title: Molecular dissection of the large mechanosensitive ion channel (MscL) of E. coli: mutants with altered channel gating and pressure sensitivity.
abstract: In the search for the essential functional domains of the large mechanosensitive ion channel (MscL) of E. coli, we have cloned several mutants of the mscL gene into a glutathione S-transferase fusion protein expression system. The resulting mutated MscL proteins had either amino acid additions, substitutions or deletions in the amphipathic N-terminal region, and/or deletions in the amphipathic central or hydrophilic C-terminal regions. Proteolytic digestion of the isolated fusion proteins by thrombin yielded virtually pure recombinant MscL proteins that were reconstituted into artificial liposomes and examined for function by the patch-clamp technique. The addition of amino acid residues to the N-terminus of the MscL did not affect channel activity, whereas N-terminal deletions or changes to the N-terminal amino acid sequence were poorly tolerated and resulted in channels exhibiting altered pressure sensitivity and gating. Deletion of 27 amino acids from the C-terminus resulted in MscL protein that formed channels similar to the wild-type, while deletion of 33 C-terminal amino acids extinguished channel activity. Similarly, deletion of the internal amphipathic region of the MscL abolished activity. In accordance with a recently proposed spatial model of the MscL, our results suggest that (i) the N-terminal portion participates in the channel activation by pressure, and (ii) the essential channel functions are associated with both, the putative central amphipathic alpha-helical portion of the protein and the six C-terminal residues RKKEEP forming a charge cluster following the putative M2 membrane spanning alpha-helix.
authors.affiliation: Department of Pharmacology, University of Western Australia, Nedlands WA 6907, Australia.
authors.forename: C C
authors.initials: CC
authors.lastname: Häse
authors.forename: A C
authors.initials: AC
authors.lastname: Le Dain
authors.forename: B
authors.initials: B
authors.lastname: Martinac
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000502:physiology*; D001483:Base Sequence; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins; D015640:Ion Channel Gating / Q000235:genetics*; D007473:Ion Channels / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007473:Ion Channels; C086596:MscL protein, E coli