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Introduction Anion exchanger 1 (AE1) (SLC4A1 gene product) is a membrane protein expressed in both kidney and red blood cells (RBCs): it exchanges extracellular bicarbonate (HCO3–) for intracellular chloride (Cl–) and participates in acid−base homeostasis. AE1 mutations in kidney α-intercalated cells can lead to distal renal tubular acidosis (dRTA). In RBC, AE1 (known as band 3) is also implicated in membrane stability: deletions can cause South Asian ovalocytosis (SAO). Methods We retrospectively collected clinical and biological data from patients harboring dRTA due to a SLC4A1 mutation and analyzed HCO3– and Cl– transports (by stopped-flow spectrophotometry) and expression (by flow cytometry, fluorescence activated cell sorting, and Coomassie blue staining) in RBCs, as well as RBC membrane stability (ektacytometry). Results Fifteen patients were included. All experience nephrolithiasis and/or nephrocalcinosis, 2 had SAO and dRTA (dRTA SAO+), 13 dominant dRTA (dRTA SAO−). The latter did not exert specific RBC membrane anomalies. Both HCO3– and Cl– transports were lower in patients with dRTA SAO+ than in those with dRTA SAO− or controls. Using 3 different extracellular probes, we report a decreased expression (by 52%, P < 0.05) in dRTA SAO+ patients by fluorescence activated cell sorting, whereas total amount of protein was not affected. Conclusion Band 3 transport function and expression in RBCs from dRTA SAO− patients is normal. However, in SAO RBCs, impaired conformation of AE1/band 3 corresponds to an impaired function. Thus, the driver of acid−base defect during dominant dRTA is probably an impaired membrane expression., Graphical abstract

Over the past few decades, studies on the red blood cell (RBC) membrane gave rise to increasingly sophisticated although divergent models of its structural organization, since investigations were often performed in denaturing conditions using detergents. To access soluble isolated RBC membrane complexes with the preservation of their interactions and conformations, we decided to apply the recent SMALP (Styrene Maleic Acid Lipid Particles) technology to RBC ghosts. Depending on the ionic strength of buffers in which ghost membranes were resuspended, the isolated proteins within SMALPs could differ on Coomassie-stained gels, but with few changes when compared to ghost membrane SDS lysates. We subsequently produced SMALPs derived from ghosts from two different blood group phenotypes, RhD-positive and RhD-negative, both types of RBC expressing the RhCE proteins but only RhD-positive cells being able to express the RhD proteins. This allowed the isolation, by size exclusion chromatography (SEC), of soluble fractions containing the Rh complex, including the RhD protein or not, within SMALPs. The use a conformation-dependent anti-RhD antibody in immunoprecipitation studies performed on SEC fractions of SMALPs containing Rh proteins clearly demonstrated that the RhD protein, which was only present in SMALPs prepared from RhD-positive RBC ghosts, has preserved at least one important conformational RhD epitope. This approach opens new perspectives in the field of the erythroid membrane study, such as visualization of RBC membrane complexes in native conditions by cryo-electron microscopy (CryoEM) or immuno-tests with conformation-dependent antibodies against blood group antigens on separated and characterized SMALPs containing RBC membrane proteins.

Anion exchanger 1 (AE1) or band 3 is a membrane protein responsible for the rapid exchange of chloride for bicarbonate across the red blood cell membrane. Nine mutations leading to single amino-acid substitutions in the transmembrane domain of AE1 are associated with dominant hereditary stomatocytosis, monovalent cation leaks, and reduced anion exchange activity. We set up a stopped-flow spectrofluorometry assay coupled with flow cytometry to investigate the anion transport and membrane expression characteristics of wild-type recombinant AE1 in HEK293 cells, using an inducible expression system. Likewise, study of three stomatocytosis-associated mutations (R730C, E758K, and G796R), allowed the validation of our method. Measurement of the rapid and specific chloride/bicarbonate exchange by surface expressed AE1 showed that E758K mutant was fully active compared with wild-type (WT) AE1, whereas R730C and G796R mutants were inactive, reinforcing previously reported data on other experimental models. Stopped-flow analysis of AE1 transport activity in red blood cell ghost preparations revealed a 50% reduction of G796R compared with WT AE1 corresponding to a loss of function of the G796R mutated protein, in accordance with the heterozygous status of the AE1 variant patients. In conclusion, stopped-flow led to measurement of rapid transport kinetics using the natural substrate for AE1 and, conjugated with flow cytometry, allowed a reliable correlation of chloride/bicarbonate exchange to surface expression of AE1, both in recombinant cells and ghosts and therefore a fine comparison of function between different stomatocytosis samples. This technical approach thus provides significant improvements in anion exchange analysis in red blood cells.

