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

The rhesus protein RhCG: a new perspective in ammonium transport and distal urinary acidification

Urinary acidification is a complex process requiring the coordinated action of enzymes and transport proteins and resulting in the removal of acid and the regeneration of bicarbonate. Proton secretion is mediated by luminal H(+)-ATPases and requires the parallel movement of NH(3), and its protonation to NH(4)(+), to provide sufficient buffering. It has been long assumed that ammonia secretion is a passive process occurring by means of simple diffusion driven by the urinary trapping of ammonium. However, new data indicate that mammalian cells possess specific membrane proteins from the family of rhesus proteins involved in ammonia/μm permeability. Rhesus proteins were first identified in yeast and later also in plants, algae, and mammals. In rodents, RhBG and RhCG are expressed in the collecting duct, whereas in humans only RhCG was detected. Their expression increases with maturation of the kidney and accelerates after birth in parallel with other acid-base transport proteins. Deletion of RhBG in mice had no effect on renal ammonium excretion, whereas RhCG deficiency reduces renal ammonium secretion strongly, causes metabolic acidosis in acid-challenged mice, and impairs restoration of normal acid-base status. Microperfusion experiments or functional reconstitution in liposomes demonstrates that ammonia is the most likely substrate of RhCG. Similarly, crystal structures of human RhCG and the homologous bacterial AmtB protein suggest that these proteins may form gas channels.Kidney International advance online publication, 6 October 2010; doi:10.1038/ki.2010.386

ProLIF: a quantitative assay for investigating integrin cytoplasmic protein interactions and synergistic membrane effects on proteoliposomes

Integrin transmembrane heterodimeric receptors control a wide range of biological interactions by triggering the assembly of large multiprotein complexes at their cytoplasmic interface. A diverse set of methods have been used to investigate cytoplasmic interactions between integrins and intracellular proteins. These predominantly consist of peptide-based pull-downs and biochemical immuno- isolations from detergent-solubilized cell lysates. However, quantitative methods to probe integrin- protein interactions in a more biologically relevant context where the integrin is embedded within a lipid bilayer have been lacking. Here we describe a technique called ProLIF (Protein-Liposome Interactions by Flow cytometry) to reconstitute recombinant integrin transmembrane domain (TMD) and cytoplasmic tail (CT) fragments on liposomes as individual ? or ? subunits or as ?? heterodimers and, using flow cytometry, to rapidly and quantitatively measure protein interactions with these membrane-embedded integrins. Importantly, the assay can analyse binding of fluorescent proteins directly from cell lysates without further purification steps. By combining integrins with membrane lipids to generate proteoliposomes, the effects of membrane composition such as PI(4,5)P2 presence on protein recruitment to the integrin CTs can be analyzed. ProLIF requires no specific instrumentation, apart from a standard flow cytometer and can be applied to measure a broad range of membrane-dependent protein-protein interactions with the potential for high-throughput/multiplex analyses

Cytosolic Guanine Nucledotide Binding Deficient Form of Transglutaminase 2 (R580a) Potentiates Cell Death in Oxygen Glucose Deprivation

Transglutaminase 2 (TG2) is a hypoxia-responsive protein that is a calcium-activated transamidating enzyme, a GTPase and a scaffolding/linker protein. Upon activation TG2 undergoes a large conformational change, which likely affects not only its enzymatic activities but its non-catalytic functions as well. The focus of this study was on the role of transamidating activity, conformation and localization of TG2 in ischemic cell death. Cells expressing a GTP binding deficient form of TG2 (TG2-R580A) with high basal transamidation activity and a more extended conformation showed significantly increased cell death in response to oxygen-glucose deprivation; however, targeting TG2-R580A to the nucleus abrogated its detrimental role in oxygen-glucose deprivation. Treatment of cells expressing wild type TG2, TG2-C277S (a transamidating inactive mutant) and TG2-R580A with Cp4d, a reversible TG2 inhibitor, did not affect cell death in response to oxygen-glucose deprivation. These findings indicate that the pro-cell death effects of TG2 are dependent on its localization to the cytosol and independent of its transamidation activity. Further, the conformational state of TG2 is likely an important determinant in cell survival and the prominent function of TG2 in ischemic cell death is as a scaffold to modulate cellular processes