ONE FACE OF A TRANSMEMBRANE HELIX IS CRUCIAL IN MECHANOSENSITIVE CHANNEL GATING

MscL is a mechanosensitive channel in bacteria that responds directly to membrane tension by opening a large conductance pore. To determine functionally important residues within this molecule, we have randomly mutagenized mscL, expressed the genes in living bacteria, and screene d for gain-of-function mutants with hampered growth, Expression of the se genes caused leakage of cytoplasmic solutes on little or no hypo-os motic stress. In excised patches, the mutant channels gated at membran e tensions that are less than that required for the gating of the wild -type MscL. Hence, the data suggest that the slowed or no-growth pheno type is caused by solute loss because of inappropriate gating of the c hannel, Most of the mutations mapped to the first transmembrane domain . When this domain is modeled as an alpha-helix, the most severe mutat ions are substitutions of smaller amino acids (three glycines and one valine) on one facet, suggesting an important role for this structure in MS channel gating.