Anion transport systems in the plasma membrane of vertebrate cells

In the case of the red blood cell, anion transport is a highly specific one-for-one exchange catalyzed by a major membrane protein known as band 3 or as capnophorin. This red cell anion-exchange system mediates the Cl-(-)HCO3- exchange responsible for most of the bicarbonate transport capacity of the blood. The rapidly expanding knowledge of the molecular biology and the transport kinetics of this specialized transport system is very briefly reviewed in Section III. Exchange diffusion mechanisms for anions are found in many cells other than erythrocytes. The exchange diffusion system in Ehrlich cells has several similarities to that in red cells. In several cell types (subsection IV-B), there is evidence that intracellular pH regulation depends on Cl-(-)HCO3- exchange processes. Anion exchange in other single cells is described in Section IV, and its role in pH regulation is described in Section VII. Anion exchange mechanism operating in parallel with, and only functionally linked to Na+-H+ or K+-H+ exchange mechanisms can also play a role in cell volume regulation as described in Section VII. In the Ehrlich ascites cell and other vertebrate cells, electroneutral anion transfer has been found to occur also by a cotransport system for cations and chloride operating in parallel with the exchange diffusion system. The cotransport system is capable of mediating secondary active chloride influx. In avian red cells, the cotransport system has been shown to be activated by adrenergic agonists and by cyclic AMP, suggesting that the cotransport is involved in regulatory processes (see subsection V-A.). In several cell types, cotransport systems are activated and play a role during volume regulation, as described in Section V and in Section VII. It is also likely that this secondary active cotransport of chloride plays a significant role for the apparently active extrusion of acid equivalents from certain cells. If a continuous influx of chloride against an electrochemical gradient is maintained by a cotransport system, the chloride disequilibrium can drive an influx of bicarbonate through the anion exchange mechanism, as described in Section VII. Finally, even the electrodiffusion of anions is shown to be regulated, and in Ehrlich cells and human lymphocytes an activation of the anion diffusion pathway plays a major role in cell volume regulation as described in Section VI and subsection VII-B.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Characterization of oxalate transport by the human erythrocyte band 3 protein

This paper describes characteristics of the transport of oxalate across the human erythrocyte membrane. Treatment of cells with low concentrations of H2DIDS (4,4'-diisothiocyanatostilbene-2,2'- disulfonate) inhibits Cl(-)-Cl- and oxalate-oxalate exchange to the same extent, suggesting that band 3 is the major transport pathway for oxalate. The kinetics of oxalate and Cl- self-exchange fluxes indicate that the two ions compete for a common transport site; the apparent Cl- affinity is two to three times higher than that of oxalate. The net exchange of oxalate for Cl-, in either direction, is accompanied by a flux of H+ with oxalate, as is also true of net Cl(-)-SO4(2-) exchange. The transport of oxalate, however, is much faster than that of SO4(2-) or other divalent anions. Oxalate influx into Cl(-)-containing cells has an extracellular pH optimum of approximately 5.5 at 0 degrees C. At extracellular pH below 5.5 (neutral intracellular pH), net Cl(-)- oxalate exchange is nearly as fast as Cl(-)-Cl- exchange. The rapid Cl(- )-oxalate exchange at acid extracellular pH is not likely to be a consequence of Cl- exchange for monovalent oxalate (HOOC-COO-; pKa = 4.2) because monocarboxylates of similar structure exchange for Cl- much more slowly than does oxalate. The activation energy of Cl(-)- oxalate exchange is about 35 kCal/mol at temperatures between 0 and 15 degrees C; the rapid oxalate influx is therefore not a consequence of a low activation energy. The protein phosphatase inhibitor okadaic acid has no detectable effect on oxalate self-exchange, in contrast to a recent finding in another laboratory (Baggio, B., L. Bordin, G. Clari, G. Gambaro, and V. Moret. 1993. Biochim. Biophys. Acta. 1148:157-160.); our data provide no evidence for physiological regulation of anion exchange in red cells.     

