Multiple transport functions of a red blood cell anion exchanger, tAE1: its role in cell volume regulation.

1. 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. 2. This inhibition does not result from an effect on the driving force for electrodiffusion but represents a direct effect on the cation pathway. 3. 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. 4. 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. 5. 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. 6. tAE1 illustrates the ability of a specific transport system to be a multifunctional protein exhibiting other transport functions when submitted to regulation.

Transport of uncharged organic solutes in Xenopus oocytes expressing red cell anion exchangers (AE1s).

When expressed in Xenopus oocytes, the trout red cell anion exchanger tAE1, but not the mouse exchanger mAE1, elicited a transport of electroneutral solutes (sorbitol, urea) in addition to the expected anion exchange activity. Chimeras constructed from mAE1 and tAE1 allowed us to identify the tAE1 domains involved in the induction of these transports. Expression of tAE1 (but not mAE1) is known to generate an anion conductance associated with a taurine transport. The present data provide evidence that (i) the capacity of tAE1 and tAE1 chimeras to generate urea and sorbitol permeability also was associated with an anion conductance; (ii) the same inhibitors affected both the permeability of solutes and anion conductance; and (iii) no measurable water transport was associated with the tAE1-dependent conductance. These results support the view that fish red blood cells, to achieve cell volume regulation in response to hypotonic swelling, activate a tAE1-associated anion channel that can mediate the passive transport of taurine and electroneutral solutes.

Expression of band 3 anion exchanger induces chloride current and taurine transport: structure-function analysis.

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.

Volume-activated Cl(-)-independent and Cl(-)-dependent K+ pathways in trout red blood cells.

1. Swelling of trout erythrocytes can be induced either by addition of catecholamine to the cell suspension, thus promoting NaCl uptake via beta-adrenergic-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 KCl co-transport. These were based upon the inhibition of Cl(-)-OH- exchange to evaluate the degree of coupling between K+ and Cl- (KCl 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 co-transport exists in the cell membrane, for which the selectivity for anions varies according to the change in cellular ionic strength induced by swelling.