Kinetic parameters of porin incorporation into black lipid membranes of oxidized cholesterol evaluated by means of alternating current

In this work we report the results of systematical investigation of the time course of porin incorporation into bilayer membranes made of oxidized cholesterol using an ac method (E. Gallucci et al., Biophys. J. 71: 824-831, 1996). This method has the advantages of continuously monitoring resistance and capacitance as a function of time during pore formation until a steady state of the phenomenon is reached. In our case the bilayer membranes are represented, as usual, by an equivalent electrical circuit consisting of one resistance and one capacitance connected in parallel. The total current passing through the membranes is easily monitored, during the insertion process and is interpreted as a probe of the process involved during channel incorporation and assembly. By means of a simple mathematical model, we provide evidence that two concurrent processes are present during the incorporation time course which can be interpreted either as insertion/deinsertion or as positive/negative cooperativity. We observe a phase transition (or similae phenomenon) which seems to take place during the insrtion process, an aspect which has been neglected in most previous investigation of insertion into bilayer membranes. Moreover, the continuous monitoring of capacitance may prove useful in tracking membrane parameters, such as thickness and dielectrical constant, that depend on the lipid and incorporated proteins.