In red cells, Rh-associated glycoprotein (RhAG) acts as an ammonia channel, as demonstrated by stopped-flow analysis of ghost intracellular pH (pHi) changes. Recently, overhydrated hereditary stomatocytosis (OHSt), a rare dominantly inherited hemolytic anemia, was found to be associated with a mutation (Phe65Ser or Ile61Arg) in RHAG. Ghosts from the erythrocytes of four of the OHSt patients with a Phe65Ser mutation were resealed with a pH-sensitive probe and submitted to ammonium gradients. Alkalinization rate constants, reflecting NH3transport through the channel and NH3diffusion unmediated by RhAG, were deduced from time courses of fluorescence changes. After subtraction of the constant value found for Rhnulllacking RhAG, we observed that alkalinization rate constant values decreased ∼50% in OHSt compared with those of controls. Similar RhAG expression levels were found in control and OHSt. Since half of the expressed RhAG in OHSt most probably corresponds to the mutated form of RhAG, as expected from the OHSt heterozygous status, this dramatic decrease can be therefore related to the loss of function of the Phe65Ser-mutated RhAG monomer.

The renal ammonium transporter RhBG and anion exchanger 1 kAE1 colocalize in the basolateral domain of α-intercalated cells in the distal nephron. Although we have previously shown that RhBG is linked to the spectrin-based skeleton through ankyrin-G and that its NH3 transport activity is dependent on this association, there is no evidence for an interaction of kAE1 with this adaptor protein. We report here that the kAE1 cytoplasmic N terminus actually binds to ankyrin-G, both in yeast two-hybrid analysis and by coimmunoprecipitation in situ in HEK293 cells expressing recombinant kAE1. A site-directed mutagenesis study allowed the identification of three dispersed regions on kAE1 molecule linking the third and fourth repeat domains of ankyrin-G. One secondary docking site corresponds to a major interacting loop of the erythroid anion exchanger 1 (eAE1) with ankyrin-R, whereas the main binding region of kAE1 does not encompass any eAE1 determinant. Stopped flow spectrofluorometry analysis of recombinant HEK293 cells revealed that the Cl(-)/HCO3 (-) exchange activity of a kAE1 protein mutated on the ankyrin-G binding site was abolished. This disruption impaired plasma membrane expression of kAE1 leading to total retention on cytoplasmic structures in polarized epithelial Madin-Darby canine kidney cell transfectants. kAE1 also directly interacts with RhBG without affecting its surface expression and NH3 transport function. This is the first description of a structural and functional RhBG·kAE1·ankyrin-G complex at the plasma membrane of kidney epithelial cells, comparable with the well known Rh·eAE1·ankyrin-R complex in the red blood cell membrane. This renal complex could participate in the regulation of acid-base homeostasis.

International audience; Urea transporter B (UT-B) is a passive membrane channel that facilitates highly efficient permeation of urea. In red blood cells (RBC), while the major function of UT-B is to transport urea, it is assumed that this protein is able to conduct water. Here, we have revisited this last issue by studying RBCs and ghosts from human variants with defects of aquaporin 1 (AQP1) or UT-B. We found that UT-B's osmotic water unit permeability (pf unit) is similar to that of AQP1. The determination of diffusional permeability coefficient (P d) allowed the calculation of the P f /P d ratio, which is consistent with a single-file water transport. Molecular dynamic simulations of water conduction through human UT-B confirmed the experimental finding. From these results, we propose an atomistic description of water–protein interactions involved in this permeation. Inside the UT-B pore, five water molecules were found to form a single-file and move rapidly along a channel by hydrogen bond exchange involving two critical threonines. We further show that the energy barrier for water located in the central region coincides with a water dipole reorientation, which can be related to the proton exclusion observed experimentally. In conclusion, our results indicate that UT-B should be considered as a new member of the water channel family.

