Mitochondrial porin incorporation into black lipid membranes: ionic and gating contribution to the total current Silvia Micelli a, * , Enrico Gallucci a , Daniela Meleleo a , Valentina Stipani a , Vittorio Picciarelli b a Dipartimento Farmaco-Biologico, Facolta ` di Farmacia, Universita ` degli Studi di Bari, v.E. Orabona 4, 70125, Bari, Italy b Dipartimento Interateneo di Fisica, Universita ` degli Studi di Bari, 70125 Bari, Italy Received 12 September 2001; received in revised form 17 December 2001; accepted 21 December 2001 Abstract We present a new ac device useful for simultaneous measurements of ionic charge movement (conductance) and gating charge displacement (capacitance) in mitochondrial porin channels incorporated in two kinds of black lipid membranes (BLMs), made up of phosphatidylinositol (charged surface) and oxidized cholesterol (neutral surface). In particular, we investigated the conductance/capacitance variations during the process of porin incorporation (VDAC) at different porin concentrations. While conductance variations are present throughout the porin concentration range investigated, a threshold value seems to be necessary in order to detect a significant capacitance variation. A clear steady state in both conductance and capacitance is reached for the phosphatidylinositol bilayer, while for the oxidized cholesterol membranes, the steady state is reached only for the conductance. The dependence of capacitance characteristics on the membrane applied voltage V m is investigated before porin incorporation and at the ionic current steady state. The results obtained confirm that before porin incorporation, there is a small dependence on V m 2 , while afterwards we find evidence of a dual exponential voltage dependence (a result similar to that found for conductance). Finally, we investigated the capacitance dependence on the radius of the hole separating the two compartments of the cell used in the measurements. In this study, performed only with oxidized cholesterol, the radius was varied from 200 to 1050 Am. We observed a significant variation in the specific capacitance in particular for smaller radii. The results were interpreted by a simple geometrical model taking into account the influence of the torus. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Capacitance; Phosphatidylinositol; Oxidized cholesterol; Gating mechanism 1. Introduction As is well known, black lipid membranes (BLM) used as model membranes can be characterized by two electrical parameters: conductance and capacitance. The first is related to the ionic current through the bilayer, while the second has been associated to the gating current due to charge rear- rangements in the membrane when voltage-dependent ion channels have been incorporated [1,2]. While conductance, especially when related to incorporation of different pro- teins, has been widely investigated due to its evident relation to channel formation, there is much less data on capacitance, even though this would provide important new information on functional properties at a molecular level. Capacitance measurements, based on the discharge through a known resistor, have been mainly limited to black lipid membranes and used to gather information on thick- ness, dielectric properties, capacitance voltage dependence and capacitance dependence on the radius of the BLM [3– 17]. In the latter case, a simple model, based on the two contributions from the bilayer in the center of the aperture and the surrounding torus, has been proposed to interpret the data [17]. New techniques for capacitance measurements have been developed and used to collect data on the capacitance variation during membrane bilayer formation [18]. Interest in the simultaneous measurements of conduc- tance/capacitance has been growing recently and data have been collected on various characteristics of gating mecha- nism as a function of the applied voltage in the auditory cells [19], of the Shaker K + channel in Xenopus oocytes 1567-5394/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII:S1567-5394(02)00003-8 Abbreviations: BLM, black lipid membrane; VDAC, voltage-dependent anion-selective channel; ac, alternating current; PI, phosphatidylinositol; OxCh, oxidized cholesterol. * Corresponding author. Tel.: +39-80-5442-775; fax: +39-80-5442-796. E-mail address: micelli@farmbiol.uniba.it (S. Micelli). www.elsevier.com/locate/bioelechem Bioelectrochemistry 57 (2002) 97 – 106