is smaller in modified cells than in control cells. However, the efflux of [35S] SO4= into a 150-mM KCl medium is 80-fold larger in modified cells than in control cells and is inhibited 99% by 10 microM H2DIDS. No detectable H+ flux is associated with SO4(=)-Cl- exchange in modified cells. In the presence of gramicidin to increase the cation permeability, the stoichiometry of SO4(=)-Cl- exchange is not distinguishable from 1:1. In modified cells loaded with SO4=, the valinomycin-mediated efflux of 86Rb+ into an Na- gluconate medium is immediately stimulated by the addition of 5 mM extracellular Cl-. Therefore, SO4(=)-Cl- exchange in modified cells causes an outward movement of negative charge, as expected for an obligatory 1:1 SO4(=)-Cl- exchange. This is the first example of an obligatory, electrogenic exchange process in band 3 and demonstrates that the coupling between influx and efflux does not require that the overall exchange be electrically neutral. The effects of membrane potential on SO4(=)-SO4= exchange and SO4(=)-Cl- exchange in modified cells are consistent with a model in which nearly a full net positive charge moves inward through the transmembrane field during the inward Cl- translocation event, and a small net negative charge moves with SO4= during the SO4= translocation event. This result suggests that, in normal cells, the negative charge on Glu 681 traverses most of the transmembrane electric field, accompanied by Cl- and the equivalent of two protein-bound positive charges.     

Interactions between diphtheria toxin entry and anion transport in Vero cells. I. Anion antiport in Vero cells

In sodium-free buffer of low ionic strength, the uptake of chloride and sulfate in Vero cells was found to occur mainly by antiport which was very sensitive to inhibition by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. Efflux of anions from the cells appeared to energize the uptake. While the uptake of Cl- occurred over a wide pH range, that of SO4(2-) showed a clear maximum at pH 6-7. The rate of efflux of 36Cl- and 35SO4(2-) was strongly increased by the presence of permeant anions in the efflux buffer. Preincubation of the cells at slightly alkaline pH strongly increased the rate of C1- efflux into buffers nominally free of permeant anions, as well as the efflux by exchange. This increase did not occur if the cells were depleted for ATP during the preincubation. Depolarization of the cells reduced the rate of efflux into buffers without permeant anions, indicating that the efflux is at least partly due to net, electrogenic, anion transport. The efflux by antiport was not affected by manipulations of the membrane potential, indicating electroneutral exchange. The uptake and efflux were increased to the same extent with increasing temperature, the activation energies were Ea = 25 kcal/mol of Cl- and Ea = 12 kcal/mol of SO4(2-). Similar anion antiport appears to occur in L, baby hamster kidney, and HeLa S3 cells.     

Net efflux of chloride from cell suspensions measured with a K+ electrode

Under appropriate conditions (presence of cation ionophores) net KCl efflux measured with a K+ electrode can be used to estimate conductive Cl- fluxes, a sensitive procedure that allows continuous recording. The procedure was tested in human red cells by demonstrating effects of ionophores and of an anion transport inhibitor, and in dissociated MDCK cells by demonstration of cAMP and volume-activated Cl- fluxes.     

Chloride-stimulated sulfate efflux in Ehrlich ascites tumor cells: evidence for 1:1 coupling

The kinetics of Cl-SO4-(2) exchange in Ehrlich ascites tumor cells was investigated in an attempt to determine the stoichiometry of this process. When tumor cells, equilibrated in Cl--free, 25 mM SO4-(2) medium are placed in SO4-(2)-free, 25 mm Cl-medium, both the net amount and rate of Cl-uptake far exceeds SO4-(2) loss.. Addition of the anion transport inhibitor SITS (4-acetamido-4,-isothiocyano-stilbene-2,2'-disulfonic acid) greatly reduces sulfate efflux (97%), but has no measurable effect on chloride uptake. Addition of furosemide, a Cl-transport inhibitor, reduces chloride uptake 94% but is without effect on sulfate efflux. These findings suggest that a chloride permeability pathway exists distinct from that utilized by SO4-(2). SITS, when added to furosemide treated cells, further reduces chloride uptake as well as inhibiting sulfate efflux, and under these experimental conditions, a linear relationship exists between SITS-sensitive, net chloride uptake and sulfate loss. The slope of this line is 1.05 (correlation coefficient = 0.996) which suggests the stoichiometry of Cl-SO4-(2) exchange is 1:1. Assuming a 1:1 stoichiometry, measurement of the initial chloride influx and initial sulfate efflux indicate that 92% of net chloride uptake is independent of sulfate efflux. Taken altogether, these results support the contention that the tumor cell possesses a permeability pathway which facilitates the exchange of one sulfate for one chloride. Under conditions where anion transport is not inhibited, this coupling is obscured by a second and quantitatively more important pathway for chloride uptake. This pathway is SITS-insensitive, although partially inhibited by furosemide.     

