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

Mechanosensitive channel large (MscL) encodes the large conductance mechanosensitive channel of the Escherichia coli inner membrane that protects bacteria from lysis upon osmotic shock. To elucidate the molecular mechanism of MscL gating, we have comprehensively substituted Gly²² with all other common amino acids. Gly²² 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 tuberculosis (Chang et al., 1998, Science. 282:2220-2226), Gly²² is buried within the constriction that closes the pore. Substituting Gly²² with hydrophilic residues decreased the threshold pressure at which channels opened and uncovered an intermediate subconducting state. In contrast, hydrophobic substitutions increased the threshold pressure. Although hydrophobic substitutions had no effect on growth, similar to the effect of an MscL deletion, channel hyperactivity caused by hydrophilic substitutions correlated with decreased proliferation. These results suggest a model for gating in which Gly²² moves from a hydrophobic, and through a hydrophilic, environment upon transition from the closed to open conformation.