Nicole Gabillat

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.

Characterization of SLC26A9, facilitation of Cl(-) transport by bicarbonate.

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.

South-east Asian ovalocytosis and the cryohydrocytosis form of hereditary stomatocytosis show virtually indistinguishable cation permeability defects

The 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.

Cation transport activity of anion exchanger 1 mutations found in inherited distal renal tubular acidosis

Anion exchanger 1 (AE1) is encoded by SLC4A1 and mediates electroneutral anion exchange across cell membranes. It is the most abundant protein in the red cell membrane, but it is also found in the basolateral membrane of renal alpha-intercalated cells, where it is required for normal urinary acidification. Recently, four point mutations in red cell AE1 have been described that convert the anion exchanger to a cation conductance. SLC4A1 mutations can also cause type 1 hypokalemic distal renal tubular acidosis (dRTA). We investigated the properties of four dRTA-associated AE1 mutations (R589H, G609R, S613F, and G701D) by heterologous expression in Xenopus laevis oocytes. Although these AE1 mutants are functional anion exchangers, unlike the red cell disease mutants, we found that they also demonstrated a cation leak. We found a large cation leak in the G701D mutant. This mutant normally requires coexpression with glycophorin A for surface membrane expression in red blood cells and oocytes. However, we found that coexpressing wild-type kidney AE1 with G701D in oocytes still caused a cation leak, consistent with heterodimerized G701D reaching the cell membrane and retaining its cation conductance property. These findings have potential structural and functional implications for AE1, and they indicate that while anion exchange and cation conductance properties are distinct, they can coexist.

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.

Consequences of point mutations in trout anion exchanger 1 (tAE1) transmembrane domains: evidence that tAE1 can behave as a chloride channel.

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 Km 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 ∼200% with respect to tAE1 and anion selectivity was impaired. Likewise, Cl− influx was increased by ∼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. J. Cell. Physiol.

Importance of several cysteine residues for the chloride conductance of trout anion exchanger 1 (tAE1).

In this study, we devised a cysteine‐focused point mutation analysis of the chloride channel function of trout anion exchanger 1 (tAE1) expressed in X. lævis oocytes. Seven cysteines, belonging to the transmembrane domain of tAE1, were mutated into serines (either individually or in groups) and the effects of these mutations on the chloride conductance of injected oocytes were measured. We showed that three cysteines were essential for the functional expression of tAE1. Namely, mutations C462S, C583S and C588S reduced Cl− conductance by 68%, 52% and 83%, respectively, when compared to wild type tAE1. These residual conductances were still inhibited by 0.5 mM niflumic acid. Western blot experiments demonstrated that C462 was involved in protein expression onto the plasma membrane. A mutant devoid of this residue was unable to express onto the plasma membrane, especially if several other cysteines were missing: consequently, the cysteine‐less mutant of tAE1 was not functional. C583 and C588 were involved in the channel function of tAE1 as shown by anion substitution experiments proving that selectivity of the mutated pore differs from the wild type one. On the contrary, they were not involved in the Cl−/HCO  3− exchange function of tAE1, as demonstrated by intracellular pH measurements. These and several complementary mutations allow us to conclude that a mutant of tAE1 containing the sole C462 can drive a marginal Cl− current; however, the minimal configuration necessary to get optimal functional expression of the tAE1 chloride channel is that of a mutant containing unaffected residues C462, C583 and C588. J. Cell. Physiol. 213: 70–78, 2007.

Southeast Asian AE1 associated renal tubular acidosis: Cation leak is a class effect

Anion Exchanger 1 (AE1) is present in the erythrocyte and also in the alpha-intercalated cell; different mutations can cause either red cell disease or distal renal tubular acidosis (dRTA). Recently, we described a cation leak property in four dRTA-causing AE1 mutants, three autosomal dominant (AD) European mutants, one autosomal recessive (AR) from Southeast Asia, G701D. G701D had a very large leak property and is unusually common in SE Asia. We hypothesized that this property might confer a survival advantage. We characterized three other AR dRTA-associated AE1 mutants found in SE Asia, S773P, Delta850 and A858D via transport experiments in AE1-expressing Xenopus oocytes. These three SE Asian mutants also had cation leaks of similar magnitude to that seen in G701D, a property that distinguishes them as a discrete group. The clustering of these cation-leaky AE1 mutations to malarious areas of SE Asia suggests that they may confer malaria resistance.