Effect of calcium ions on kinetic of porin pore formation in BL membranes

Porins are a major class of proteins found in the outer membrane of enteric bacteria and mitochondria. These proteins form water-filled transmembrane channels and the pore appears to be formed by a-sheet rather than b-helices. Porin molecules from mitochondria were reconstituted in bilayers of phosphatidylinositol dissolved (1 %) in n-decane and the kinetic of pore formation has been studied by means of alternate current (f = 1Hz). Ionic solutions bathing the membrane were KC1 1 M and the effect of calcium ions (1-10 mM) on pore formation has been tested. After addition of porins to the aqueous phases bathing a completely black membrane the transmembrane electrical current increases with time, the behaviour is S-shaped and the steady state value is reached after many minutes depending on porin concentration. Porin already present in aqueous solutions prior to the formation of the membrane resulted in a delay in channel formation and a slower kinetic was obtained. The rate of porin incorporation decreased in the presence of calcium ions and decreasing porin concentration. The kinetic of pore formation depends on the applied voltage. Experimental data were fitted with a four-parameter logistic equation. In control conditions transmembrane current was calculated considering the changes of membrane electrical resistance and capacitance during porin incorporation.