大肠杆菌机械敏感性离子通道MscS失活特性分析

Analysis of inactivation characteristics of Escherichia coli mechanosensitive ion channel MscS

摘要:

[目的]细菌机械敏感性离子通道MscS能够在细菌周围环境渗透压急剧降低时,打开并释放胞内内容物,平衡内外渗透压差,使细菌存活。鉴于其广泛分布在各种细菌中,而在哺乳动物中未发现其同源体,MscS被认为是一种新型抗生素靶点。MscS一个独特的开放特征是具有失活特性,即在持续的机械刺激条件下,MscS从开放状态进入一种非离子通透的失活状态,从而避免因通道持续开放引起大量内容物流失导致细菌死亡。该研究的目的是鉴定影响MscS失活的关键氨基酸,为靶向MscS的药物设计提供思路。[方法]采用分子克隆方法制备MscS Cyto-helix(P166−I170)半胱氨酸突变体,利用巯基化合物MTSET⁺结合半胱氨酸从而对其侧链基团进行修饰,并通过低渗刺激实验,检测表达MscS半胱氨酸突变体的大肠杆菌分别在无或有MTSET⁺处理下,低渗刺激诱发通道开放后的存活率筛选显著影响通道功能的突变体。利用电生理膜片钳方法检测突变体在MTSET⁺处理前后通道失活特性的变化,结合定点突变手段进一步探讨失活机制。[结果]MTSET⁺处理导致表达半胱氨酸突变体G168C-MscS的大肠杆菌在低渗刺激后存活率极大降低;G168C- MscS在结合MTSET⁺后失去失活特性,保持持续开放,是导致细菌胞内内容物大量流失并死亡的重要原因;酪氨酸突变G168Y-MscS、亮氨酸突变G168L-MscS和赖氨酸突变G168K-MscS的失活特性与野生型WT-MscS一致,而天冬氨酸突变G168D、缬氨酸突变G168V和异亮氨酸突变G168I的失活速率显著降低,尤其是G168I-MscS失去失活特性,表明MscS 168位点是影响通道失活的关键位点,并且通道失活特性与该位点氨基酸侧链基团的大小及电荷性质相关。[结论]G168位点甘氨酸是影响MscS通道失活的关键氨基酸。

Abstract:

[Objective]The mechanosensitive channel of small conductance (MscS) in bacteria releases solutes and water when a hypo-osmotic shock raises the pressure in the cells,thereby enabling the survival of bacteria.Given its wide distribution in various bacteria and no homologues found in mammals,MscS is considered a novel antibiotic target.A hallmark of MscS is that it enters a tension-insensitive inactivated state upon prolonged mechanical stimulation,thereby avoiding the loss of a large amount of cell content and preventing cell death.This study aims to identify the key residues related to the inactivation of MscS,which is expected to serve as a reference for the development of MscS-targeting drugs.[Methods] The cysteine mutants of MscS Cyto-helix (P166−I170) were prepared with molecular cloning method.The thiol compound MTSET⁺ binds to cysteine and thus modify cysteine's side chain group.In this study,osmotic downshock assay was used to examine the viability of Escherichia coli expressing cysteine mutants of MscS Cyto-helix (P166−I170) upon hypotonic stimulation without or with MTSET⁺ treatment and screened for cysteine mutant that significantly affected the channel function.The inactivation of MscS mutants before and after MTSET⁺ treatment was examined by electrophysiological experiments.In addition,the inactivation mechanism of MscS was further explored by eletrophysiology combined with site-directed mutagenesis.[Results] MTSET⁺ led to a great decrease in the survival rate of E.coli expressing G168C-MscS upon hypotonic stimulation.G168C-MscS lost its inactivation property after binding to MTSET⁺ and remained open,resulting in great loss of intracellular contents and bacterial death.The inactivation properties of G168Y-MscS,G168L-MscS,and G168K-MscS mutants were consistent with WT-MscS,while the inactivation rates of the three mutants G168D,G168V,and G168I were significantly reduced,especially G168I-MscS which lost the inactivation properties.Therefore,MscS G168 affected channel inactivation,and the channel inactivation characteristics were related to the size and charge of the residue side chain group at 168 site.[Conclusion]MscS G168 is a key residue that affects the inactivation of MscS.