ABSTRACT

Mechanosensitive (MS) ion channels are widespread mechanisms for cellular mechanosensation that can be directly activated by increasing membrane tension. The well-studied MscS family of MS ion channels is found in bacteria, archaea, and plants. MscS-Like (MSL)1 is localized to the inner mitochondrial membrane of Arabidopsis thaliana, where it is required for normal mitochondrial responses to oxidative stress. Like Escherichia coli MscS, MSL1 has a pore-lining helix that is kinked. However, in MSL1 this kink is comprised of two charged pore-lining residues, R326 and D327. Using single-channel patch-clamp electrophysiology in E. coli, we show that altering the size and charge of R326 and D327 leads to dramatic changes in channel kinetics. Modest changes in gating pressure were also observed while no effects on channel rectification or conductance were detected. MSL1 channel variants had differing physiological function in E. coli hypoosmotic shock assays, without clear correlation between function and particular channel characteristics. Taken together, these results demonstrate that altering pore-lining residue charge and size disrupts normal channel state stability and gating transitions, and led us to propose the “sweet spot” model. In this model, the transition to the closed state is facilitated by attraction between R326 and D327 and repulsion between R326 residues of neighboring monomers. In the open state, expansion of the channel reduces inter-monomeric repulsion, rendering open state stability influenced mainly by attractive forces. This work provides insight into how unique charge-charge interactions can be combined with an otherwise conserved structural feature to help modulate MS channel function.

摘要

机械敏感（MS）离子通道是细胞机械传感的广泛机制，可以通过增加膜张力直接激活。在细菌，古细菌和植物中发现了经过充分研究的MS离子通道MscS家族。MscS-​​Like（MSL）1位于拟南芥的内部线粒体膜上，在这里正常的线粒体对氧化应激的反应是必需的。像大肠杆菌MscS一样，MSL1具有一个扭曲的孔衬螺旋。但是，在MSL1中，该纽结由两个带电的孔衬残基R326和D327组成。在大肠杆菌中使用单通道膜片钳电生理，我们发现改变R326和D327的大小和电荷会导致通道动力学发生巨大变化。还观察到门控压力的适度变化，而未检测到对通道整流或电导的影响。MSL1通道变异体在大肠杆菌中具有不同的生理功能低渗休克试验，功能与特定通道特征之间无明显关联。综上所述，这些结果表明，改变孔隙衬里的残留电荷和大小会破坏正常的通道状态稳定性和门控过渡，并导致我们提出“最佳点”模型。在该模型中，R326和D327之间的吸引以及相邻单体的R326残基之间的排斥促进了向封闭状态的过渡。在打开状态下，通道的扩张会减少单体间的排斥，从而使打开状态的稳定性主要受到吸引力的影响。这项工作提供了有关如何将独特的电荷相互作用与其他保守结构特征结合以帮助调制MS通道功能的见解。