Pathways for flip-flop of mono- and di-anionic phospholipids in the erythrocyte membrane.
The inward translocations (flip), from the outer to the inner membrane leaflet of human erythrocytes, of di-anionic NBD-labeled phospholipids containing as a head group phosphate esters of glycolate, butyrate and hydroxyethanesulfonate are slow processes (k = 0.005-0.008 h-1, 37 degrees C) at pH 7.4. A decrease of pH highly stimulates the flip. A major role of the anion exchanger (AE1), band 3, in this flip is indicated by (a) the strong inhibition of the flip (55-85%) by stilbene disulfonates and other inhibitors of anion transport, (b) the stimulation and loss of pH dependence of the flip after modification of band 3 by Woodward's reagent K and NaBH4, and (c) the stimulation of the flip after proteolytic cleavage of band 3 by papain. The flip of mono-anionic NBD-phospholipids with phosphate esters of glycerol, glycol, methanol, butanol and benzyl alcohol is much faster than that of their dianionic analogs (k = 0.04 to > 3.0 h-1, 37 degrees C). It is inhibited by stilbene disulfonates to a decreasing extent (35 to 0%) and is not affected by several reversible inhibitors of anion exchange. This indicates a minor component of band-3-mediated flip and a major component of nonmediated flip. The outward translocations (flop), from the inner to outer membrane leaflet, of both mono- and di-anionic phospholipids are very fast (1.0-5.9 h-1), ATP-dependent and inhibitable by vanadate, fluoride, SH-reagents or Mg(2+)-depletion of cells and thereby likely to be largely mediated by a 'floppase'. The stationary distributions of the NBD-labeled anionic phospholipids are asymmetric to an extent (outer to inner leaflet ratio 2-9) correlating with the ratio of the rates of the outward and the inward translocation. Thus, asymmetry is largely abolished by blockage of the floppase-mediated translocation.
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Istituto di Fisiologia Generale e Chimica Biologica, Sassari, Italy.
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D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000097:blood; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D001457:Anion Exchange Protein 1, Erythrocyte / Q000037:antagonists & inhibitors / Q000378:metabolism; D000838:Anions; D001692:Biological Transport / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D010206:Papain / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000097:blood; D010743:Phospholipids / Q000097:blood*
C078632:1-acyl-2-(12-((7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl)phosphatidylcholine; D001457:Anion Exchange Protein 1, Erythrocyte; D000838:Anions; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D010713:Phosphatidylcholines; D010743:Phospholipids; D010206:Papain; D000251:Adenosine Triphosphatases; D009327:4-Chloro-7-nitrobenzofurazan; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid