The Selectivity of MscS is Determined by the Cytoplasmic Domain

Abstract

The mechanosensitive channel of small conductance (MscS) is a heptameric pressure sensitive channel expressed in the inner membrane of E. coli. This channel possesses three transmembrane helices and a large water-filled cytoplasmic cage which comprises more than 50% of the total protein. This cytoplasmic domain is conserved throughout the MscS channel family and in MscS has been shown to be a dynamic structure with an active role in channel gating. It has also been suggested that the cytoplasmic domain determines the weak anion selectivity exhibited by MscS. In order to address this question this study reconstituted wild type MscS and single residue mutants of the cytoplasmic vestibule in azolectin liposomes (protein:lipid 1:10000). Patch clamp recordings of these channels were then performed in the presence of asymmetric solutions of KCl (600/200 mM) and BaCl2 (50/200 mM). The MscS mutants studied were R184E, R185E, E187R and E227A. Both E187R and E227A mutants show reduced selectivity characterised by a lower anion-cation permeability ratio. These residues are likely to determine selectivity by binding cations. From these data it is clear that charged residues in the cytoplasmic domain of MscS determine its selectivity. This is interesting because unlike K+, Na+ and Ca2+ channels the selectivity of MscS is not determined by residues in the pore region but residues situated in the large water-filled cytoplasmic domain.