PORIN INCORPORATION KINETICS IN PHOSPHATIDYLINOSITOL AND OXIDIZED CHOLESTEROL MEMBRANES

Conductivity of planar lipid bilayer membranes can be modulated by insertion of protein molecules (1). Mithocondrial porins form water filled pores in the outer membrane and these channels act selectively on the permeation of hydrophilic solutes. Aims of this research were: 1) the reconstitution of porin channels in planar lipid bilayer membranes, 2) the analysis and the comparison of porin incorporation kinetics in different lipid membranes: phosphatidylinositol (PI) and oxidized cholesterol, 3) the study of protein concentration effect on the incorporation kinetics, 4) the analysis of the channel reconstitution dependence on the applied voltage. The artificial membranes were formed, with lipid in n-decane (1% w/w), brushing the solution on a small circular hole between two compartments, 4 ml volume, of a teflon experimental chamber. The two compartments were filled with KCl 1M solution and continuously stirred. Pt electrodes in bathing solutions were connected to a current to voltage converter and to a sinusoidal voltage source (Vs=VS sin2nt; n=1Hz) so that the voltage at the converter (Vl) resulted directly related to the electrical current crossing the membrane (2). Experiments were performed at room temperature: 24 ± 1 °C. Membrane electrical conductances and capacitances were higher in oxidized cholesterol than in PI membranes. Addition of porin to the solutions bathing a completely black membrane induced an increase of membrane current with time indicating protein fusion and channel formation into the lipid matrix. In PI membranes the time course of current was S-shaped, irrespective of porin addition before or after membrane formation, but in the first case a delay in channel formation and a slower kinetics were observed (3). In oxidized cholesterol membranes the current time behaviours were S-shaped when porin was added after membrane formation but resulted of exponential type when porin was present before membrane formation. In this second case faster kinetics were observed and the incorporation started with an higher conductance value. The time lag between porin addition and the first current variation was shorter in oxidized cholesterol than in PI membranes. For a same porin concentration the incorporation kinetics were faster and higher steady levels were reached in oxidized cholesterol than in PI membranes. In both types of membranes porin incorporation rate decreased lowering porin concentration; five kinetics of porin incorporation (protein added after membrane formation) in oxidized cholesterol membranes are reported in figure. Besides the porin concentration also the applied voltage affects the incorporation kinetics and the channel formation. At a given porin concentration faster kinetics are recorded and higher steady levels are reached increasing the voltage to a critical value after which incorporation is less effective, the kinetics are slower and accompanied by lower steady levels. The voltage dependence of functional pore reconstitution was more evident in PI than in oxidized cholesterol membranes. In oxidized cholesterol membrane, at the steady state, nonlinear current-voltage characteristics were obtained. The conductance increases with the voltage to reach a critical value after which inactivation occurs. At a given applied voltage higher membrane conductances were calculated increasing porin concentration. Significative differences in the current-voltage relations were obtained depending on the absence or the presence of porin at the membrane formation. In this latter case the membranes had higher conductance values but, at the tested voltages, the range of variability was smaller. The nature of the lipid matrix can be considered responsible of the different protein incorporation; in particular the surface charge of the lipid bilayer membrane 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 planar bilayer lipid membranes. These observations suggest the possibility to insert protein channels in lipid layers built on a solid substrate finalized to realize organic semiconductors to be used as biosensor or transducers. Work supported in part by CNR grant 92.02202.CT14. 1) G. Monticelli - "Pore formation in planar lipid bilayer membranes" Proc. X School on Biophysics of Membrane Transport, Szczyrk (Poland) 1990, 1: 315-327. 2) G. Monticelli, E. Gallucci and S. Micelli - "Experimental data on incorporation of porin molecules in lipid bilayers" Proc. X School on Biophysics of Membrane Transport, Szczyrk (Poland) 1990, 1: 328-343. 3) G. Monticelli, E. Gallucci and S. Micelli - "Pore formation in lipid membranes" Proc. Int. Cong. Memb. and Memb. Processes, Chicago 1990, 1: 175-177