Characterization of chloride transport pathways in cultured human keratinocytes.

In human keratinocytes, mediated transport of Cl- was found to occur mainly by two mechanisms: an anion exchange and an electrically conductive pathway. The contribution of the anion exchange, which accounted for about 50% of overall Cl- efflux, was assessed either by its sensitivity to inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and by means of Cl- substitution experiments. The anion exchange exhibited a saturation behaviour over the range 10-135 mM Cl-; Cl- was more efficient than HCO3-, Br- and NO3- in increasing Cl- efflux rate, whereas SO4(2-) and I- inhibited Cl- efflux. The electrically conductive Cl- pathway, which accounted for about 40% of total Cl- efflux, was inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and was at least partially sensitive to variation of the plasma membrane potential. The Cl- channel was insensitive to elevation in the intracellular concentration of either cyclic AMP and calcium ions. Indomethacin, an inhibitor of the cyclooxygenase, failed to reduce Cl- efflux, whereas nordihydroguaiaretic acid (NDGA), an inhibitor of the lipoxygenase, induced 50% inhibition of Cl- efflux. These results support the conclusion that endogenous production of lipoxygenase-derived arachidonic acid metabolite(s) might be responsible for high basal Cl- permeability in human keratinocytes.     

Catecholamine-stimulated ion transport in duck red cells. Gradient effects in electrically neutral [Na + K + 2Cl] Co-transport

The transient increase in cation permeability observed in duck red cells incubated with norepinephrine has been shown to be a linked, bidirectional, co-transport of sodium plus potassium. This pathway, sensitive to loop diuretics such as furosemide, was found to have a [Na + K] stoichiometry of 1:1 under all conditions tested. Net sodium efflux was inhibited by increasing external potassium, and net potassium efflux was inhibited by increasing external sodium. Thus, the movement of either cation is coupled to, and can be driven by, the gradient of its co-ion. There is no evidence of trans stimulation of co- transport by either cation. The system also has a specific anion requirement satisfied only by chloride or bromide. Shifting the membrane potential by varying either external chloride (at constant internal chloride) or external potassium (at constant internal potassium in the presence of valinomycin and DIDs [4,4'-diisothiocyano- 2,2'-disulfonic acid stilbene]), has no effect on nor-epinephrine- stimulated net sodium transport. Thus, this co-transport system is unaffected by membrane potential and is therefore electrically neutral. Finally, under the latter conditions-when Em was held constant near EK and chloride was not at equilibrium-net sodium extrusion against a substantial electrochemical gradient could be produced by lowering external chloride at high internal concentrations, thereby demonstrating that the anion gradient can also drive co-transport. We conclude, therefore, that chloride participates directly in the co- transport of [Na + K + 2Cl].     

External anions regulate stilbene-sensitive proton transport in placental brush border vesicles

