Structural and functional studies of bacterial mechanosensitive channels

Bacterial mechanosensitive (MS) channels represent the most primitive form of MS channels gating solely in response to changes in bilayer tension. The two main families of bacterial MS channels are MscS and MscL. In concert these channels are imperative to obviate the effects of toxic downshocks in external osmolarity. While osmoprotection is the best characterised physiological role for these channels the genetic diversity of this family hints at as yet undiscovered physiological functions. This thesis explores one such possibility, showing that MscS expression provides a selective advantage in the presence of cell wall attack with knockout E. coli strains of MscS having increased susceptibility to cell wall targeting antibiotics. This thesis also shows, using the Ca2+ sensitive photoprotein aequorin, that on gating these channels become not only a gateway for solute efflux but also a conduit for ion entry. In particular, influx of Ca2+ may represent a physiologically relevant signal but more importantly this finding may pave the way for a high-throughput screen for novel MS channel activators which would be of potential value as lead compounds for antibiotics. Furthermore, this thesis demonstrates that in the presence of divalentcations (Ca2+ & Ba2+) MscS exhibits increased anion selectivity, rectification and eight distinct long-lived subconducting states at hyperpolarising membrane potentials. In an attempt to identify the structural basis of these subconducting states the first single residue MscS mutants that display altered anion selectivity are reported. This selectivity, in contrast to voltage-gated K+, Na+ and Ca2+ channels, is not determined by residues in the pore region but rather by charged residues in the cytoplasmic domain and is likely conserved throughout the MscS family