Interaction Between The Transmembrane Domain TM3 And The Cytoplasmic Domain Cyto-helix Of The Mechanosensitive Channel MscS And Its Function

The ability to sense and respond to the intracellular or extracellular mechanical stimulus is critical for all life.Mechanosensation is involved in diverse physiological processes,including bacterial division,plant growth,blood pressure control,touching,hearing and pain sensation.Mechanosensitive channels,a specialized class of membrane-spanning protein complexes,could transduce a variety of mechanical stimuli into electrochemical signals by changing their molecular conformation,and play an important role in mechanotransduction.The study of the gating mechanism of mechanosensitive channels is essential for the process of mechanotransduction.Compared to the mechanosensitive channels in eukaryotes,prokaryotic mechanosensitive channels could serve as an excellent model for experimental analysis due to their simple structure and clear function.Among them,Mechanosensitive channel of Small conductane(MscS)from Escherichia coli is an example.When the extracellular osmolarity is dramatically decreased,MscS senses membrane tension resulting from swelling and functions as safety valves to alleviate cell turgor by releasing osmolytes and,thereby,prevents cell lysis.MscS opens a large pore(?lnS)in direct response to tension in lipid bilayer.X-ray crystallography shows that MscS is a homoheptamer complex,and each subunit composed of 286 amino acids contains three transmembrane domains(TM1,TM2 and TM3)and a large cytoplasmic domain.Seven highly conserved TM3 helices line the pore of MscS,and play an important role in gating MscS.The cytoplasmic domain encloses a large vestibule that is perforated by seven lateral portals and an axial portal formed by the ? barrel.The cytoplasmic domain is considered to have a role in channel ion selectivity,opening conductance and adaptation.Previous studies have showed that during MscS opening,both TM3 and the cytoplasmic domain undergo large conformational changes.However,whether the cytoplasmic domain could interact with TM3 and how the gating of MscS could be regulated by the interaction between the cytoplasmic domain and TM3 remain unclear.We suggest that the two important domains are not isolated,instead,they interact with each other and regulate the opening of MscS commonly.In the crystal structure of MscS,TM3b helices located at the membrane/cytoplasmic interface serve as a bridge connecting the transmembrane domain and the cytoplasmic domain.In the cytoplasmic domain,an a helix is highly conserved and spatially closed to TM3b helix,and we refer the a helix as Cyto-helix.Here,we have investigated whether Cyto-helix interacts with TM3b and how such interhelical interaction regulates the opening of MscS by using the physiological experiments,electrophysiological single channel recording,cysteine disulfide trapping and calcein release assay.Considering the wild type E.coli MscS contains no endogenous cysteines,we generated a mutant library in which every amino acid in the TM3b helix and Cyto-helix had been sequentially replaced with cysteine and adopted the method of disulfide trapping and thiol reagents modifications of the substituted cysteines to study how the cysteine mutant influence the function of MscS.Through cysteine scanning,we found that when both amino acid of N117 in TM3b helix and the amino acid of N167 in Cyto-helix were mutated into cysteine simutaneously,E.coli cells expressing N117CN167C MscS failed to survive upon hypoosmotic shock.The electrophysiological result demonstrated that the double cysteine mutant N117CN167C MscS lost activity.Western Blot showed that when cells expressing N117CN167C MscS were osmotically downshocked in the presence of an oxidizer copper phenanthroline,multiple oligomers appeared,suggesting that disulfide cross-linking formed between N117C and N167C.We suggest that during MscS opening,N117C and N167C get closer to each other,and the formed disulfide bridges between N117C and N167C inhibited the opening of N117CN167C MscS.To test this hypothesis,we adopted the method of histidine coordinated by metal ions.The presence of Zn2+ could theoretically coordinate the histidine residues within the complex,and therefore lead to what was effectively cross linking at the specific sites mutated.The activity of N117HN167H MscS decreased after the treatment of Zn2+,suggesting that N117HN167H MscS could be coordinated by Zn2+.We further adopted the method of electrostatic attracting from opposite charges to validate our conclusion.N117R carries positive charge,and N167E carries negative charge.We found that the conductance of N117RN167E MscS was significantly decreased,suggesting that the attraction between N117R and N167E inhibit the full opening of N117RN167E MscS.The method of combination with GOF(gain-of-function)mutant also validate our conclusion that the interaction between N117 and N167 influence the function of MscS.L109S MscS is a strong GOF mutant,and opens spontaneously without mechanical force.The result showed that the conductance of L109SN117CN167C MscS is smaller than that of L109S MscS,suggesting that the existence of N117CN167C inhibited the channel fully opening.Moreover,the activity of L109SN117CN167C MscS almost disappeared after the oxidizer promotes the cross-linking of cysteines,which is consistent with our conclusion.Our study showed that during MscS opening,N117 and N167 get closer,and interact with each other.If N117 cross linked with N167,the channel activity will be suppressed.According to the results,we proposed a gating model for MscS.During MscS opening,TM3b helix and Cyto-helix firstly get closer to each other and then move apart,which provides insight into the gating mechanism of MscS.