Channel reconstitution in planar lipid bilayer membranes

Porins are a major class of proteins found in the outer membrane of gram-negative bacteria and mitochondria. These molecules form water-filled channels and act selectively on the permeation of hydrophilic solutes. Pore forming properties have been studied in reconstitution experiments with planar lipid bilayer membrane. The artificial membranes were formed with phosphatidylinositol (PI) in n-decane brushing the solution on a small circular hole of a teflon cell. PI was extracted from ox brain and porins were purified by mitochondria of different tissues of mammalia. Ionic soluctions were KCl 0.5-1 M and in some experiments CaCl2 1-10 mM was added to both compartments. Porin incorporation has been studied by means of alternate current (f = 1 Hz) recording the voltage after a current to voltage converter in series with the membrane. Electrical capacitance of the system was measured at 1 kHz at some times during porin incorporation. The membrane capacitance resulted 307.5 ± 24.1 (n = 5) and 225.3 ± 3.8 (n = 7) nF/cm2 in KC1 0.5 and 1 M respectively. The electrical resistance was calculated by using data from measurements in alternate current and from direct current measurements too. The addition of porins to the aqueous phases separated by a completely black membrane resulted in an increase of transmembrane current. As time passed the current amplitude increased, indicating protein fusion and pore formation into the lipid matrix. The current time behaviour was S-shaped. A time lag was observed between membrane formation and the first appreciable membrane current variation when porin was already present in aqueous solution prior to the formation of the membrane. This time varied in different experiments and decreased the higher were porin concentration values. Faster incorporation kinetics were observed increasing porin concentration or the applied voltage. The presence of calcium in bathing solution reduced the dependence on the voltage. Experimental data were fitted with a four-parameter logistic function and the different curves compared using the obtained constants (maximum dVl/dt, steady state value, etc.). Analytical time derivative of electrical potential was calculated from the obtained function and the transmembrane current calculated.