Scrambling of Phospholipids Activates Red Cell Membrane Cholesterol

Abstract

Cholesterol is predicted to associate more strongly with the outer than the inner leaflet of plasma membrane bilayers based on the relative in vitro affinities of their phospholipids. Complex formation with the high affinity species (especially saturated sphingomyelins) is said to reduce the chemical activity (escape potential or fugacity) of the sterol. We therefore tested the hypothesis that scrambling the sidedness of plasma membrane phospholipids of intact cells will increase the chemical activity of outer surface cholesterol. Upon activating the plasma membrane scramblase in intact human red cells by introducing ionomycin to raise cytoplasmic Ca ++ , phosphatidylserine became exposed and, concomitantly, the chemical activity of exofacial cholesterol was increased. (This was gauged by its susceptibility to cholesterol oxidase and its rate of transfer to cyclodextrin.) Similar behavior was observed in human fibroblasts. Two other treatments known to activate cell surface cholesterol (namely, exposure to glutaraldehyde and to low ionic strength buffer) also brought phosphatidylserine to the cell surface but by a Ca ++ -independent mechanism. Given that phospholipid scrambling is important in blood coagulation and apoptosis, the concomitant activation of cell surface cholesterol could contribute to these and other pathophysiological signaling processes.It has been observed that increasing the cholesterol in the mammalian PM 1 beyond the physiological level evokes a sharp rise in its susceptibility to attack by CO and in its transfer both to extracellular cyclodextrin and the endoplasmic reticulum (1,2). These findings have been explained by the following hypothesis (2,3): Cholesterol forms complexes of varied strength and stoichiometry with different bilayer PLs. The complexed sterol has a lower chemical activity (escape potential or fugacity) than uncomplexed (free) cholesterol; for example, that added in excess of the binding capacity of the PL. It is the high chemical activity of the uncomplexed sterol that accounts for its increased interaction with CO and cyclodextrin. Physiologically, the high fugacity of excess cholesterol provides a homeostatic mechanism that keeps the PM sterol level near the capacity of its PL partners, ~0.8 mol per mol phospholipid (4). [Note that the concept of cholesterol chemical activity of interest here differs distinctly from that of active cholesterol, which refers to the attributes of biologically functional sterols (5).] † This work was supported in part by National Institutes of Health grant HL 28448.* To whom correspondence should be addressed at ‡ Dept. of Pathology, Rush University Medical Center, 1653 West Congress Parkway, Chicago, IL 60612. Tel: 312-942-5256; Fax: 312-563-3115; E-mail: ylange@rush.edu. 1 The abbreviations used are: CO, cholesterol oxidase; DIDS, 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; ER, endoplasmic reticulum; HBS, 150 mM NaCl-5 mM histidine, pH 7.5; MBCD, methyl-β-cyclodextrin; PBS, 150 mM NaCl-5 mM NaPi, pH 7.5; PC, phosphatidylc...

Stability and Stoichiometry of Bilayer Phospholipid–Cholesterol Complexes: Relationship to Cellular Sterol Distribution and Homeostasis

“…These data are in accord with the abundance of phospholipids with saturated chains and phosphorylcholine head groups in the outer leaflet of the RBC bilayer (66–68). The relatively high cholesterol oxidase sensitivity of the cytoplasmic leaflet of this membrane (not tested here) also agrees with its phospholipid composition (41, 69–71). The apparent Q value for the RBC (namely, 11) was lower than that predicted from the sum of the phospholipids at their outer and inner leaflets.…”

Cholesterol homeostasis and the escape tendency (activity) of plasma membrane cholesterol

“…That ABCA1 resides in liquid-disordered regions of the plasma membrane rather than in rafts is also consistent with its preferential export of uncomplexed (high activity) cholesterol [135]. Of additional relevance is the observation that ABCA1 promotes the translocation of phosphatidylserine to the exofacial leaflet of the plasma membrane [133], since this too would increase the activity of cell surface cholesterol [85]. A different (slower and more complex) system by which cholesterol export could be capped at the plasma membrane equivalence point would be through regulation of the expression of ABCA1 by 27-hydroxycholesterol [122], provided that the production of this oxysterol turns out to reflect the active excess of cholesterol, as discussed above.…”

“…This hypothesis has been tested by determining whether scrambling the transverse distribution of the phospholipids in the plasma membrane alters the activity of the cholesterol in the outer leaflet. When the phospholipids of the two leaflets of plasma membrane bilayers were allowed to equilibrate by activating the membrane scramblase through an elevation in cytoplasmic Ca ++ , the susceptibility of cholesterol to cholesterol oxidase and its rate of capture by extracellular cyclodextrin both increased [69,85]. A plausible inference is that the movement of the less avid inner-leaflet phospholipids to the outer leaflet reduced its sterol complexation and thereby raised the activity of its cholesterol.…”

Linking phospholipid flippases to vesicle-mediated protein transport

“…the ER). Consistent with this possibility, scrambling of erythrocyte plasma membrane phospholipids has been shown to increase the escape tendency of cholesterol as measured by the rate of transfer to cyclodextrin [95]. Why would cells evolve a mechanism to downregulate the activity of P4-ATPases?…”