Anion-dependent cation transport in erythrocytes

A selective survey of the literature reveals at least three major anion-dependent cation transport systems, defined as Na+ + Cl-, K+ + Cl- and Na+ + K+ + Cl- respectively. In human red cells, kinetic data on the fraction of K+ and Na+ influx inhibitable by bumetanide are presented to indicate an Na+:K+ stoichiometry of 1:2. For LK sheep red cells the large Cl- -dependent K+ leak induced by swelling is shown to share many characteristics with that induced by N-ethylmaleimide (NEM) treatment. NEM has complex effects, both inhibiting and then activating Cl- -dependent K+ fluxes dependent on NEM concentration. The alloantibody anti-L can prevent the action of NEM. In human red cells NEM induces a large Cl- -dependent specific K+ flux, which shows saturation kinetics. Its anion preference is Cl- greater than Br- greater than SCN- greater than I- greater than NO3- greater than MeSO4-. This transport pathway is not inhibited by oligomycin or SITS, although phloretin and high concentrations of furosemide and bumetanide (over 0.3 mM) do inhibit. Quinine (0.5 mM) is also an inhibitor. It is concluded that at least two distinct Cl- -dependent transport pathways for K+ are inducible in mammalian red cells, although the evidence for their separation is not absolute.     

3H]bumetanide binding to duck red cells. Correlation with inhibition of (Na + K + 2Cl) co-transport

Bumetanide is a potent inhibitor of cation-chloride co-transport systems in many cell types, including duck red cells. We studied equilibrium binding of [3H]bumetanide to intact duck red cells under a number of conditions known to affect (Na + K + 2Cl) co-transport in these cells. Saturable [3H]bumetanide binding to duck red cells is markedly stimulated by addition of norepinephrine or cell shrinkage, conditions which similarly stimulate co-transport. In the presence of norepinephrine and saturating concentrations of extracellular sodium, potassium, and chloride for the co-transporter, we found approximately 1000 [3H]bumetanide-binding sites/red cell, and measurement of 24Na+ influx on the same cells yielded a turnover number of approximately 4000/s for the co-transporter. 24Na+ influx was negatively correlated with the amount of bound [3H]bumetanide, and both saturable binding and inhibition of influx were half-maximal at approximately 10(-7) M [3H]bumetanide. Binding of [3H]bumetanide to duck red cells is stimulated in a saturable manner by increasing extracellular sodium and potassium. Chloride has a biphasic effect on [3H]bumetanide binding; increasing [Cl-]o (by replacement of methylsulfate) from 0 to 32 mM markedly enhances binding, whereas further increasing [Cl-]o to 160 mM inhibits binding. This behavior is similar to that reported for bumetanide inhibition of duck red cell (Na + K + 2Cl) co-transport (Haas, M., and McManus, T. J. (1983) Am. J. Physiol. 245, C235-C240; Haas, M., and McManus, T. J. (1982) Biophys. J. 37, 214a) and [3H]bumetanide binding to membranes from dog kidney outer medulla (Forbush, B. III, and Palfrey, H. C. (1983) J. Biol. Chem. 258, 11787-11792).     

Differential inhibition of AE1 and AE2 anion exchangers by oxonol dyes and by novel polyaminosterol analogs of the shark antibiotic squalamine.

Oxonol and polyaminosterol drugs were examined as inhibitors of recombinant mouse AE1 and AE2 anion exchangers expressed in Xenopus laevis oocytes and were compared as inhibitors of AE1-mediated anion flux in red cells and in HL-60 cells that express AE2. The oxonols WW-781, diBA(5)C4, and diBA(3)C4 inhibited HL-60 cell Cl-/Cl- exchange with IC50 values from 1 to 7 microM, 100-1000 times less potent than their IC50 values for red cell Cl-/anion exchange. In Xenopus oocytes, diBA(5)C4 inhibited AE1-mediated Cl- efflux several hundred times more potently than that mediated by AE2. Several novel squalamine-related polyaminosterols were also evaluated as anion exchange inhibitors. In contrast to diBA(5)C4, polyaminosterol 1361 inhibited oocyte-expressed AE2 8-fold more potently than AE1 (IC50 0.6 versus 5.2 microM). The 3-fold less potent desulfo-analog, 1360, showed similar preference for AE2. It was found that 1361 also partially inhibited Cl- efflux from red cells, whereas neither polyaminosterol inhibited Cl efflux from HL60 cells. Thus, the oxonol diBA(5)C4 is >100-fold more potent as an inhibitor of AE1 than of AE2, whereas the polyaminosterols 1360 and 1361 are 8-fold more potent as inhibitors of AE2 than of AE1. Assay conditions and cell type influenced IC50 values for both classes of compounds.     

