ANNEXIN-V AND VESICLE MEMBRANE ELECTROPORATION

The method of membrane electroporation (ME) has been used as an analyt ical tool to quantify the effect of membrane curvature on transient el ectric pore formation, and on the adsorption of the protein annexin V (M-r = 35,800) to the outer surface of unilamellar lipid vesicles (of radii 25 less than or equal to a/nm less than or equal to 200). Relaxa tion kinetic studies using optical membrane probes of the diphenylhexa triene type suggest that electric pore formation is induced by ionic i nterfacial polarization causing entrance of the (more polarizable) wat er into the lipid bilayer membrane yielding (hydrophobic and hydrophil ic) pore states with a mean stationary pore radius r(p)=0.35 (+/-0.05) nm. Extent and rate of ME, compared at the same induced transmembrane voltage, were found to decrease both with increasing vesicle radius a nd with increasing protein concentration. This 'inhibitory' effect of annexin V is apparently allosteric and saturates at about [AN(T)](sat) = 4 mu M annexin V for vesicles of a = 100 nm at 1 mM total lipid con centration, 0.13 mM total Ca2+ concentration and at T = 293 K. Data an alysis in terms of Gibbs area-difference-elasticity energy suggests th at the bound annexin V reduces the gradient of the lateral pressure ac ross the membrane. At [AN(T)](sat), about 20% of the vesicle surface i s covered by the bound protein, but it is only 0.01% of the surface of the outer lipid leaflet in which a part of the protein, perhaps the a romatic residue of the tryptophan (W 187), is inserted. Insertion lead s to a denser packing of the lipid molecules in the outer membrane lea flet. As a consequence, the radius of the electropores in the remainin g membrane part, not covered by annexin V decreases (r(p)/nm = 0.37, 0 .36 and 0.27) with increasing adsorption of the protein ([AN(T)] = 0, 2 and 4 mu M, respectively).