Assessment of potential stimuli for mechano-dependent gating of MscL: effects of pressure, tension, and lipid headgroups
Assessment of potential stimuli for mechano-dependent gating of MscL: effects of pressure, tension, and lipid headgroups
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DOI：
10.1021/bi0509649
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发表时间：
2005-09-13
期刊：
影响因子：
2.9
通讯作者：
Blount, P
中科院分区：
文献类型：
--
作者：
Moe, P;Blount, P
MScL 是一种大电导的机械敏感通道，充当“紧急泄压阀”，保护细菌免受急性低渗应激。尽管人们普遍认为，MscL 蛋白和足够的膜基质对于该通道的功能是必要且充分的，但膜的确切作用尚未阐明。在这里，我们通过在确定的脂质双层中体外重建 MscL 蛋白来解决膜基质的作用。我们将拉普拉斯定律应用于可视化的膜片，我们可以测量膜片的曲率，如先前研究中所述。在这里，通过比较具有不同曲率的贴片，我们证明 MscL 通道感知膜内的张力，并且穿过它的压力作为刺激没有起到可检测的作用。此外，门控仅在接近达到最小曲率半径时发生，这表明侧向张力而不是膜曲率是重要的生物物理参数。最后，我们通过测量特定头基对通道门控所需的膜张力的影响来检查特定头基的贡献。我们发现，向非天然膜添加阴离子脂质或内源脂质都不会影响激活曲线的左移。事实上，大肠杆菌膜的主要内源脂质磷脂酰乙醇胺导致通道在较高张力阈值下活动，表明这种脂质通过膜生物物理特性的变化而不是通过 MscL 特异性相互作用来改变活性。
MscL is a mechanosensitive channel of large conductance that serves as an "emergency relief valve", protecting bacteria from acute hypoosmotic stress. Although it is well-accepted that the MscL protein and an adequate membrane matrix are necessary and sufficient for the function of this channel, the exact role of the membrane has yet to be elucidated. Here, we address the role of the membrane matrix through in vitro reconstitution of the MscL protein in defined lipid bilayers. We have applied Laplace's law to visualized membrane patches where we can measure patch curvature as described in previous studies. Here, by comparing patches with different curvatures, we demonstrate that the MscL channel senses tension within the membrane and that the pressure across it plays no detectable role as a stimulus. In addition, gating only occurs when the smallest radius of curvature is nearly achieved, suggesting that the lateral tension rather than membrane curvature is the important biophysical parameter. Finally, we have examined the contribution of specific headgroups by measuring their effect on the membrane tension required to gate the channel. We have found that the addition of neither anionic nor endogenous lipids to a non-native membrane effected a leftward shift in the activation curve. In fact, the major endogenous lipid of the Escherichia coli membrane, phosphatidylethanolamine, led to a channel activity at a higher tension threshold, suggesting that this lipid effects altered activity through changes in the biophysical properties of the membrane, rather than through an MscL-specific interaction.