Molecular dissection of the large mechanosensitive ion channel (MscL) of E. coli: mutants with altered channel gating and pressure sensitivity.
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.
Häse
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CC
Department of Pharmacology, University of Western Australia, Nedlands WA 6907, Australia.
Le Dain
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AC
Martinac
B
B
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
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007473:Ion Channels; C086596:MscL protein, E coli