Victor Chang Cardiac Research Institute, Darlinghurst, Australia.
Biophys J. 2011 Apr 6;100(7):1635-41. doi: 10.1016/j.bpj.2011.02.016.
High hydrostatic pressure (HHP) present in natural environments impacts on cell membrane biophysical properties and protein quaternary structure. We have investigated the effect of high hydrostatic pressure on G22E-MscL, a spontaneously opening mutant of Escherichia coli MscL, the bacterial mechanosensitive channel of large conductance. Patch-clamp technique combined with a flying-patch device and hydraulic setup allowed the study of the effects of HHP up to 90 MPa (as near the bottom of the Marianas Trench) on the MscL mutant channel reconstituted into liposome membranes, in addition to recording in situ from the mutant channels expressed in E. coli giant spheroplasts. In general, against thermodynamic predictions, hydrostatic pressure in the range of 0.1-90 MPa increased channel open probability by favoring the open state of the channel. Furthermore, hydrostatic pressure affected the channel kinetics, as manifested by the propensity of the channel to gate at subconducting levels with an increase in pressure. We propose that the presence of water molecules around the hydrophobic gate of the G22E MscL channel induce hydration of the hydrophobic lock under HHP causing frequent channel openings and preventing the channel closure in the absence of membrane tension. Furthermore, our study indicates that HHP can be used as a valuable experimental approach toward better understanding of the gating mechanism in complex channels such as MscL.
高静水压力（HHP）存在于自然环境中，会影响细胞膜的生物物理性质和蛋白质四级结构。我们研究了高静水压力对 G22E-MscL 的影响，G22E-MscL 是大肠杆菌 MscL 的自发开放突变体，MscL 是细菌的机械敏感通道大电导。膜片钳技术结合飞行片装置和液压装置允许在脂质体膜中对重组的 MscL 突变通道进行高达 90 MPa（接近马里亚纳海沟底部）的 HHP 影响的研究，此外还可以从在大肠杆菌巨球体中表达的突变通道原位记录。一般来说，与热力学预测相反，0.1-90 MPa 范围内的静水压力通过有利于通道的开放状态而增加通道的开放概率。此外，静水压力会影响通道动力学，表现为通道在亚电导水平下的门控倾向随着压力的增加而增加。我们提出，水分子存在于 G22E MscL 通道的疏水门周围，在 HHP 下会导致疏水锁的水合作用，从而频繁打开通道，并防止在没有膜张力的情况下关闭通道。此外，我们的研究表明，HHP 可作为一种有价值的实验方法，用于更好地理解 MscL 等复杂通道的门控机制。
