This work presents a functional analysis of mutations in two bacterial mechanosensitive channels, MscL and MscS using bacterial growth phenotyping combined with electrophysiological and structural analyses. The introduction of aromatic caps at the ends of lipid facing helices in MscL compromises the osmotic rescuing function of the channel and changes gating parameters. The characteristic absence of aromatic residues at membrane interfaces is critical for MscL function, as the opening transition is associated with a strong helical reorientation. According to the current model of MscS, the pore-forming TM3 helices are predicted to separate, tilt, and straighten upon channel opening. This dynamic transition has been examined using a cysteine scan of this region and MTS accessibility experiments. Both cell viability assays and electrophysiological data support the hypothesis of a helical separation. Conductance measurements in gate mutants suggest that the pore lumen narrows toward the periplasmic end, consistent with the current model.