Gramicidin-induced enhancement of transbilayer reorientation of lipids in the erythrocyte membrane

Abstract

Incorporation of the channel-forming antibiotic gramicidin into the membrane of human erythrocytes highly (up to 30-fold) enhances rates of reorientation (flip) of lysophosphatidylcholine and palmitoylcarnitine to the inner membrane layer after their primary incorporation into the outer layer. Despite the high increase of flip rates by gramicidin, the asymmetric orientation of the inner membrane layer phospholipids phosphatidylethanolamine and phosphatidylserine is stable as demonstrated by the lack of accessibility of these lipids toward cleavage by exogenous phospholipase A2. On the other hand, gramicidin enhances the rate of cleavage of outer membrane layer phosphatidylcholine by phospholipase A2, which indicates changes in the packing of phosphatidylcholine following gramicidin binding. The increase of flip becomes detectable when about 10(5) copies of gramicidin per cell have been bound (gramicidin to membrane phospholipid ratio of 1:2000). This is a 1000-fold higher concentration than that required for an increase of K+ permeability mediated by the gramicidin channel. Acceleration of flip is thus not simply correlated with channel formation. The enhancement of flip is markedly dependent on structural details of gramicidin. Formylation of its four tryptophan residues abolishes the effect. Even at high concentrations of formylated gramicidin at which the extents of binding of native and of formylated gramicidin to the membrane are comparable, no flip acceleration is produced. Enhancement of flip by gramicidin occurs after a temperature-dependent lag phase. At 37 degrees C, flip rates begin to increase within a few minutes and at 25 degrees C, only after 3 h. This lag phase is most likely not due to limitations by the rate of binding of gramicidin to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between gramacidin conformation dependent induction of phospholipid transbilayer movement and hexagonal HII phase formation in erythrocyte membranes

“…rein mediated process [1]. Since non-bilayer lipid structures can facilitate transbilayer transport of lipids in model systems [2,3], the possibility should be considered that protein-induced (transient) non-bilayer lipid structures are responsible for The hydrophobic peptid¢ antibiotic gramicidin A is an attractive model to systematically investigate the interrelationships between pcptide-induced transbilayer movements of lipids and membrane organization [5,6]. Gramicidin inserts spontaneously into the hydrophobic core of the membrane and forms, at low concentrations by N-to N-terminal dlmerizafion of fl6.3 hefices cationselective channels [7,8].…”

“…This process is accompanied by an increase in permeation of intermediate sized solutes but not of high molecular weigh! dextrans [5]. Above a ratio of 1 : 80 part of the lipid of the ery throcyte membrane becomes organized i~; the hexagonal H n phase [6] resulting in cell lysis.…”

“…Above a ratio of 1 : 80 part of the lipid of the ery throcyte membrane becomes organized i~; the hexagonal H n phase [6] resulting in cell lysis. These effects are mechanistically related as formylation of the four tryptophans of the peptide or even a single Trp-, Phe substitution, affects all of these properties likewise [5,6,9].…”

“…45 mCi/mmol) in the outer membrane layer of erythrocytes and measuring the gramicidin-dependent re.orientation of lysophosphatidylcholiae from the outer to the inner membrane layer by quantifying the increase in the fraction of lipid probe that becomes non-extractable by albumin [5,14]. Non-specific leak formati,~n was measured by suspending erythrocytes in isotonic itepes-buffered saline (at 37" C, hematocrit 10%) comair~ing 40 mM mannitol or sucrose to protect the celts against colloid-osmotic lysis as a result of specific ehannd-mediated salt uptake by the cells.…”

“…Non-specific leak formati,~n was measured by suspending erythrocytes in isotonic itepes-buffered saline (at 37" C, hematocrit 10%) comair~ing 40 mM mannitol or sucrose to protect the celts against colloid-osmotic lysis as a result of specific ehannd-mediated salt uptake by the cells. Rates of influx of the solutes via non-specific leaks was monitored by the time-dependent increase of hemolysis [5]. The extent of binding of 14C-labeled gramiddin (spec.act.…”