Department of Molecular Physiology and Biophysics, Faculty of Medical Sciences, University of Fukui, 910-1193 Fukui, Japan.
Department of Molecular Physiology and Biophysics, Faculty of Medical Sciences, University of Fukui, 910-1193 Fukui, Japan
Proc Natl Acad Sci U S A. 2018 Dec 18;115(51):13117-13122. doi: 10.1073/pnas.1812282115. Epub 2018 Dec 3.
Molecular mechanisms underlying channel-membrane interplay have been extensively studied. Cholesterol, as a major component of the cell membrane, participates either in specific binding to channels or via modification of membrane physical features. Here, we examined the action of various sterols (cholesterol, epicholesterol, etc.) on a prototypical potassium channel (KcsA). Single-channel current recordings of the KcsA channel were performed in a water-in-oil droplet bilayer (contact bubble bilayer) with a mixed phospholipid composition (azolectin). Upon membrane perfusion of sterols, the activated gate at acidic pH closed immediately, irrespective of the sterol species. During perfusion, we found that the contacting bubbles changed their shapes, indicating alterations in membrane physical features. Absolute bilayer tension was measured according to the principle of surface chemistry, and inherent bilayer tension was ∼5 mN/m. All tested sterols decreased the tension, and the nonspecific sterol action to the channel was likely mediated by the bilayer tension. Purely mechanical manipulation that reduced bilayer tension also closed the gate, whereas the resting channel at neutral pH never activated upon increased tension. Thus, rather than conventional stretch activation, the channel, once ready to activate by acidic pH, changes the open probability through the action of bilayer tension. This constitutes a channel regulating modality by two successive stimuli. In the contact bubble bilayer, inherent bilayer tension was high, and the channel remained boosted. In the cell membrane, resting tension is low, and it is anticipated that the ready-to-activate channel remains closed until bilayer tension reaches a few millinewton/meter during physiological and pathological cellular activities.
已经广泛研究了通道-膜相互作用的分子机制。胆固醇作为细胞膜的主要成分，既可以与通道特异性结合，也可以通过改变膜的物理特性来参与其中。在这里，我们研究了各种固醇（胆固醇、表胆固醇等）对典型钾通道（KcsA）的作用。使用混合磷脂组成（偶氮鞘磷脂）的油水滴双层（接触气泡双层）进行 KcsA 通道的单通道电流记录。在固醇的膜灌注过程中，酸性 pH 下的激活门立即关闭，而与固醇种类无关。在灌注过程中，我们发现接触气泡改变了它们的形状，表明膜物理特性发生了变化。根据表面化学原理测量绝对双层张力，固有双层张力约为 5 mN/m。所有测试的固醇都降低了张力，并且固醇对通道的非特异性作用可能是通过双层张力介导的。减少双层张力的纯机械操作也会关闭门，而中性 pH 下的静息通道在张力增加时永远不会激活。因此，通道不是通过传统的拉伸激活，而是通过双层张力的作用改变其开放概率，从而激活。这构成了通过两个连续刺激调节通道的方式。在接触气泡双层中，固有双层张力较高，通道保持增强。在细胞膜中，静息张力较低，预计在生理和病理细胞活动期间，当双层张力达到几毫牛顿/米时，准备激活的通道将保持关闭状态。