The mechanism for HCO3-(-)independent proton permeability in microvillus membrane vesicles (MVV) isolated from human placenta was examined by using the entrapped pH indicator 6-carboxyfluorescein (6CF). Proton fluxes (JH) across MVV were determined in response to induced pH and anion gradients from the time course of 6CF fluorescence, the MVV buffer capacity, and the 6CF vs. pH calibration. In the absence of anions, JH was 12 +/- 2 nequiv s-1 (mg of protein)-1 (pHin 7.4, pHout 6.0, MVV voltage-clamped with K+/valinomycin, 23 degrees C), corresponding to a proton permeability coefficient of 0.02 cm/s, with an activation energy of 9.1 +/- 0.3 kcal/mol. JH was inhibited 20% by dihydro-4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (H2DIDS) with KI = 8 microM [( Cl-]out = 0 mM). For a 0.5-unit pH gradient JH increased from 1.5 to 4.6 nequiv s-1 (mg of protein)-1 as the internal MVV pH was increased (5.5-7.5). External Cl-, Br-, and I- (but not SO4(2-) and PO4-) increased JH 1.3-2.5-fold for both inwardly and outwardly directed pH gradients with KD = 1.0 +/- 0.4 mM (Br-) and greater than 100 mM (Cl-). This increase was blocked by 100 microM H2DIDS but not by amiloride or furosemide. Internal Cl- did not alter JH induced by pH gradients nor were proton fluxes induced by anion gradients in the absence of a pH gradient. Experiments in which JH was driven by membrane potentials (induced by valinomycin and K+ gradients) indicated that proton transport was voltage-sensitive. These experiments demonstrate a stilbene-sensitive electrogenic proton transport mechanism in MVV that is regulated allosterically by anions at an external binding site.     

A sensitive technique for the determination of anion exchange activities in brush-border membrane vesicles. Evidence for two exchangers with different affinities for HCO3- and SITS in rat intestinal epithelium

A large percentage (up to 70%) of 36Cl- influx in brush-border membrane vesicles from rat small intestine under equilibrium exchange conditions was found to be mediated by SITS-inhibitable anion exchange. This Cl-/anion exchange could be measured 10-15-times more sensitive by determining the uptake of trace amounts of 125I- driven by a large Cl- gradient (in greater than out) generated by passing the vesicles through an anion-exchange column. Voltage clamping of the vesicle membrane with K+ and valinomycin did not effect the chloride driven 125I- uptake, showing that the 'overshooting' I- uptake was not mediated by an electrical diffusion potential, as might be generated by the Cl- gradient in the presence of a chloride channel. The Cl-/anion exchange was further characterized in brush-border membrane vesicles from both rat ileum and jejunum by studying the inhibitory action of various anions on the Cl- driven I- uptake. NO3-, Cl-, SCN- and formate at 2 mM could inhibit Cl-/I- exchange for more than 80%. The ileal brush-border membrane vesicles displayed a clear heterogeneity with respect to the inhibitory action of SO2-(4), SITS and HCO-3 on Cl-/I- exchange. Approximately 30% of the Cl-/I- exchange was insensitive to SO2-(4) and showed a relatively low sensitivity to SITS (IC50 = 1 mM) but could be inhibited for 80% by 2 mM HCO-3. Presumably this component represents Cl-/OH- or Cl-/HCO-3 exchange. The residual 70% showed a high sensitivity to SO2-(4) (IC50 = 0.5 mM) and SITS (IC50 = 2.5 microM) but was less sensitive to HCO-3. This part of the exchange activity showed inhibition characteristics very similar to the Cl-/I- exchange in the jejunal vesicles. The latter process was also inhibited for 80% by 2 mM oxalate. As discussed in this paper both exchangers may be involved in the electroneutral transport of NaCl across the apical membrane of the small intestinal villus cell.     

Voltage dependence of DIDS-insensitive chloride conductance in human red blood cells treated with valinomycin or gramicidin

Net K and Cl effluxes induced by valinomycin or by gramicidin have been determined directly at varied external K, denoted by [K]o, in the presence and absence of the anion transport inhibitors DIDS (4,4'-diiso- thiocyano-2,2'-disulfonic acid stilbene), and its less potent analogue SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid). The results confirm that pretreatment with 10 microM DIDS, or 100 microM SITS, for 30 min at 23 degrees C inhibits conductive Cl efflux, measured in the continued presence of the inhibitors at 1 mM [K]o, by only 59-67%. This partial inhibition by 10 microM DIDS at 1 mM [K]o remains constant when the concentration of DIDS, or when the temperature or pH during pretreatment with DIDS, are increased. Observations of such partial inhibition previously prompted the postulation of two Cl conductance pathways in human red blood cells: a DIDS-sensitive pathway mediated by capnophorin (band 3 protein), and a DIDS-insensitive pathway. The present experiments demonstrate that at [K]o corresponding to values of EK between -35 and 0 mV the DIDS- insensitive component of net Cl efflux is negligible, being < or = 0.1 muMol/g Hb/min, both with valinomycin (1 microM) and with gramicidin (0.06 microgram/ml). At lower [K]o, where EK is below approximately -35 mV, the DIDS-insensitive fraction of net Cl efflux increases to 2.6 muMol/g Hb/min with valinomycin (1 microM), and to 4.8 muMol/g Hb/min with gramicidin (0.06 microgram/ml). With net fluxes determined from changes in mean cell volume, and with membrane potentials measured from changes in the external pH of unbuffered red cell suspensions, a current-voltage curve for DIDS-insensitive Cl conductance has been deduced. While specific effects of varied [K]o on net Cl efflux are unlikely but cannot strictly be ruled out, the results are consistent with the hypothesis that DIDS-insensitive Cl conductance turns on at an Em of approximately -40 mV.     

