Electro-optics of membrane electroporation in diphenylhexatriene-doped lipid bilayer vesicles

• Published in: Biophysical chemistry Vol. 58; no. 1; pp. 109 - 116
• Main Authors: Kakorin, S., Stoylov, S.P., Neumann, E.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 16.01.1996
• Subjects: Chemical Phenomena; Chemistry, Physical; Diphenylhexatriene; Diphenylhexatriene - chemistry; Diphenylhexatriene - pharmacology; Electrochemistry; Electroporation; Electroporation - methods; Fluorescent Dyes - chemistry; Fluorescent Dyes - pharmacology; Kinetics; Lipid Bilayers - chemistry; Lipids; Mathematical Computing; Membrane Potentials - drug effects; Membrane Potentials - physiology; Membranes; Optics and Photonics; Turbidity dichroism; Vesicles; Turbidity dichroism; Electroporation; Lipids; Diphenylhexatriene; Vesicles; Membranes
• ISSN: 0301-4622; 1873-4200
• DOI: 10.1016/0301-4622(95)00090-9

The electric (linear) dichroisms observed in the membrane electroporation of salt-filled lipid bilayer vesicles (diameter Φ = 2 a = 0.32 μm; inside [NaCl] = 0.2 M) in isotonic aqueous 0.284 M sucrose-0.2 mM NaCl solution indicate orientation changes of the anisotropic light scattering centers (lipid head groups) and of the optical transition moments of the membrane-inserted probe 1,6-diphenyl-1,3,5-hexatriene (DPH). Both the turbidity dichroism and DPH absorbance dichroism show peculiar features: (1) at external electric fields E ≥ E sat the time course of the dichroism shows a maximum value (reversal): E sat = 4.0 (±0.2) MV m −1, T = 293 K (20°C), (2) this reversal value is independent of the field strength for E ≥ E sat, (3) the dichroism amplitudes exhibit a maximum value E max = 3.0 (±0.5) MV m −1, (4) for the pulse duration of 10 μs there is one dominant visible normal mode, the relaxation rate increases up to τ −1 ≈ 0.6 × 10 6 s −1 at E sat and then decreases for E > E sat. The data can be described in terms of local lipid phase transitions involving clusters L n of n lipids in the pore edges according to the three-state scheme C ⇌ HO ⇌ HI, C being the closed bilayer state, HO the hydrophobic pore state and HI the hydrophilic or inverted pore state with rotated lipid and DPH molecules. At E ≥ E sat, further transitions HO ⇌ HO ∗ and HI ⇌ HI ∗ are rapidly coupled to the C ⇌ HO transition, which is rate-limiting. The vesicle geometry conditions a cosθ dependence of the local membrane field effects relative to the E direction and the data reflect cosθ averages. The stationary induced transmembrane voltage Δϕ( θ, λ m) = − 1.5 aEf( λ m| cosθ| does not exceed the limiting value Δϕ sat = − 0.53 V, corresponding to the field strength E m,sat = − Δϕ sat d = 100 MV m −1 (10 3 kV cm −1), due to increasing membrane conductivity λ m. At E = E sat, f( λ m) = 0.55, λ m = 0.11 mS m −1. The lipid cluster phase transition model yields an average pore radius of r p = 0.35 (±0.05) nm of the assumed cylindrical 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 of DPH molecules in pore states approaches a saturation value; the fraction of DPH molecules in HI pores is 12 (±2)% and that in HO pores is 48 (±2)%. The percentage of membrane area P ≈ ( λ m λ i ) × 100 % of conductive openings filled with the intravesicular medium of conductance λ i = 2.2 S m − linearly increases from P ≈ 0% ( 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 basis of fluorescence imaging data for sea urchin eggs give the same order 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 exhibit a sigmoid field dependence. Therefore our data suggest that it is only the HI and HI ∗ pore states which are conductive. It is noted that the various peculiar features of the dichroism data cannot be described by simple whole particle deformation.