Analysis of inactivation characteristics of Escherichia coli mechanosensitive ion channel MscS

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.