No abstract

Most, but not all, cell types release intracellular organic solutes (e.g. taurine) in response to cell swelling to achieve cell volume regulation. Although this efflux is blocked by classical inhibitors of the electroneutral anion exchanger band 3 (AE1), it is thought to involve an anion channel. The role of band 3 in volume‐dependent taurine transport was determined by expressing, in Xenopus oocytes, band 3 from erythrocytes which do (trout) or do not (mouse) release taurine when swollen. AE1 of both species elicited anion exchange activity, but only trout band 3 showed chloride channel activity and taurine transport. Chimeras constructed from trout and mouse band 3 allowed the identification of some protein domains critically associated with channel activity and taurine transport. The data provide evidence that swelling‐induced taurine movements occur via an anion channel which is dependent on, or controlled by, band 3. They suggest the involvement of proteins of the band 3 (AE) family in cell volume regulation.

SLC26 family members are anionic transporters involved in Cl^- and HCO₃^- absorption or secretion in epithelia. SLC26A9, preferentially expressed in the lung, is a poorly characterized member of this family. In this study, we investigated the transport properties of human SLC26A9 to determine its functional and pharmacological characteristics. SLC26A9 protein expression results in the appearance of an anionic current exhibiting an apparently linear current/voltage relationship and increases in ³⁶Cl influxes and effluxes. The sequences of conductivity, Cl^- >I^- > NO₃^- ≧ gluconate > SO₄ ^2- and selectivity (P_x/P_CI), I^- > NO₃^- > Cl^- > gluconate > SO₄^2- are found. Cl^- channel inhibitors DIDS and NS 3623 inhibit SLC26A9 associated currents while the specific CFTR inhibitor (CFTR(inh)-172) or glybenclamide has little effect. Elevation of intracellular cAMP (a CFTR activator) is also ineffective whereas increasing intracellular calcium blocks the SLC26A9 associated currents. The HCO₃^- conductance mediated by the SLC26A9 protein expression is low and no intracellular pHi changes are detectable under conditions favoring a Cl^-/HCO₃^- exchange. However, the presence of HCO₃^-/CO₂ stimulates the Cl^--transporting activity of SLC26A9 in Xenopus laevis oocytes or SLC26A9-transduced COS-7 cells. As an important initial step in characterizing SLC26A9 function, we conclude that SLC26A9 is a Cl^- channel and we suggest that HCO₃^- acts as a modulator of the channel. SLC26A9 physiological role in airway epithelia and its potential interaction with CFTR remain to be elucidated.

The anion exchanger 1 (AE1) is encoded by the SLC4A1 gene and catalyzes the electroneutral anion exchange across cell plasma membrane. It is the most abundant transmembrane protein expressed in red cell where it is involved in CO 2 transport. Recently, 4 new point mutations of SLC4A1 gene have been described leading to missense mutations in the protein sequence (L687P, D705Y, S731P, or H734R). These point mutations were associated with hemolytic anemia, and it was shown that they confer a cation transport feature to the human AE1. Facing this unexpected property for an electroneutral anion exchanger, we have studied the transport features of mutated hAE1 by expression in xenopus oocytes. Our results show that the point mutations of hAE1 convert the electroneutral anion exchanger to a cation conductance: the exchangers are no longer able to exchange Cl ؊ and HCO 3 ؊ , whereas they transport Na ؉ and K ؉ through a conductive mechanism. These data shed new light on transport mechanisms showing the tiny difference, in terms of primary sequence, between an electroneutral exchange and a conductive pathway. (Blood.

It was previously shown that expressed in Xenopus oocyte the mouse (mAE1) and the trout (tAE1) anion exchanger behave differently: both elicit anion exchange activity but only tAE1 induces a transport of organic solutes correlated with a chloride channel activity. The present data, obtained by measurement of Xenopus oocyte membrane permeability and conductance, provide evidence that tAE1 also induces a large increase in Na+ and K+ permeability inhibited by several AE1 inhibitors. This inhibition does not result from an effect on the driving force for electrodiffusion but represents a direct effect on the cation pathway. As a control, expression of cystic fibrosis transmembrane conductance regulator (CFTR) having, once stimulated by 3‐isobutyl‐1‐methylxanthine (IBMX), the same anion conductance magnitude as tAE1 did not induce any cation movement. Chloride exchange, channel activity and cation transport induced by anion exchanger expression are inhibited by free or covalently bound H2DIDS as well. This covalent inhibition is reversed by the point mutation of Lys‐522, the covalent binding site of H2DIDS to the protein. These data reveal that tAE1 itself acts both as an anion exchanger and as a channel of broad selectivity. All results obtained by expression of AE1 isoforms in Xenopus oocytes and those obtained in erythrocytes are consistent with the proposal that, in nucleated erythrocytes, tAE1 functions as the swelling‐activated osmolyte anion channel involved in cell volume regulation. In contrast AE1 from mammalian red cells, which do not regulate their volume, lacks swelling‐activated osmolyte channel properties. tAE1 illustrates the ability of a specific transport system to be a multifunctional protein exhibiting other transport functions when submitted to regulation.

