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

Abstract

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.Hypotonically swollen cells recover their volume (regulatory volume decrease) by releasing intracellular solutes (ions and organic compounds) and obligated water. Taurine, an amino acid that is present in various cells at concentrations of up to 40 mM, is often the primary organic osmolyte involved in regulatory volume decrease. But, in some cells, polyols, such as sorbitol and myoinositol, play an important role (1). Volumeactivated amino acid and polyol efflux mechanisms share two characteristics (1): (i) they are mediated by passive, Naindependent transport pathways having the characteristics of anion channels and (ii) they are similarly affected by various anion transport blockers, suggesting that transport of these structurally unrelated compounds may be mediated by a single pathway.Fish erythrocytes respond to hypotonic swelling by inducing a loss of K ϩ and Cl Ϫ and also a transport of taurine and polyols (2-8) similarly affected by various anion transport blockers (5, 7-9). Recent observations (10) show that: (i) expression of the trout anion exchanger AE1 (tAE1) in Xenopus oocyte elicits, in addition to anion exchange activity, both anion conductance and appearance of a passive taurine transport; (ii) a very tight correlation exists between expression of anion conductance and taurine transport; (iii) compounds that inhibit anion conductance also inhibit taurine transport; (iv) expression of the anion exchanger (mAE1) of mouse erythrocyte, a cell that does not release organic osmolytes in response to swelling, elicits anion exchange activity but neither conductance nor taurine transport. Taken together, these results indicate that the tAE1-induced channel mediates a taurine transport and suggest that, in fish red blood cells, the volume-activated taurine transport is associated with the anion exchanger (band 3 protein).Hyposmotic swelling of trout red blood cells induces not only taurine transport (4) but also a transport of sorbitol that is inhibited...

Evidence for Up-regulation of the Endogenous Na-K-2Cl Co-transporter by Molecular Interactions with the Anion Exchanger tAE1 Expressed in Xenopus Oocyte

“…Effect of 10 Ϫ6 M bumetanide and Cl Ϫ or Na ϩ removal was also measured on Rb influx in tAE1-expressing oocyte. The Rb influx in Cl Ϫ -free medium was done in isosmotic NO 3 Ϫ -containing MBS, as published previously (14). The Na ϩ -free medium was MBS in which N-methyl-D-glucamine was substituted for Na ϩ (see "Experimental Procedures" for media composition).…”

“…All of the AE polypeptides can be divided into two main domains of about the same size: an N-terminal cytoplasmic domain interacting with cytoskeleton and a membrane spanning domain, which is responsible for ion translocation, with a short C-terminal end in the cytoplasm. By expression studies of tAE1 in Xenopus oocytes, we have demonstrated previously that this normally electroneutral anion exchanger is able to form an anion conductive path-way permeable to different charged or neutral osmolytes such as urea, choline, sorbitol, Na ϩ , and K ϩ (13)(14)(15). In physiological conditions, these transport properties of tAE1 are activated by a decrease in intracellular ionic strength in swollen erythrocytes, and they are involved in the cell regulatory volume decrease response (16,17).…”

Volume Expansion Stimulates p72 and p56 in Skate Erythrocytes

“…In erythrocytes of a number of species, the volume-stimulated taurine efflux appears to occur by a transport pathway that involves band 3 (2,3). Oocyte expression of band 3 cloned from trout, but not mouse, results not only in a Cl Ϫ exchange activity, but also in a swelling-activated pathway for a number of solutes including taurine (4). This latter finding strongly supports a role for band 3 as either the volumeactivated transporter or a component of a volume-activated taurine transport pathway.…”

“…Inclusion of the cytoplasmic domain of band 3 in this assay showed that only Syk and Lyn can directly phosphorylate the cytoplasmic domain of band 3. Upon cell volume expansion, Syk activity was increased as assessed by three different assays (immune complex assay measuring autophosphorylation, assay of the level of phosphotyrosine of the immunoprecipitated kinase, and assay of level of 32 P in the kinase immunoprecipitated from cells prelabeled with 32 PO 4 and then volume-expanded). The tyrosine kinase Lyn was also stimulated by volume expansion, most notably when analyzed by the latter two methods.…”

Leaky Cl − –HCO 3 − exchangers: cation fluxes via modified AE1

“…In fact, tAE1 will also allow significant transport of organic osmolytes (e.g. taurine) and even significant Na and K fluxes (Fievet et al 1995(Fievet et al , 1998. Physiologically this multifunctional property of tAE1 plays a role in cell volume regulation in response to swelling (Guizouarn et al 2001).…”