MOLECULAR DISSECTION OF THE LARGE MECHANOSENSITIVE ION-CHANNEL (MSCL)OF ESCHERICHIA-COLI - MUTANTS WITH ALTERED CHANNEL GATING AND PRESSURE SENSITIVITY

In the search for the essential functional domains of the large mechan osensitive ion channel (MscL) of E. coli, we have cloned several mutan ts of the mscL gene into a glutathione S-transferase fusion protein ex pression system. The resulting mutated MscL proteins had either amino acid additions, substitutions or deletions in the amphipathic N-termin al region, and/or deletions in the amphipathic central or hydrophilic C-terminal regions. Proteolytic digestion of the isolated fusion prote ins 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 we re poorly tolerated and resulted in channels exhibiting altered pressu re sensitivity and gating. Deletion of 27 amino acids from the C-termi nus 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 t he MscL abolished activity. In accordance with a recently proposed spa tial model of the MscL, our results suggest that (i) the N-terminal po rtion participates in the channel activation by pressure, and (ii) the essential channel functions are associated with both, the putative ce ntral amphipathic cr-helical portion of the protein and the six C-term inal residues RKKEEP forming a charge cluster following the putative M 2 membrane spanning alpha-helix.