Catecholamine-induced transport systems in trout erythrocyte. Na+/H+ countertransport or NaCl cotransport?

It has previously been shown (Baroin, A., F. Garcia-Romeu, T. Lamarre, and R. Motais. 1984a, b. Journal of Physiology. 350:137, 356:21; Mahé, Y., F. Garcia-Romeu, and R. Motais. 1985. European Journal of Pharmacology. 116:199) that the addition of catecholamines to an isotonic suspension of nucleated red blood cells of the rainbow trout first stimulates a cAMP-dependent, amiloride-sensitive Na+/H+ exchange. This stimulation seems to be transient. It is followed by a more permanent activation of a coupled entry of Na+ and Cl-, which is inhibited by amiloride but also by inhibitors of band 3 protein (DIDS, furosemide, niflumic acid). The coupled entry of Na+ and Cl- could therefore result from the parallel and simultaneous exchange of Na+out for H+in (via the cAMP-dependent Na+/H+ antiporter) and Cl- out for HCO3- in (via the anion exchange system located in band 3 protein). However, in view of the following arguments, it had been proposed that NaCl uptake does not proceed by the double-exchanger system but via an NaCl cotransport: (a) Na+ entry requires Cl- as anion (in NO3- medium, the Na uptake is strongly inhibited, whereas NO3- is an extremely effective substitute for Cl- in the anion exchange system); (b) Na uptake is not significantly affected by the presence of HCO3- in the suspension medium despite the fact that in red cells, Cl-/HCO3- exchange occurs more readily than the exchanges of Cl- for basic equivalents in a theoretically CO2-free medium (the so-called Cl-/OH- exchanges). The purpose of the present paper was a reassessment of the two models by using monensin, an ionophore allowing Na+/H+ exchange. From this study, it appears that NaCl entry results from the simultaneous functioning of the Na+/H+ antiporter and the anion exchange system. The apparent Cl dependence is explained by the fact that, in these erythrocytes, NO3- clearly inhibits the turnover rate of the Na+/H+ antiporter. As Na+/H+ exchange is the driving component in the salt uptake process, this inhibition explains the Cl requirement for Na entry. The lack of stimulation of cell swelling by bicarbonate is explained by the fact that the rate of anion exchange in a CO2-free medium (Cl-/OH- exchange) is roughly equivalent to that of Na+/H+ exchange and thus in practice is not limiting to the net influx of NaCl through the two exchangers.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Sulfate transport in human neutrophils

The mechanism by which SO4(2-) is transported across the plasma membrane of isolated human neutrophils was investigated. Unlike the situation in erythrocytes, SO4(2-) and other divalent anions are not substrates for the principal Cl-/HCO3- exchange system in these cells. At an extracellular concentration of 2 mM, total one-way 35SO4(2-) influx and efflux in steady-state cells amounted to approximately 17 mumol/liter of cell water per min. The intracellular SO4(2-) content was approximately 1 mM, approximately 25-fold higher than the passive distribution level. Internal Cl- trans stimulated 35SO4(2-) influx. Conversely, 35SO4(2-) efflux was trans stimulated by external Cl- (Km approximately 25 mM) and by external SO4(2-) (Km approximately 14 mM), implying the presence of a SO4(2-)/Cl- countertransport mechanism. The exchange is noncompetitively inhibited by 4-acetamido-4'-isothiocyanostilbene-2,2' -disulfonate (SITS) (Ki approximately 50 microM) and competitively blocked by alpha-cyano-4-hydroxycinnamate (Ki approximately 230 microM) and by ethacrynate (Ki approximately 7 microM); furosemide and probenecid also suppressed activity. The carrier exhibits broad specificity for a variety of monovalent (NO3- approximately Cl- greater than Br- greater than formate- greater than I- approximately p-aminohippurate-) and divalent WO4(2-) greater than oxalate2- greater than SO4(2-) greater than MoO4(2-) greater than SeO4(2-) greater than AsO4(2-) anions. There was little, if any, affinity for HCO3-, phosphate, or glucuronate. The influx of SO4(2-) is accompanied by an equivalent cotransport of H+, the ion pair H+ + SO4(2-) being transported together in exchange for Cl-, thereby preserving electroneutrality. These findings indicate the existence of a separate SO4(2-)/Cl- exchange carrier that is distinct from the neutrophil's Cl-/HCO3- exchanger. The SO4(2-) carrier shares several properties in common with the classical inorganic anion exchange mechanism of erythrocytes and with other SO4(2-) transport systems in renal and intestinal epithelia, Ehrlich ascites tumor cells, and astroglia.     

