Mitochondrial porin incorporation in different black lipid membranes and its gating mechanism studied by means of alternating current

The kinetics of pore formation by mitochondrial porin in black lipid membranes of phosphatidylinositol and oxidized cholesterol was investigated as a function of external applied voltage. During the insertion process, the total current passing through the membrane is easily monitored by means of an ac device, and is interpreted as a probe of the process involved during channel incorporation and assembly. The time course of the total current flowing through the membrane was well-described by means of a mathematical model, that gives kinetic parameters describing two concurrent processes which can be interpreted as positive/negative cooperativity. In the growth rate of pore incorporation we observe a process similar to a phase transition taking place at a critical time, an aspect which has been neglected in most previous investigations of insertion into model membranes. The behavior around the instant of transition phase is compatible with what is typically expected in other kinetics growth processes. By using data obtained under steady state conditions, with a well-defined number of pores inserted into the membranes, the conductance measurements provided indirect indications of two possible gating mechanisms. Moreover, our measurement device makes it easy to acquire information on the capacitance characteristics of black membranes. While before porin incorporation we do not observe a significant capacitance dependence on the external applied voltage, afterwards we find evidence of a dual-exponential voltage dependence of capacitance, a result similar to that found for conductance.