Hydrophilicity of a single residue within MscL correlates with increased channel mechanosensitivity

Mechanosensitive channel large (MscL) encodes the large conductance mechano sensitive channel of the Escherichia coil inner membrane that protects bact eria from lysis upon osmotic shock. To elucidate the molecular mechanism of MscL gating, we have comprehensively substituted Gly(22) with all other co mmon amino acids. Gly(22) was highlighted in random mutagenesis screens of E. coli MscL (Ou et al., 1998, Proc. Nat. Acad. Sci. USA. 95:11471-11475). By analogy to the recently published MscL structure from Mycobacterium tube rculosis (Chang et al., 1998, Science. 282:2220-2226), Gly(22) is buried wi thin the constriction that closes the pore. Substituting Gly(22) With hydro philic residues decreased the threshold pressure at which channels opened a nd uncovered an intermediate subconducting state. In contrast, hydrophobic substitutions increased the threshold pressure. Although hydrophobic substi tutions had no effect on growth, similar to the effect of an MscL deletion, channel hyperactivity caused by hydrophilic substitutions corrrelated with decreased proliferation. These results suggest a model for gating in which Gly(22) moves from a hydrophobic, and through a hydrophilic, environmment upon transition from the closed to open conformation.