title: C-terminal charged cluster of MscL, RKKEE, functions as a pH sensor.
abstract: The RKKEE cluster of charged residues located within the cytoplasmic helix of the bacterial mechanosensitive channel, MscL, is essential for the channel function. The structure of MscL determined by x-ray crystallography and electron paramagnetic resonance spectroscopy has revealed discrepancies toward the C-terminus suggesting that the structure of the C-terminal helical bundle differs depending on the pH of the cytoplasm. In this study we examined the effect of pH as well as charge reversal and residue substitution within the RKKEE cluster on the mechanosensitivity of Escherichia coli MscL reconstituted into liposomes using the patch-clamp technique. Protonation of either positively or negatively charged residues within the cluster, achieved by changing the experimental pH or residue substitution within the RKKEE cluster, significantly increased the free energy of activation for the MscL channel due to an increase in activation pressure. Our data suggest that the orientation of the C-terminal helices relative to the aqueous medium is pH dependent, indicating that the RKKEE cluster functions as a proton sensor by adjusting the channel sensitivity to membrane tension in a pH-dependent fashion. A possible implication of our results for the physiology of bacterial cells is briefly discussed.
authors.affiliation: School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Anna
authors.initials: A
authors.lastname: Kloda
authors.forename: Alexandre
authors.initials: A
authors.lastname: Ghazi
authors.forename: Boris
authors.initials: B
authors.lastname: Martinac
mesh_terms: D002462:Cell Membrane / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry*; D040542:Mechanotransduction, Cellular; D010455:Peptides / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D055672:Static Electricity
chemical_list: D029968:Escherichia coli Proteins; D007473:Ion Channels; C086596:MscL protein, E coli; D010455:Peptides