In this study, we have shown that, when expressed in Xenopus oocytes, trout anion exchanger 1 (tAE1) was able to act as a bifunctional protein, either an anion exchanger or a chloride conductance. Point mutations of tAE1 were carried out and their effect on Cl- conductance and Cl- unidirectional flux were studied. We have shown that mutations made in transmembrane domain 7 had dramatic effects on tAE1 function. Indeed, when these residues were mutated, either individually or together (mutants E632K, D633G, and ED/KG), Cl- conductance was reduced to 28-44% that of wild-type tAE1. Moreover, ion substitution experiments showed that anion selectivity was altered. However, the exchanger function was unchanged, as evidenced by the fact that Cl- influx and K(m) were identical for each of these mutants and similar to the wild-type protein parameters. By contrast, mutations made in the C-terminal domains of the protein (R819M, Q829K) affected both transport functions. Cl- conductance was increased by approximately 200% with respect to tAE1 and anion selectivity was impaired. Likewise, Cl- influx was increased by approximately 260% and was no longer saturable. These and other mutations carried out in transmembrane domains 7, 8, 12-14 of tAE1 allow us to demonstrate without doubt that, in addition to its anion exchanger activity, tAE1 can also function as a chloride channel. Above all, this work led us to identify amino acids involved in this double function organization.

SummaryThe hereditary stomatocytoses are a group of dominantly inherited conditions in which the osmotic stability of the red cell is compromised by abnormally high cation permeability. This report demonstrates the very marked similarities between the cryohydrocytosis form of hereditary stomatocytosis and the common tropical condition south‐east Asian ovalocytosis (SAO). We report two patients, one showing a novel cryohydrocytosis variant (Ser762Arg in SLC4A1) and a case of SAO. Both cases showed a mild haemolytic state with some stomatocytes on the blood film, abnormal intracellular sodium and potassium levels which were made markedly abnormal by storage of blood at 0°C, increased cation ‘leak’ fluxes at 37°C and increased Na+K+ pump activity. In both cases, the anion exchange function of the mutant band 3 was destroyed. Extensive electrophysiological studies comparing the cation leak and conductance in Xenopus laevis oocytes expressing the two mutant genes showed identical patterns of abnormality. These data are consistent with the cryohydrocytosis form of hereditary stomatocytosis and we conclude that the cation leak in SAO is indistinguishable from that in cryohydrocytosis, and that SAO should be considered to be an example of hereditary stomatocytosis.

SUMMARY1. Swelling of trout erythrocytes can be induced either by addition of catecholamine to the cell suspension, thus promoting NaCl uptake via fiadrenergic-stimulated Na+-H+ exchange (isotonic swelling) or by suspending red blood cells in a hypotonic medium (hypotonic swelling). In both cases cells tend to regulate their volume by losing K+, but the characteristics of the volume-activated K+ pathways are different: after hormonally induced swelling the K+ loss is strictly Cl-dependent; after hypotonic swelling the K+ loss is essentially Cl-independent.2. In order to determine the nature of these volume regulatory pathways (i.e. whether the net K+ loss was conductive or was by electroneutral K+-H+ exchange or KCl co-transport), studies were performed to analyse ion fluxes and associated electrical phenomena. The cell membrane potential and intracellular ionic activities of volume-regulating and volume-static cells were measured by impalement with conventional microelectrodes and double-barrelled ion-sensitive microelectrodes.3. The information gained from the electrical and ion flux studies leads to the conclusion that both Cl--independent and Cl--dependent K+ loss proceed via electrically silent pathways.4. Experiments were designed to distinguish between electroneutral K+-H+ exchange or KCI co-transport. These were based upon the inhibition of Cl--OHexchange to evaluate the degree of coupling between K+ and Cl-(KCI stoichiometry, pH change). The experimental observations are consistent with the fact that both Cl--independent and Cl--dependent K+ loss are mediated by coupled K+-anion co-transport and not by K+-H+ exchange.5. On the basis of previous data, we suggest that only one type of K+-anion cotransport exists in the cell membrane, for which the selectivity for anions varies according to the change in cellular ionic strength induced by swelling.