Self-inhibition of chloride transport in Ehrlich ascites tumor cells

Previous studies have shown that mediated Cl- transport which occurs by at least two processes (Cl- -dependent cation cotransport and Cl- self-exchange) becomes progressively inhibited when extracellular Cl- exceeds about 60 mM (Hoffmann et al., 1979). To account for this type of kinetic behavior, that is, self-inhibition, an anion transport system possessing two sites, a high affinity transport site and a lower affinity modifier site is suggested (Dalmark, 1976). In the present experiments we have attempted to determine which of the mediated transport pathways is susceptible to self-inhibition by studying the dependence of the steady state Cl- flux on the extracellular Cl- concentration and how DIDS, an inhibitor of Cl- self-exchange, and H + affect this relationship. Addition of DIDS to Ehrlich cells results in inhibition of Cl- transport at every Cl- concentration tested (40-150 mM). Moreover, the Cl- flux/Cl- concentration relationship no longer exhibits self-inhibition, suggesting that this phenomenon is a characteristic of the Cl- self-exchanger rather than of the Cl- -dependent cation cotransport system. Lowering the extracellular pH (pHo) from 7.35 to 5.30 stimulates Cl- transport by a process that saturates with respect to [H +]. Half-maximal stimulation occurs at pHo 6.34. A comparison of the kinetic parameters, Ks and Jmax, calculated from the ascending limb of the Cl- flux/Cl- concentration curve at pHo 7.30 to those at pHo 5.50 show that the values for Ks are almost identical (23.6 mM and 21.3 mM, respectively), while the values for Jmax [22.2 mEq/Kg dry wt) X min] differ by only 15%. This finding along with the observation that DIDS completely blocks H + stimulation of Cl- transport is compatible with the suggestion that H + interact at the modifer site of the Cl- self-exchanger and thereby prevents self-inhibition.     

Saturation behavior of ascites tumor cell chloride exchange in the presence of gluconate.

Steady state Cl- flux across the Ehrlich mouse ascites cell membrane was studied when gluconate replaced Cl- in the external medium. Saturation behavior was observed; K 1/2 was 23.9 mM Cl- and V was 758 micromol.g-1 dry weight.h-1. The cells lost K+, Cl- and H2O, consistent with relative impermeability to gluconate, and the Cl- efflux rate coefficient was elevated. The results indicate that a major portion of Cl- exchange occurs as a membrane transport process and suggest that the process is sensitive to intracellular Cl- levels.     

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.     

Functional carboxyl groups in the red cell anion exchange protein. Modification with an impermeant carbodiimide

Anion exchange in human red blood cell membranes was inactivated using the impermeant carbodiimide 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide (EAC). The inactivation time course was biphasic: at 30 mM EAC, approximately 50% of the exchange capacity was inactivated within approximately 15 min; this was followed by a phase in which irreversible exchange inactivation was approximately 100-fold slower. The rate and extent of inactivation was enhanced in the presence of the nucleophile tyrosine ethyl ester (TEE), suggesting that the inactivation is the result of carboxyl group modification. Inactivation (to a maximum of 10% residual exchange activity) was also enhanced by the reversible inhibitor of anion exchange 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) at concentrations that were 10(3)-10(4) times higher than those necessary for inhibition of anion exchange. The extracellular binding site for stilbenedisulfonates is essentially intact after carbodiimide modification: the irreversible inhibitor of anion exchange 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) eliminated (most of) the residual exchange activity: DNDS inhibited the residual (DIDS-sensitive) Cl- at concentrations similar to those that inhibit Cl- exchange of unmodified membranes: and Cl- efflux is activated by extracellular Cl-, with half-maximal activation at approximately 3 mM Cl-, which is similar to the value for unmodified membranes. But the residual anion exchange function after maximum inactivation is insensitive to changes of extra- and intracellular pH between pH 5 and 7. The titratable group with a pKa of approximately 5.4, which must be deprotonated for normal function of the native anion exchanger, thus appears to be lost after EAC modification.     

