Different Transport Mechanisms in Plant and Human AMT/Rh-type Ammonium Transporters

The conserved family of AMT/Rh proteins facilitates ammonium transport across animal, plant, and microbial membranes. A bacterial homologue, AmtB, forms a channel-like structure and appears to function as an NH3 gas channel. To evaluate the function of eukaryotic homologues, the human RhCG glycoprotein and the tomato plant ammonium transporter LeAMT1;2 were expressed and compared in Xenopus oocytes and yeast. RhCG mediated the electroneutral transport of methylammonium (MeA), which saturated with Km = 3.8 mM at pHo 7.5. Uptake was strongly favored by increasing the pHo and was inhibited by ammonium. Ammonium induced rapid cytosolic alkalinization in RhCG-expressing oocytes. Additionally, RhCG expression was associated with an alkali-cation conductance, which was not significantly permeable to NH4+ and was apparently uncoupled from the ammonium transport. In contrast, expression of the homologous LeAMT1;2 induced pHo-independent MeA+ uptake and specific NH4+ and MeA+ currents that were distinct from endogenous currents. The different mechanisms of transport, including the RhCG-associated alkali-cation conductance, were verified by heterologous expression in appropriate yeast strains. Thus, homologous AMT/Rh-type proteins function in a distinct manner; while LeAMT1;2 carries specifically NH4+, or cotransports NH3/H+, RhCG mediates electroneutral NH3 transport

Functional Reconstitution into Liposomes of Purified Human RhCG Ammonia Channel

BACKGROUND: Rh 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 FINDINGS: An 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/SIGNIFICANCE: This 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

Permeability of phospholipid membranes and human red blood cell membranes to hydrogen peroxide

Resumen del Conference paper presentado a SfRBM 28th Annual ConferenceHydrogen peroxide (H2O2) is an oxygen-derived oxidant involved in multiple redox processes in the cell, ranging from physiological signaling pathways to oxidative damage reactions when it is found at higher concentrations. In the vascular system, H2O2 is metabolized mainly by red blood cells (RBC) due to their very efficient antioxidant systems and high membrane permeability. However, the information regarding H2O2 transport in the human RBC membrane is limited, as neither the exact value of the permeability coefficient (Pm) nor the permeation mechanisms are known. To explore whether H2O2 permeates through the lipid fraction or protein channels, we studied H2O2 solubility in organic solvents and its permeability in lipid membranes, in order to compare with the RBC membrane. Through measurements of partition constants, we found that H2O2 is 14 and 122000 times less soluble in octanol and hexadecane than in water, anticipating a large thermodynamic barrier to H2O2 permeation by lipid membranes. The Pm in phospholipid membranes of different compositions, determined using the catalase-latency method, varied from 4×10-4 to 5×10-3 cm s-1, at 37°C. On the other hand, in human RBC we determined a Pm of 1.6×10-3 cm s-1. After obtaining these results, we evaluated the potential role of aquaporins as H2O2 transporters by checking the effect of aquaporin inhibitors in H2O2 consumption by RBC, and also by studying H2O2 permeability in RBC devoid of either aquaporin 1 or aquaporin 3. Surprisingly, we could not detect any differences in H2O2 permeability in any case. Altogether, these results provide new information on lipid membrane permeability to H2O2 and a new value for the Pm in human RBC, which was previously unknown. Additionally, they indicate that H2O2 is not transported by aquaporins in human RBC membranes, suggesting simple diffusion or a still unidentified membrane protein as a more probable pathway.ANII: ANII: FMV_1_2019_15559

The permeability of human red blood cell membranes to hydrogen peroxide is independent of aquaporins

Hydrogen peroxide (H2O2) not only is an oxidant but also is an important signaling molecule in vascular biology, mediating several physiological functions. Red blood cells (RBCs) have been proposed to be the primary sink of H2O2 in the vasculature because they are the main cellular component of blood with a robust antioxidant defense and a high membrane permeability. However, the exact permeability of human RBC to H2O2 is neither known nor is it known if the mechanism of permeation involves the lipid fraction or protein channels. To gain insight into the permeability process, we measured the partition constant of H2O2 between water and octanol or hexadecane using a novel double-partition method. Our results indicated that there is a large thermodynamic barrier to H2O2 permeation. The permeability coefficient of H2O2 through phospholipid membranes containing cholesterol with saturated or unsaturated acyl chains was determined to be 4 × 10−4 and 5 × 10−3 cm s−1, respectively, at 37 °C. The permeability coefficient of human RBC membranes to H2O2 at 37 °C, on the other hand, was 1.6 × 10−3 cm s−1. Different aquaporin-1 and aquaporin-3 inhibitors proved to have no effect on the permeation of H2O2. Moreover, human RBCs devoid of either aquaporin-1 or aquaporin-3 were equally permeable to H2O2 as normal human RBCs. Therefore, these results indicate that H2O2 does not diffuse into RBCs through aquaporins but rather through the lipid fraction or a still unidentified membrane protein.ANII: FCE_2017_136043ANII: FMV_2019_155597CSIC: I+D_2014_C632-348CSIC: 2018_4

Detergent-free isolation of native red blood cell membrane complexes

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The ammonia transporter RhCG modulates urinary acidification by interacting with the vacuolar proton-ATPases in renal intercalated cells

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Mice Expressing RHAG and RHD Human Blood Group Genes

International audienceAnti-RhD prophylaxis of haemolytic disease of the fetus and newborn (HDFN) is highly effective, but as the suppressive mechanism remains uncertain, a mouse model would be of interest. Here we have generated transgenic mice expressing human RhAG and RhD erythrocyte membrane proteins in the presence and, for human RhAG, in the absence, of mouse Rhag. Human RhAG associates with mouse Rh but not mouse Rhag on red blood cells. In Rhag knockout mice transgenic for human RHAG, the mouse Rh protein is "rescued" (re-expressed), and coimmunoprecipitates with human RhAG, indicating the presence of hetero-complexes which associate mouse and human proteins. RhD antigen was expressed from a human RHD gene on a BAC or from RHD cDNA under control of β-globin regulatory elements. RhD was never observed alone, strongly indicative that its expression absolutely depends on the presence of transgenic human RhAG. This first expression of RhD in mice is an important step in the creation of a mouse model of RhD allo-immunisation and HDFN, in conjunction with the Rh-Rhag knockout mice we have developed previously