Pore formation in BLM studied by means of alternate current

Pores in biomembranes can be created through exogenous agents which act on the membrane. Many cytolytic peptides, proteins and toxins function by creating nonselective and selective pores within the plasma membrane of a targeted cell. Native proteins or exogenous agents can be tested or reconstituted into lipid bilayer membranes of different composition providing valuable information on their properties when acting as a channel or a pore. The incorporation kinetics of these substances in planar black lipid membranes can be studied by means of alternate current. Membranes are formed brushing a solution of a lipid in solvent on a small circular hole (1.3 mm diameter) in the wall between two compartments of a Teflon chamber. After blacking of the membrane the channel forming molecules are added to one or both compartments separated by the membrane. Pt electrodes are inserted in both aqueous compartments switched in series with a sinusoidal source (n=1 Hz) and a current to voltage converter. Voltage time behaviour at the converter is monitored with a storage oscilloscope and recorded on a strip chart recorder. With this experimental set up the membrane is in series with the circuit of the measuring device, constituted by a parallel resistance and capacitance, and the input sinusoidal voltage (Vssinwt; w = 2pn) is applied to the whole series. Electrical capacitance of the system can be measured (at 1 KHz) at some times during molecule incorporation into membrane. Without membrane the system has been tested with KCl 1M solution in both compartments. The stray capacitance was less than 100 pF and the resistance was 12.6 ± 0.0 (n = 40) Kohm when working with Vs = 40 mV. The value of this calculated electrical resistance did not vary assuming the capacitance in the range 1-200 pF. The phase angle was 0.553 ± 0.000 (n = 40) rad. Membranes were formed from phosphatidylinositol in n-decane (1% w/v). With KCl 1M the membrane electrical resistance and capacitance were 35.8 ± 1.3 (n = 80) Mohm and 3.5 ± 0.1 (n = 80) nF respectively with a phase angle of 0.627 ± 0.016 (n = 80) rad. Because of the different order of magnitude of electrical parameters, with or without the membrane, a simplified equivalent electrical circuit, a two element series of parallel resistance and capacitance (the membrane and the measuring device), has been used to analyze the observed phenomena.