Volume-sensitive K influx in human red cell ghosts

K influx into resealed human red cell ghosts increases when the ghosts are swollen. The influx demonstrates properties similar to volume-sensitive K fluxes present in other cells. The influx is, for the most part, insensitive to the nature of the major intracellular cation and therefore is not a K-K exchange. The influx is much greater when the major anion is Cl than when the major anion is NO3; Cl stimulates the flux and, at constant Cl, NO3 inhibits it. Increase in the influx rate is rapid when shrunken ghosts are swollen or when NO3 is replaced by Cl. The volume-sensitive K influx requires intracellular MgATP at low concentrations, and ATP cannot be replaced by nonhydrolyzable ATP analogues. The volume-sensitive influx is inhibited by Mg2+ and by high concentrations of vanadate, but is stimulated by low concentrations of vanadate. It is not modified by cAMP, the removal of Ca2+ by EGTA, substances that activate protein kinase C, or by inhibition of phosphatidylinositol kinase. The influx is inhibited by neomycin and by trifluoperazine.     

Na+, Cl− cotransport in Ehrlich ascites tumor cells activated during volume regulation (regulatory volume increase)

Ehrlich ascites cells were preincubated in hypotonic medium with subsequent restoration of tonicity. After the initial osmotic shrinkage the cells recovered their volume within 5 min with an associated KCl uptake. The volume recovery was inhibited when NO-3 was substituted for Cl-, and when Na+ was replaced by K+, or by choline (at 5 mM external K+). The volume recovery was strongly inhibited by furosemide and bumetanide, but essentially unaffected by DIDS. The net uptake of Cl- was much larger than the value predicted from the conductive Cl- permeability. The undirectional 36Cl flux, which was insensitive to bumetanide under steady-state conditions, was substantially increased during regulatory volume increase, and showed a large bumetanide-sensitive component. During volume recovery the Cl- flux ratio (influx/efflux) for the bumetanide-sensitive component was estimated at 1.85, compatible with a coupled uptake of Na+ and Cl-, or with an uptake via a K+,Na+,2Cl- cotransport system. The latter possibility is unlikely, however, because a net uptake of KCl was found even at low external K+, and because no K+ uptake was found in ouabain-poisoned cells. In the presence of ouabain a bumetanide-sensitive uptake during volume recovery of Na+ and Cl- in nearly equimolar amounts was demonstrated. It is proposed that the primary process during the regulatory volume increase is an activation of an otherwise quiescent, bumetanide-sensitive Na+,Cl- cotransport system with subsequent replacement of Na+ by K+ via the Na+/K+ pump, stimulated by the Na+ influx through the Na+,Cl- cotransport system.     

Chloride activity and its control in skeletal and cardiac muscle

Ion-selective microelectrodes have been used to compare the mechanisms controlling intracellular Cl- activity in skeletal and cardiac muscle. In frog Sartorius skeletal muscle fibres, Cl- levels are low (about 3 mM) and are determined mainly passively. The effect of any Cl- transport system will be quickly short-circuited through the high membrane Cl- conductance. In contrast, the sheep-heart Purkinje fibre, like other cardiac tissues, contains higher than passive levels of intracellular Cl- (20-30 mM). Many Cl- movements occur, not through Cl- channels (the permeability for Cl- is low), but by a Cl- -HCO3- countertransport system. High internal Cl- levels are achieved by an exchange of extracellular Cl- for intracellular HCO3-, which acidifies the fibre by 0.3 pH. Anion exchange in heart differs from that proposed for other excitable cells in that it is not specialized to compensate for an intracellular acidosis. Instead, it can prevent the fibres from becoming too alkaline by promoting a bicarbonate efflux and a chloride influx whenever internal bicarbonate levels rise. Possible reasons for this are briefly discussed.     

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.     

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.     

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.     

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.