Chinese hamster ovary cell mutants deficient in an anion exchanger functionally similar to the erythroid band 3

Studies in Chinese hamster ovary cells demonstrate the presence of an anion exchanger, which has some of the properties of the band 3 transporter in erythrocytes. 1) Extracellular chloride is a competitive inhibitor of sulfate influx and stimulates sulfate efflux, suggesting that the mechanism of uptake is SO2-(4)/Cl- exchange. 2) The anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibits sulfate uptake in a dose-dependent manner. Half-maximal inhibition is achieved at 0.06 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. 3) Low extracellular pH markedly stimulates sulfate uptake. A 6-fold decrease in the apparent Km is observed at pHout 5.5 as compared to pHout 7.5. However, studies carried out over a broad range of extracellular SO2-(4) concentrations indicate the presence of three components of this transport activity in Chinese hamster ovary cells: two high affinity low capacity systems, one in the range 0.5 microM less than [SO2-(4)]out less than 50 microM and one in the range 50 microM less than [SO2-(4)]out less than 150 microM, and a low affinity, high capacity system (at [SO2-(4)]out greater than 150 microM). These properties have not been previously reported for the erythroid band 3 transporter. The availability of mutants deficient in these activities has enabled us to carry out studies which suggest that the high affinity systems are functionally independent of the low affinity system, but that all systems are dependent on the same anion exchange protein. Studies in a mutant which lacks all components of the transport activity indicates that the anion exchanger may be instrumental in the regulation of the intracellular pH in Chinese hamster ovary cells.     

Ionic events during the volume response of human peripheral blood lymphocytes to hypotonic media. I. Distinctions between volume-activated Cl- and K+ conductance pathways

Human peripheral blood lymphocytes (PBL), when placed into hypotonic media, first swell and then shrink back to their original volumes because of a rapid KCl leakage via volume-activated K+ and anion permeation pathways. By using gramicidin, a cation channel-forming ionophore, cation transport through the cell membrane can be shunted so that the salt fluxes and thus the volume changes are limited by the rate of the net anion movements. The "gramicidin method," supplemented with direct measurements of volume-induced ion fluxes, can be used to assess the effects of drugs and of various treatments on cation and anion permeabilities. It is demonstrated that quinine and cetiedil are much more effective blockers of volume-induced K+ transport than of Cl- transport, while dipyridamole, DIDS, and NIP-taurine inhibit only volume-induced Cl- movement. Oligomycins block both cation and anion transport pathways, oligomycin A being more effective in inhibiting K+ transport and oligomycin C preferentially blocking Cl- movement. Ca depletion of PBL abolishes volume-induced K+ transport but has no effect on Cl- transport. Repletion of cell calcium by ionophore A23187 immediately restores rapid K+ transport without significantly affecting volume-induced Cl- transport. These observations, taken together with other reported information, can be best explained by a model in which cell swelling activates independent Cl- and K+ conductance pathways, the latter being similar in properties to the Ca2+-activated K+ transport observed in various cell membranes.     

