Pore formation in lipid bilayer membranes made of phosphatidylinositol and oxidized cholesterol followed by means of alternating current.

The kinetics of porin incorporation into black lipid membranes (BLM) made of phosphatidylinositol (PI) or oxidized cholesterol (Ox Ch) were studied by means of alternating current; the set-up was able to acquire resistance and capacitance simultaneously by means of a mixed double-frequency approach at 1 Hz and 1 KHz, respectively. Conductance was dependent on the interaction between protein-forming pores and lipids. For PI membranes below a porin concentration of 12.54 ng/ml, there was no membrane conductivity, whereas at 200 ng/ml a steady-state value was reached. Different behavior was displayed by Ox Ch membranes, in which a concentration of 12.54 ng/ml was sufficient to reach a steady state. The incorporation kinetics when porin was added after membrane formation were sigmoidal. When porin was present in the medium before membrane formation, the kinetics were sigmoidal for PI membranes but became exponential for Ox Ch membranes. Furthermore, for BLM made of PI, the conductance-versus-porin concentration relationship is sigmoidal, with a Hill coefficient of 5.6 +/- 0.07, which is functional evidence corroborating the six-channel repeating units seen previously. For BLM made of Ox Ch, this relationship followed a binding isotherm curve with a Hill coefficient of 0.934 +/- 0.129.