Formation and properties of aqueous leaks induced in human erythrocytes by electrical breakdown.

Leaks were induced in human erythrocytes by brief (tau = 1-40 microseconds) discharges of high electric fields (3-20 kV/cm). Leak permeabilities were characterized by measuring (a) net and tracer fluxes of K+ and nonelectrolytes under protection of the cells against colloid-osmotic lysis, or (b) rates of colloid osmotic lysis in various salt solutions. The induced permeabilities are essentially stable for hours at 0-2 degrees C. Leak permeability P increases exponentially with the breakdown voltage ED according to a function of the general type P = bED. The basis b varies with the pulse length. A log-linear presentation reveals a biphasic linear relationship with a break at which the slope (= log b) decreases markedly. Elevated ionic strengths of the suspension medium during the electric discharge enhance leak formation. Leak permeability exhibits an apparent activation energy of 29 +/- 5 kJ/mol, indicative of diffusion through aqueous pathways. Somewhat differing equivalent pore radii emerge from measurements with different probes: 0.6-0.8 nm from tracer fluxes of polyols (Mr = 3600, ED = 4-7 kV/cm) and 0.8-1.9 nm from osmotic protection studies with polyethylene glycols (Mr = 200-3300, ED = 6-10 kV/cm). These numbers and the non-monoexponential increase of leak permeability with the field strength suggest a dual mechanism for the increase of leak permeability: an increase of the number of pores at low breakdown voltage and an additional increase of pore size at higher voltage. Estimated numbers of pores range from 1 to 10 per cell, which suggests dynamic fluctuating structural defects to be involved. The leaks discriminate small monovalent inorganic ions in the sequence of free solution mobility. Organic anions are discriminated according to size and charge. Common properties of these electrically induced defects and of chemically induced leaks (diamide, periodate, t-butylhydroperoxide) in the erythrocyte membrane suggest close similarities in the molecular organization.

Dielectric breakdown of the erythrocyte membrane enhances transbilayer mobility of phospholipids.

Dielectric breakdown of erythrocytes is shown to result in a loss of asymmetry of phosphatidylethanolamine and in a markedly enhanced transbilayer mobility of exogenous lysophosphatidylcholine. The effect is much more pronounced in non-resealed cells than in cells resealed after the breakdown. A casual relationship between the structural defects in the lipid phase, indicated by these results, and fusion by dielectric breakdown is discussed.

Concomitant changes of membrane leak permeability and phospholipid dynamics in erythrocytes subjected to chemical and physical membrane perturbation.

A mild selective oxidant of erythrocyte membrane SH-groups producing SS-bonds and inducing reversible crosslinking of spectrin, as well as strong less specific oxidants which produce a more extensive modification of membrane proteins and peroxidation of lipids and dielectric breakdown of the membrane induce formation of structural defects acting as aqueous pores and reorientation sites for phospholipids. The coupling of a process requiring hydrophilic structures (leak permeability) and a process also involving hydrophobic constituents (flip of phospholipids) suggests that the membrane lipid domain is involved in the effects.