Anion exchanger 1 (AE1) or band 3 is a membrane protein responsible for the rapid exchange of chloride for bicarbonate across the red blood cell membrane. Nine mutations leading to single amino-acid substitutions in the transmembrane domain of AE1 are associated with dominant hereditary stomatocytosis, monovalent cation leaks, and reduced anion exchange activity. We set up a stopped-flow spectrofluorometry assay coupled with flow cytometry to investigate the anion transport and membrane expression characteristics of wild-type recombinant AE1 in HEK293 cells, using an inducible expression system. Likewise, study of three stomatocytosis-associated mutations (R730C, E758K, and G796R), allowed the validation of our method. Measurement of the rapid and specific chloride/bicarbonate exchange by surface expressed AE1 showed that E758K mutant was fully active compared with wild-type (WT) AE1, whereas R730C and G796R mutants were inactive, reinforcing previously reported data on other experimental models. Stopped-flow analysis of AE1 transport activity in red blood cell ghost preparations revealed a 50% reduction of G796R compared with WT AE1 corresponding to a loss of function of the G796R mutated protein, in accordance with the heterozygous status of the AE1 variant patients. In conclusion, stopped-flow led to measurement of rapid transport kinetics using the natural substrate for AE1 and, conjugated with flow cytometry, allowed a reliable correlation of chloride/bicarbonate exchange to surface expression of AE1, both in recombinant cells and ghosts and therefore a fine comparison of function between different stomatocytosis samples. This technical approach thus provides significant improvements in anion exchange analysis in red blood cells.

International audience; Zidi-Yahiaoui N, Callebaut I, Genetet S, Le Van Kim C, Cartron JP, Colin Y, Ripoche P, Mouro-Chanteloup I. Functional analysis of human RhCG: comparison with E. coli ammonium transporter reveals similarities in the pore and differences in the vestibule. Am J Physiol Cell Physiol 297: C537–C547, 2009. First published June 24, 2009; doi:10.1152/ajpcell.00137.2009.—Rh glycoproteins are members of the ammonium transporter (Amt)/methylamine permease (Mep)/Rh family facilitating movement of NH3 across plasma membranes. Homology models constructed on the basis of the experimental structures of Escherichia coli AmtB and Nitrosomonas europaea Rh50 indicated a channel structure for human RhA (RhAG), RhB (RhBG), and RhC (RhCG) glycoproteins in which external and internal vestibules are linked by a pore containing two strictly conserved histidines. The pore entry is constricted by two highly conserved phenylalanines, “twin-Phe.” In this study, RhCG function was investigated by stopped-flow spectrofluorometry measuring kinetic pH variations in HEK293E cells in the presence of an ammonium gradient. The apparent unitary NH3 permeability of RhCG was determined and was found to be close to that of AmtB. With a site-directed mutagenesis approach, critical residues involved in Rh NH3 channel activity were highlighted. In the external vestibule, the importance of both the charge and the conformation of the conserved aspartic acid was shown. In contrast to AmtB, individual mutations of each phenylalanine of the twin-Phe impaired the function while the removal of both resulted in recovery of the transport activity. The impact of the mutations suggests that, although having a common function and a similar channel structure, bacterial AmtB and human Rh vary in several aspects of the NH3 transport mechanisms.

RhBG, a human member of the Amt/Mep/Rh/superfamily of ammonium transporters, has been shown to facilitate NH(3) transport and to be anchored to the basolateral plasma membrane of kidney epithelial cells, via ankyrin-G. We showed here that triple alanine substitution of the (419)FLD(421) sequence, which links the cytoplasmic C-terminal domain of RhBG to ankyrin-G, not only disrupted the interaction of RhBG with the spectrin-based skeleton but also delayed its cell surface expression, decreased its plasma membrane stability, and abolished its NH(3) transport function in epithelial cell lines. Similarly, we demonstrated that both anchoring to the membrane skeleton and ammonium transport activity are regulated by the phosphorylation status of the C-terminal tail of RhBG. Tyrosine 429, which belongs to the previously reported YED basolateral targeting signal of RhBG, was demonstrated to be phosphorylated in vitro using purified Src and Syk kinases and ex vivo by analyzing the effect of pervanadate treatment on wild-type RhBG or Y429A mutants. Then, we showed that Y429D and Y429E mutations, mimicking constitutive phosphorylation, abolished NH(3) transport and enhanced Triton X-100 solubilization of RhBG from the cell membrane. In contrast, the nonphosphorylated/nonphosphorylatable Y429A and Y429F mutants behaved the same as wild-type RhBG. Conversely, Y/A or Y/F but not Y/E or Y/D mutations of residue 429 abolished the exclusive basolateral localization of RhBG in polarized epithelial cells. All these results led to a model in which targeting and ammonium transport function of RhBG are regulated by both phosphorylation and membrane skeleton binding of the C-terminal cytoplasmic domain.

