COMPARISON OF PORIN INCORPORATION KINETICS IN PLANAR BLACK LIPID MEMBRANES OF OXIDIZED CHOLESTEROL AND PHOSPHATIDYLINOSITOL

Porin molecules (the mitochondrial outer membrane proteins) when reconstituted into planar lipid bilayer membranes (BLMs) form voltage-dependent channels. In this work we studied the roles of protein concentration and of the applied voltage on the porin incorporation kinetics in: 1) negatively charged BLMs of phosphatidylinositol (PI), 2) BLMs of oxidized cholesterol (OxCh). The porin, purified from heart beef mitochondria, was kindly given by Prof. F. Palmieri. BLMs were obtained as previously reported (Proc. X School Biophys. Memb. Transp. 1:328-343, Poland 1990). Bathing media were KCI 1 M; at membrane black, the porin was added to the stirred aqueous solutions (24±2 °C) and the channel formation studied by means of alternate current. The conductances and capacitances of BLMs were: PI) 2.27±0.09 μS/cm2, 251.0±9.3 nF/cm2 (n=61); OxCh) 4.77±0.53 μS/cm2, 422.0±23.5 nF/cm2 (n=30). With PI BLMs the addition of porin yields an S-shaped increase of the current under a fixed set of experimental parameters (applied voltage, salt concentration, temperature). Besides the porin concentration also the applied voltage affects the incorporation kinetics. We observed that increasing the voltage, between 20-40 mV, the magnitude of the conductance increases too; at lower and higher voltages the kinetics of incorporation are lower. The S-shaped kinetics is preserved at all the porin concentrations and voltages tested. Porin incorporation kinetics in OxCh membranes follow an hyperbolic function and, as for PI, the incorporation is a function of the porin concentration and of the applied voltage. The increase and the behaviour were different, in that PI BLMs needed as much as ten times the porin concentration to reach the maximum value of the membrane current as compared to OxCh BLMs. The differences between the two types of kinetics persist when the protein is already present in bathing solutions before the membrane formation either in PI or in OxCh BLMs. However the incorporation kinetics in OxCh start with an elevated conductance and continue to increase through hyperbolic curves whereas in PI BLMs the S-shaped kinetics are shifted on the right. It could be assumed that the protein incorporation into the two different BLMs is governed by the different lipid matrix. In particular, the surface charge of the BLM and the interface potential, due to the local accumulation of ions at the membrane-solution interface, play a key role in the incorporation of porin in PI BLMs.