PORIN PORE FORMATION: DEPENDENCE ON CONCENTRATION AND VOLTAGE IN BLM

The mitochondrial outer membrane protein called “porin”, when reconstituted into planar lipid bilayer membranes (BLM) forms channels and generates voltage-dependent ion conductances In this report the roles of protein concentration and of the applied voltage on the porin incorporation kinetics in a negatively charged BLM of phosphatidylinositol (PI) have been studied. BLM were obtained by the usual technique previously reported (Proc. X School Biophys. Memb. Transp. 1: 328, Poland, 1990). The porin, from heart beef mitochondria, was kindly given by Prof. F. Palmieri. Bathing media were KC1 1M and, at membrane black, the porin was added to the stirred aqueous solutions (24 ± 2° C). In all the experiments porin incorporation has been studied by means of alternate current. The membrane conductance and capacitance were 2.27 ± 0.09 μS/cm2 (n=61) and 251.0 ± 9.3 nF/cm2 (n=61) respectively. Addition of porin to the solutions in contact with BLM, yields an increase of the current under a fixed set of experimental parameters (as applied voltage, salt concentration, temperature). This implies that a cooperative process is involved in the incorporation of the porin in PI membrane. 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. To a first approximation the S-shaped kinetics is preserved at all the porin concentrations and voltages tested. At least two features characterize the porin incorporation in PI BLMs: a) the surface charge of the BLM and the resulting interface potential due to the local accumulation of the ions at the membrane-solution interface; b) the hydrocarbon interior of the bilayer.