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Pathways for flip-flop of mono- and di-anionic phospholipids in the erythrocyte membrane

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Abstract

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°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°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 banadate, fluoride, SH-reagents or Mg2+-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.

Keywords

Erythrocyte membrane
Anionic phospholipid
Flip-flop
Anion transport
Band 3
Flippase

Abbreviations

DENSA, 5-(N-decyl)aminonaphtalene-2-sulfonate
DIDS, 4,4′-diisothiocyanostilbene-2,2′-disulfonate
DNDS, 4,4′-dinitros-tilbene-2,2′-disulfonate
LPM, lysophosphatidylmethanol
NBD-PC, 1-oleoyl-2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino hexanoyl]-sn-glycero-3- phosphocholine
NPP, 4-nitrophenylphosphate
PA, phosphatidic acid
PB, phosphatidylbutanol
PBA, phosphatidylhydroxybutyric acid
PBz, phosphatidylbenzylalcohol
PC, phosphatidylcholine
PE, phosphatidylethanolamine
PESA, phosphatidylhydroxyethanesulfonic acid
PG, phosphatidylglycerol
PGc, phosphatidylglycol
PGcA, phosphatidylglycolic acid
PM, phosphatidylmethanol
PS, phosphatidylserine
WRK, Woodward's reagent K

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