title: 2,2,2-Trifluoroethanol changes the transition kinetics and subunit interactions in the small bacterial mechanosensitive channel MscS.
abstract: 2,2,2-Trifluoroethanol (TFE), a low-dielectric solvent, has recently been used as a promising tool to probe the strength of intersubunit interactions in membrane proteins. An analysis of inner membrane proteins of Escherichia coli has identified several SDS-resistant protein complexes that separate into subunits upon exposure to TFE. One of these was the homo-heptameric stretch-activated mechanosensitive channel of small conductance (MscS), a ubiquitous component of the bacterial turgor-regulation system. Here we show that a substantial fraction of MscS retains its oligomeric state in cold lithium-dodecyl-sulfate gel electrophoresis. Exposure of MscS complexes to 10-15 vol % TFE in native membranes or nonionic detergent micelles before lithium-dodecyl-sulfate electrophoresis results in a complete dissociation into monomers, suggesting that at these concentrations TFE by itself disrupts or critically compromises intersubunit interactions. Patch-clamp analysis of giant E. coli spheroplasts expressing MscS shows that exposure to TFE in lower concentrations (0.5-5.0 vol %) causes leftward shifts of the dose-response curves when applied extracellularly, and rightward shifts when added from the cytoplasmic side. In the latter case, TFE increases the rate of tension-dependent inactivation and lengthens the process of recovery to the resting state. MscS responses to pressure ramps of different speeds indicate that in the presence of TFE most channels reside in the resting state and only at tensions near the activation threshold does TFE dramatically speed up inactivation. The effect of TFE is reversible as normal channel activity returns 15-30 min after a TFE washout. We interpret the observed midpoint shifts in terms of asymmetric partitioning of TFE into the membrane and distortion of the bilayer lateral pressure profile. We also relate the increased rate of inactivation and subunit separation with the capacity of TFE to perturb buried interhelical contacts in proteins and discuss these effects in the framework of the proposed gating mechanism of MscS.
authors.affiliation: Department of Biology, University of Maryland, College Park, Maryland.
authors.forename: Bradley
authors.initials: B
authors.lastname: Akitake
authors.forename: Robin E J
authors.initials: RE
authors.lastname: Spelbrink
authors.forename: Andriy
authors.initials: A
authors.lastname: Anishkin
authors.forename: J Antoinette
authors.initials: JA
authors.lastname: Killian
authors.forename: Ben
authors.initials: B
authors.lastname: de Kruijff
authors.forename: Sergei
authors.initials: S
authors.lastname: Sukharev
mesh_terms: D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D007473:Ion Channels / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D040542:Mechanotransduction, Cellular / Q000187:drug effects / Q000502:physiology*; D011485:Protein Binding; D021122:Protein Subunits; D014270:Trifluoroethanol / Q000008:administration & dosage*
chemical_list: D029968:Escherichia coli Proteins; D007473:Ion Channels; C469542:MscS protein, E coli; D021122:Protein Subunits; D014270:Trifluoroethanol