Unidirectional incorporation of a bacterial mechanosensitive channel into liposomal membranes
Unidirectional incorporation of a bacterial mechanosensitive channel into liposomal membranes
复制标题
DOI：
10.1096/fj.15-275198
复制
发表时间：
2015-10-01
期刊：
影响因子：
4.8
通讯作者：
Martinac, Boris
中科院分区：
文献类型：
--
作者：
Nomura, Takeshi;Cox, Charles D.;Martinac, Boris
细菌小电导机械敏感通道(MSCs)在保护细菌细胞抵抗低渗休克中起着至关重要的作用。利用脂质体重建法对MSCs的功能特性进行了广泛的研究。这是一种广泛使用的实验范式，对于机械敏感通道尤其重要，因为可以不受细胞骨架的影响来探测通道活动。使用此范例遇到的一个永久问题是未知的渠道定位。在这里，我们利用膜片钳电生理学、共聚焦显微镜和连续介质力学模拟的强大组合，研究了使用2种离子通道重建方法形成的脂质体中MSCs的取向。利用先前确定的MSCs的电生理和药理学特性，我们能够确定在脂质体中，MSCs采用与天然膜中相同的取向，而与脂类成分无关。这些结果有力地支持了这一观点，即这些特定的方法导致了膜离子通道的均匀掺入，并告诫不要仅使用刺激类型来假设机械敏感通道的取向。
The bacterial mechanosensitive channel of small conductance (MscS) plays a crucial role in the protection of bacterial cells against hypo-osmotic shock. The functional characteristics of MscS have been extensively studied using liposomal reconstitution. This is a widely used experimental paradigm and is particularly important for mechanosensitive channels as channel activity can be probed free from cytoskeletal influence. A perpetual issue encountered using this paradigm is unknown channel orientation. Here we examine the orientation of MscS in liposomes formed using 2 ion channel reconstitution methods employing the powerful combination of patch clamp electrophysiology, confocal microscopy, and continuum mechanics simulation. Using the previously determined electrophysiological and pharmacological properties of MscS, we were able to determine that in liposomes, independent of lipid composition, MscS adopts the same orientation seen in native membranes. These results strongly support the idea that these specific methods result in uniform incorporation of membrane ion channels and caution against making assumptions about mechanosensitive channel orientation using the stimulus type alone.