Formation of flip sites for phospholipids by introduction of channel-forming antibiotics into the membrane of human erythrocytes.

Incorporation into the erythrocyte membrane of channel-forming antibiotics, such as the polyene amphotericin B or the polypeptide gramicidin A, highly accelerates the transbilayer reorientation (flip) of exogenously incorporated lysolecithin. The first enhancement of flip is obtained when about 2 X 10(5) copies per cell are incorporated of each of the antibiotics. An up to 40-fold increase is obtained at about 2 X 10(6) copies per cell. Conversely, pimaricin, a polyene antibiotic which does not form channels probably because it cannot span the lipid bilayer, does not enhance flip. Moreover, the N-formylated analogue of gramicidin, which does not form channels either, does not enhance flip. The results clearly demonstrate the specificity of the flip enhancing effect and indicate a requirement of a focal perturbation of the outer and the inner membrane layer to induce flip by the channel formers. Besides flip enhancement antibiotics increase the rate of cleavage of outer membrane layer phosphatidylcholine by phospholipase A2. Moreover, amphotericin increases accessibility of inner membrane layer phosphatidylethanolamine to the lipase, whereas gramicidin does not enhance accessibility of this phospholipid. This indicates a more general perturbation of the inner membrane lipid domain by the polyenes.