Laboratory of Physical and Structural Biology, Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland, USA.
Biophys J. 2010 Jul 7;99(1):134-43. doi: 10.1016/j.bpj.2010.03.070.
Single channels of Bacillus anthracis protective antigen, PA(63), were reconstituted into planar lipid membranes and their inhibition by cationic aminopropylthio-beta-cyclodextrin, AmPrbetaCD, was studied. The design of the highly efficient inhibitor, the sevenfold symmetrical cyclodextrin molecule chemically modified to add seven positive charges, was guided by the symmetry and predominantly negative charge of the PA(63) pore. The protective action of this compound has been demonstrated earlier at both single-molecule and whole-organism levels. In this study, using noise analysis, statistics of time-resolved single-channel closure events, and multichannel measurements, we find that AmPrbetaCD action is bimodal. The inhibitor, when added to the cis side of the membrane, blocks the channel reversibly. At high salt concentrations, the AmPrbetaCD blockage of the channel is well described as a two-state Markov process, in which both the on- and off-rates are functions of the salt concentration, whereas the applied voltage affects only the off-rate. At salt concentrations smaller than 1.5 M, the second mode of AmPrbetaCD action on the channel is discovered: addition of the inhibitor enhances voltage gating, making the closed states of the channel more favorable. The effect depends on the lipid composition of the membrane.
单个枯草芽孢杆菌保护性抗原（PA(63)）通道被重构成平面脂膜，并研究了阳离子氨基丙基硫基-β-环糊精（AmPrβCD）对其的抑制作用。这种高效抑制剂的设计，即七倍对称的环糊精分子被化学修饰以增加七个正电荷，是基于 PA(63) 孔的对称性和主要带负电荷的性质。该化合物的保护作用在单分子和整个生物体水平上都得到了早期证明。在这项研究中，我们使用噪声分析、时间分辨单通道关闭事件的统计和多通道测量，发现 AmPrβCD 的作用是双模态的。当抑制剂被添加到膜的顺式侧时，通道可以被可逆地阻断。在高盐浓度下，AmPrβCD 对通道的阻断可以很好地用两态 Markov 过程来描述，其中 ON 和 OFF 速率都是盐浓度的函数，而施加的电压仅影响 OFF 速率。在盐浓度小于 1.5 M 时，发现了 AmPrβCD 对通道的第二种作用模式：添加抑制剂增强了电压门控，使通道的关闭状态更加有利。这种效应取决于膜的脂质组成。
