Extensive electroporation abolishes experimentally induced shape transformations of erythrocytes: a consequence of phospholipid symmetrization?

As shown in earlier work (M.M. Henszen et al., Mol. Membr. Biol. 14 (1997) 195-204), exposure of erythrocytes to single brief electric field pulses (5-7 kV cm(-1)) enhances the transbilayer mobility of phospholipids and produces echinocytes which can subsequently be transformed into stomatocytes in an ATP-dependent process. These shape transformations arise from partly reversible changes of the transbilayer disposition of phospholipids, in agreement with the bilayer couple concept. Extensive membrane modification by repetitive (</=20) field pulses followed by 20 h incubation at 37 degrees C is now shown to produce discocytic cells which are resistant to many established shape-transforming treatments, including (A) single electric field pulses, Ca(2+) incorporation and exposure to membrane active amphiphiles, but also (B) metabolic depletion, binding of band 3 ligands, alkaline pH and contact with glass surfaces. The suppression of type A effects can readily be interpreted by a complete symmetrization of the phospholipids in extensively field pulse-modified cells which prevents shape transformations related to the asymmetric disposition of the phospholipids. This symmetrization could be further substantiated by more direct determinations of the transbilayer distribution of phospholipids. Suppression of shape transformations of type B may indicate an involvement of phospholipid asymmetry in these processes on a yet unknown mechanistic basis. Alternatively we discuss field pulse-induced alterations of the disposition of peripheral proteins or of the conformation of integral membrane proteins as mechanisms interfering with shape transformations of type B.