The Overhydrated Stomatocytic (OHSt) erythrocytes of four patients, characterized by the F65S mutation (Bruce et al, Blood 2009) located in the pore of the ammonia channel Rh Associated Glycoprotein (RhAG), were studied. We have previously demonstrated that the equivalent substitution (F74L) in the non-erythroid analogue RhCG resulted in a reduction of the ammonia influx of 50% in transfected HEK293 cells.Ghosts prepared by hypotonic lysis of OHSt and control (Ctl) erythrocytes, resealed in the presence of a pH-sensitive probe (pyranine), exhibited echinocytic morphology. Images of 1667 (Ctl) and 1998 (OHSt) echinocytes, visualized by light microscopy, allowed the determination of average ghost diameters: 5.81±0.2 µm (Ctl) and 5.83±0.06 µm (OHSt). RhAG densities, as determined by flow cytometry, were similar for Ctl and OHSt (78 000 to 80 000 copies/cell).Ammonium permeability (P’NH3) was measured by following the internal compartment alkalinisation, after submitting ghosts to inward ammonium gradients. Alkalinisation rate constants (k) were deduced from stopped-flow analysis of pH variations at 15°C, allowing the determination of P’NH3 by using the simplified equation P’NH3=k.r/3 (r: radius of spheric ghosts). The P’NH3 values were significantly different between Ctl and OHSt samples (1.7±0.11 vs 0.71±0.004 µm.s−1).Additionally, water permeability was measured in the same OHSt resealed ghosts but containing 8mM 6-carboxyfluorescein (6-CF), after submitting them to an osmotic gradient (150mosm/kg/H2O mannitol). The osmotic water permeability (Pf) values deduced from the fluorescence quenching at 15°C were similar between Ctl and OHSt samples (0.035±0.005 vs 0.035±0.04 cm.s−1).In conclusion, the F65S mutation of RhAG induces a reduction of ammonia flux resulting in an alteration of pHi regulation. The decrease of ammonia conductance through the mutated RhAG can result from loss of hydrophobicity and/or from structural modifications inside the pore of the channel.

BackgroundRh glycoproteins (RhAG, RhBG, RhCG) are members of the Amt/Mep/Rh family which facilitate movement of ammonium across plasma membranes. Changes in ammonium transport activity following expression of Rh glycoproteins have been described in different heterologous systems such as yeasts, oocytes and eukaryotic cell lines. However, in these complex systems, a potential contribution of endogenous proteins to this function cannot be excluded. To demonstrate that Rh glycoproteins by themselves transport NH(3), human RhCG was purified to homogeneity and reconstituted into liposomes, giving new insights into its channel functional properties.Methodology/principal findingsAn HA-tag introduced in the second extracellular loop of RhCG was used to purify to homogeneity the HA-tagged RhCG glycoprotein from detergent-solubilized recombinant HEK293E cells. Electron microscopy analysis of negatively stained purified RhCG-HA revealed, after image processing, homogeneous particles of 9 nm diameter with a trimeric protein structure. Reconstitution was performed with sphingomyelin, phosphatidylcholine and phosphatidic acid lipids in the presence of the C(12)E(8) detergent which was subsequently removed by Biobeads. Control of protein incorporation was carried out by freeze-fracture electron microscopy. Particle density in liposomes was a function of the Lipid/Protein ratio. When compared to empty liposomes, ammonium permeability was increased two and three fold in RhCG-proteoliposomes, depending on the Lipid/Protein ratio (1/300 and 1/150, respectively). This strong NH(3) transport was reversibly inhibited by mercuric and copper salts and exhibited a low Arrhenius activation energy.Conclusions/significanceThis study allowed the determination of ammonia permeability per RhCG monomer, showing that the apparent Punit(NH3) (around 1x10(-3) microm(3)xs(-1)) is close to the permeability measured in HEK293E cells expressing a recombinant human RhCG (1.60x10(-3) microm(3)xs(-1)), and in human red blood cells endogenously expressing RhAG (2.18x10(-3) microm(3)xs(-1)). The major finding of this study is that RhCG protein is active as an NH(3) channel and that this function does not require any protein partner.