Dynamics of the holes in human erythrocyte membrane ghosts.

Abstract

Osmotic lysis of human erythrocytes creates in each membrane a single large hole which subsequently contracts to a size determined largely by the buffer, typically 7-140 A in radius. Cations, but not anions, stimulated the rate and extent of hole size reduction with simple saturation behavior; the potency of Ca2+ exceeded M&+, which greatly surpassed Na+. The hole size was minimal at pH 5.5. Hole size change was greatly accelerated by increasing temperature between 0 and 50 “C. Whatever its size, the hole radius was stable to 21-2 A over several days at 0 “C. Cationic amphipaths such as chlorpromazine promoted hole closure in a salt-dependent fashion, while anionic amphipaths such as 2,4-dinitrophenolate or hexanoate opposed hole closure. Hole size thus appears to be determined by a balance between the force expanding the hole (electrostatic repulsion among anionic groups on the membrane) and the force driving hole closure (perhaps the hydrophobic effect of the lipid/ water interface at the hole perimeter and/or membrane elasticity). Hole size was initially completely reversible; however, the extent to which holes were able to expand and contract gradually diminished in parallel until the oles became fixed at an ambient size. The rate of this process increased directly with temperature, pH, and the presence of hexanol, and inversely with cation concentration. A 30% reduction in membrane cholesterol greatly accelerated the loss of hole sealing. Glutaraldehyde at 0.5 l ~ l ~ (0.005%) blocked hole closure (but not hole expansion) without cross-linking membrane proteins significantly. Once lost, the ability to expand hole size could be regenerated by osmotically stressing sucrosefilled ghosts. We infer that hole size dynamics. reflect metastable configurations of the membrane bilayer which can be reversibly altered, dissipated, and restored by experimental manipulations. Applications of the hemolytic hole as a continuously tunable molecular filter are suggested.