NONMEDIATED FLIP-FLOP OF ANIONIC PHOSPHOLIPIDS AND LONG-CHAIN AMPHIPHILES IN THE ERYTHROCYTE-MEMBRANE DEPENDS ON MEMBRANE-POTENTIAL

The nonmediated inward translocation (flip) of the anionic fluorescent N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- (NBD-)labeled phospholipid pho sphatidylmethanol (PM) from the outer to the inner membrane leaflet of human erythrocytes and vice versa depends on membrane potential. Inte restingly, inside-positive potentials due to chloride gradients and th e native chloride conductance of the cells resulted in an increase of the flip rates. This flip enhancement could be suppressed by addition of gramicidin D, which increases cation conductance, or 4,4'-diisothio cyanatostilbene-2,2'-disufonate (DIDS), which inhibits anion conductan ce. Conversely, inside negative potentials established by an outward-d irected K+ gradient in the presence of gramicidin on DIDS-treated cell s resulted in a decrease of flip rate. Flip rate exhibited an exponent ial dependence on membrane potential. The opposite effects of the posi tive and negative potentials were obtained for the outward translocati on (flop) from the inner to the outer membrane leaflet. Similar potent ial dependencies were found for the nonmediated flip of anionic NBD-la beled phosphatidic acid (PA) and 2-(N-decyl)aminonaphthalene-6-sulfoni c acid (2,6-DENSA) following blockage of the band-3-mediated component of flip, The membrane potential also influences the stationary distri bution of the anionic lipids between the inner and outer leaflets. The distribution is shifted to the inner leaflet by increasingly positive potentials and to the outer leaflet by increasingly negative potentia ls. It is concluded that nonmediated flip-flop of the anionic phosphol ipids and the long-chain sulfonate represents electrogenic translocati on of the unprotonated charged lipids across the hydrophobic barrier.