Micelli Silvia, Gallucci Enrico, Meleleo Daniela, Stipani Valentina, Picciarelli Vittorio
Dipartimento Farmaco-Biologico, Facoltà di Farmacia, Università degli Studi di Bari, v.E. Orabona 4, 70125, Bari, Italy.
Bioelectrochemistry. 2002 Sep;57(2):97-106. doi: 10.1016/s1567-5394(02)00003-8.
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 microm. 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.
我们展示了一种新型交流设备，它可用于同时测量掺入两种由磷脂酰肌醇（带电表面）和氧化胆固醇（中性表面）制成的黑色脂质膜（BLM）中的线粒体孔蛋白通道的离子电荷移动（电导）和门控电荷位移（电容）。特别是，我们研究了在不同孔蛋白浓度下孔蛋白掺入（电压依赖性阴离子通道，VDAC）过程中的电导/电容变化。在所研究的孔蛋白浓度范围内均存在电导变化，但似乎需要一个阈值才能检测到显著的电容变化。对于磷脂酰肌醇双层膜，电导和电容均达到清晰的稳态，而对于氧化胆固醇膜，仅电导达到稳态。在孔蛋白掺入之前和离子电流稳态时，研究了电容特性对膜施加电压V(m)的依赖性。所得结果证实，在孔蛋白掺入之前，对V(m)(2)的依赖性较小，而之后我们发现了双指数电压依赖性的证据（这一结果与电导的情况相似）。最后，我们研究了电容对测量中所用细胞两个隔室之间分隔孔半径的依赖性。在这项仅使用氧化胆固醇进行的研究中，半径从200微米变化到1050微米。我们观察到比电容有显著变化，特别是对于较小的半径。通过考虑圆环影响的简单几何模型对结果进行了解释。