Chloride net efflux from intact erythrocytes under slippage conditions. Evidence for a positive charge on the anion binding/transport site

Tracer chloride and potassium net efflux from valinomycin-treated human erythrocytes were measured into media of different chloride concentrations, Clo, at 25 degrees C and pH 7.8. Net efflux was maximal [45-50 mmol (kg cell solids)-1 min-1] at Clo = 0. It decreased hyperbolically with increasing Clo to 14-16 mmol (kg cell solids)-1 min- 1. Half-maximal inhibition occurred at Clo = 3 mM. In the presence of the anion exchange inhibitor DNDS, net efflux was reduced to 5 mmol (kg cell solids)-1 min-1, independent of Clo. Of the three phenomenological components of net efflux, the Clo-inhibitable (DNDS-inhibitable) component was tentatively attributed to "slippage," that is, net transport mediated by the occasional return of the empty transporter. The Clo-independent (DNDS-inhibitable) component was tentatively attributed to movement of chloride through the anion transporter without the usual conformational change of the transport site on the protein ("tunneling"). These concepts of slippage and tunneling are shown to be compatible with a model that describes the anion transporter as a specialized single-site, two-barrier channel that can undergo conformational changes between two states. Net chloride efflux when the slippage component dominated (Clo = 0.7 mM) was accelerated by a more negative (inside) membrane potential. It appears that the single anion binding-and-transport site on each transporter has one net positive charge and that is neutralized when a chloride ion is bound.     

Enhancement of conductive anion permeability in cultured cells by cetiedil

Cetiedil, a drug that is reported to block K+-channels, substantially increases the conductive C1- permeability of Chinese hamster ovary (CHO) cells. The permeability was monitored by volume changes in cells treated with gramicidin to increase the cation permeability. Under this circumstance, increases in Cl- conductances result in volume changes detectable by electronic sizing, with the direction determined by the gradients of the permeating ions. In NaCl or KCl media, swelling occurs, but in N-methylglucamine chloride, shrinking. The increases in Cl- conductance could also be measured as an increased 36Cl- flux or by changes in membrane potential (measured by fluorescence of a potential-sensitive dye) toward the Cl- equilibrium potential. The effect of cetiedil was concentration dependent, with maximal effect at 50 microM. The anion specificity for the conductance was NO3- greater than Cl- = Br- much greater than SO4-2 or isethionate. A number of other drugs that influence transport activities had no effect on Cl- conductance. The cetiedil effect on Cl- conductance was observed in one other cell line, but was absent in several other cell types. The cetiedil-induced Cl- conductance in CHO cells appears to involve a different pathway than that induced by exposure to hypotonic medium.     

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.     

The interaction of intracellular Mg2+ and pH on Cl- fluxes associated with intracellular pH regulation in barnacle muscle fibers

The intracellular dialysis technique was used to measure unidirectional Cl- fluxes and net acid extrusion by single muscle fibers from the giant barnacle. Decreasing pHi below normal levels of 7.35 stimulated both Cl- efflux and influx. These increases of Cl- fluxes were blocked by disulfonic acid stilbene derivatives such as SITS and DIDS. The SITS-sensitive Cl- efflux was sharply dependent upon pHi, increasing approximately 20-fold as pHi was decreased from 7.35 to 6.7. Under conditions of normal intracellular Mg2+ concentration, the apparent pKa for the activation of Cl- efflux was 7.0. We found that raising [Mg2+]i, but not [Mg2+]o, had a pronounced inhibitory effect on both SITS-sensitive unidirectional Cl- fluxes as well as on SITS-sensitive net acid extrusion. Increasing [Mg2+]i shifted the apparent pKa of Cl- efflux to a more acid value without affecting the maximal flux that could be attained. This relation between pHi and [Mg2+]i on SITS-sensitive Cl- efflux is consistent with a competition between H ions and Mg ions. We conclude that the SITS-inhibitable Cl- fluxes are mediated by the pHi-regulatory transport mechanism and that changes of intracellular Mg2+ levels can modify the activity of the pHi regulator/anion transporter.     

