ELECTROOPTICS OF MEMBRANE ELECTROPORATION IN DIPHENYLHEXATRIENE-DOPEDLIPID BILAYER VESICLES

The electric (linear) dichroisms observed in the membrane electroporat ion of salt-filled lipid bilayer vesicles (diameter O = 2a = 0.32 mu m ; inside [NaCl] = 0.2 M) in isotonic aqueous 0.284 M sucrose-0.2 mM Na Cl solution indicate orientation changes of the anisotropic light scat tering centers (lipid head groups) and of the optical transition momen ts of the membrane-inserted probe 1,6-diphenyl-1,3,5-hexatriene (DPH). Both the turbidity dichroism and DPH absorbance dichroism show peculi ar features: (1) at external electric fields E greater than or equal t o E(sat) the time course of the dichroism shows a maximum value (rever sal): E(sat) = 4.0 (+/- 0.2) MV m(-1), T = 293 K (20 degrees C), (2) t his reversal value is independent of the field strength for E greater than or equal to E(sat), (3) the dichroism amplitudes exhibit a maximu m value E(max) = 3.0 (+/- 0.5) MV m(-1), (4) for the pulse duration of 10 mu s there is one dominant visible normal mode, the relaxation rat e increases up to tau(-1) approximate to 0.6 X 10(6) s(-1) at E(sat) a nd then decreases for E > E(sat). The data can be described in terms o f local lipid phase transitions involving clusters L(n) of n lipids in the pore edges according to the three-state scheme C reversible arrow HO = HI, C being the closed bilayer state, HO the hydrophobic pore st ate and HI the hydrophilic or inverted pore state with rotated lipid a nd DPH molecules. At E greater than or equal to E(sat) further transit ions HO reversible arrow HO and HI reversible arrow HI* are rapidly c oupled to the C reversible arrow HO transition, which is rate-limiting . The vesicle geometry conditions a cos theta dependence of the local membrane field effects relative to the (E) over right arrow direction and the data reflect cos theta averages. The stationary induced transm embrane voltage Delta phi(theta, lambda(m)) = - 1.5aEf(lambda(m))/cos theta/ does not exceed the limiting value Delta phi(sat) = - 0.53 V, c orresponding to the field strength E(m),(sat) = - Delta phi(sat)/d = 1 00 MV m(-1) (10(3) kV cm(-1)), due to increasing membrane conductivity lambda(m). At E = E(sat), f(lambda(m)) = 0.55, lambda(m) = 0.11 mS m( -1). The lipid cluster phase transition model yields an average pore r adius of (r) over bar(p) = 0.35 (+/- 0.05) nm of the assumed cylindric al pore of thickness d = 5 nm, suggesting an average cluster size of [ n] = 12 (+/-2) lipids per pore edge. For E > E(sat) the total number o f DPH molecules in pore states approaches a saturation value; the frac tion of DPH molecules in HI pores is 12 (+/-2)% and that in HO pores i s 48 (+/-2)%. The percentage of membrane area P approximate to (lambda (m)/lambda(i)) X 100% of conductive openings filled with the intravesi cular medium of conductance lambda(i) = 2.2 S m(-1) linearly increases from P approximate to O% (E = 1.8 MV m(-1)) to P = 0.017% (E = 8.5 MV m(-1)). Analogous estimations made by Kinosita et al. (1993) on the b asis of fluorescence imaging data for sea urchin eggs give the same or der of magnitude for P (0.02 - 0.2%). The increase in P with the field strength is collinear with the increase in concentration of HI and HI states with the field strength, whereas the HO and HO* states exhibi t a sigmoid field dependence. Therefore our data suggest that it is on ly the HI and HI pore states which are conductive. It is noted that t he various peculiar features of the dichroism data cannot be described by simple whole particle deformation.