The role of external applied voltage on kinetic parameters of porin incorporation into black lipid membranes of oxidized cholesterol

The time course of porin incorporation into black lipid membranes of oxidized cholesterol, as a function of on external applied voltage, was studied by means of an ac method previously reported (E. Gallucci et al., Biophys. J. 71: 824-831, 1996). The kinetic parameters was derived by means of a simple mathematical model before described (D. Meleleo et al., SIF, 1998, Р 53). At the different voltages Vs examined, the kinetics remained unmodified in its cooperative behavior. The K₁ or growth rate parameter describing porin insertion or positive cooperativity seems to be independent of the applied voltage. The K2 or growth rate parameter related to porin deinsertion or negative cooperativity is strongly dependent on applied voltage. In particular K₂ decreases with increasing applied voltages (Vs). The v(tm) or value of the maximum depolarization rate, and the Vll(tm) or value of the output voltage when the derivative v(tm) reaches the maximum, both increase with applied voltage (Vs). The kinetic values found indicated that mitochondrial porin is not driven by potential in the membrane, but its insertion depends (for fixed porin concentration, temperature and pH) on the lipid-protein affinity. This result supports previous authors' findings of porin insertion into mitochondrial membranes and asymmetric lipid bilayers (H. Freitag et al., Eur.J. Biochem. 74:483-492, 1993; S.M. Gasser et al.,J. Biol. Chem. 25: 3427- 3430,1983, A. Viese et al., Biophys. J., 70:321-329,1996). On the other hand, as K₂ was found to be related with the conductance at the end of porin incorporation, its correlation with Vs reflects the voltage- dependence characteristics of the porin channel.