Sulfate transport in toad skin: evidence for mitochondria-rich cell pathways in common with halide ions

1. In short-circuited toad skin preparations exposed bilaterally to NaCl-Ringer's containing 1 mM SO2(-4), influx of sulfate was larger than efflux showing that the skin is capable of transporting sulfate actively in an inward direction. 2. This active transport was not abolished by substituting apical Na+ for K+. 3. Following voltage activation of the passive Cl- permeability of the mitochondria-rich (m.r.) cells sulfate flux-ratio increased to a value predicted from the Ussing flux-ratio equation for a monovalent anion. 4. In such skins, which were shown to exhibit vanishingly small leakage conductances, the variation of the rate coefficient for sulfate influx (y) was positively correlated with the rate coefficient for Cl- influx (x), y = 0.035 x - 0.0077 cm/sec (r = 0.9935, n = 15). 5. Addition of the phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine to the serosal bath of short-circuited preparations resulted in a significant stimulation of the passive Cl- and SO2(-4) permeabilities. 6. It is suggested that SO2(-4) and Cl- ions are transported along the same pathway of the m.r. cells. Depending on the transport mode of the apical Cl- transport system, electro-diffusion, active transport (sulfate:bicarbonate exchange) and self-exchange diffusion take place. Irrespective of the mechanism of transport, sulfate is probably transported as a monovalent anion species.     

Sulfate-chloride exchange transport in a glioma cell line

Transport of SO4(2-) was studied in the glioma cell line LRM55 to determine whether it is mediated by the Cl-/HCO3- exchanger or the K+/Cl- cotransporter previously described in these cells (Wolpaw, E.W. and Martin, D.L. (1984) Brain Res. 297, 317-327). 35SO4(2-) influx was saturable with SO4(2-). External SO4(2-) stimulated 35SO4(2-) efflux, indicating an exchange mechanism. External Cl- was a competitive inhibitor of 35SO4(2-) influx. Internal Cl- stimulated 35SO4(2-) influx and external Cl- stimulated 35SO4(2-) efflux, indicating that Cl- is an exchange substrate for the SO4(2-) carrier. Also, SO4(2-) flux was sensitive to SITS, DIDS and furosemide. However, saturating external SO4(2-) did not inhibit 36Cl- influx and did not inhibit 36Cl- efflux via the Cl-/HCO3- exchanger. Moreover, K+ did not stimulate 36Cl- efflux via the Cl-/HCO3- exchanger. Moreover, K+ did not stimulate 35SO4(2-) influx as it does Cl- influx. These findings indicate that SO4(2-) transport into these cells is mediated by an exchange carrier distinct from both the Cl-/HCO3- exchanger and the K+/Cl- cotransporter. While Cl- is an alternative substrate for the SO4(2-) porter, this carrier is responsible for only a minor fraction of total Cl- flux in these cells.     

Anion exchanger is present in both luminal and basolateral renal membranes

Binding of the anion-exchange inhibitor 3H2-labeled 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) to highly purified luminal and basolateral beef kidney tubular membranes was characterized. Specific binding of [3H2]DIDS is present in both luminal and basolateral membranes. Scatchard analysis revealed a Kd for [3H2]DIDS of 5.5 microM and 19.3 microM and a maximal number of binding sites of 10.9 nmol and 31.7 nmol DIDS/mg protein in basolateral and luminal membranes, respectively. To assess the role of this putative anion exchanger on transport we measured 35SO4 uptake by luminal and basolateral membranes. In both luminal and basolateral membranes sulfate uptake was significantly greater in the presence of an outward-directed Cl gradient, OH gradient or HCO3 gradient than in the absence of these gradients. There was an early anion-dependent sulfate uptake of five to ten times the equilibrium uptake at 60 min. The sulfate taken in could be released by lysis of the vesicles indicating true uptake and not binding of sulfate. No significant difference in SO4 uptake was found in the presence and in the absence of valinomycin, indicating that the anion exchanger is electroneutral. The anion-dependent sulfate uptake was completely inhibited by either DIDS or furosemide in both luminal and basolateral membranes. Dixon analysis of HCO3-dependent SO4 uptake by luminal membranes in the presence of different concentrations of DIDS revealed a Ki for DIDS of 20 microM. The similar values of the Kd for [3H2]DIDS binding and the Ki for DIDS inhibition of SO4 uptake might suggest an association between DIDS binding and the inhibition of SO4 transport. In addition, an inward-directed Na gradient stimulated sulfate uptake in luminal but not in basolateral membranes. The Na-dependent sulfate uptake in luminal membranes was also inhibited by DIDS. We conclude that, in addition to the well-known Na-dependent sulfate uptake in luminal membranes, there exists an anion exchanger in both basolateral and luminal membranes capable of sulfate transport.