The Hollow Domain of the Mechanosensitive Channel MscS is a Sensor of Cytoplasmic Crowding

Abstract

Mechanosensitive channel MscS is a ubiquitous osmolyte release valve found in walled cells, from bacteria to higher plants. In excised patches, applied tension of 5-8 mN/m activates E. coli MscS, followed by adaptive closure and complete inactivation, both of which are strongly promoted by crowding agents in the cytoplasm and by depolarizing voltages. Previous genetic screens1 and models derived from the crystal structure suggest allosteric coupling between the cytoplasmic ‘cage’ domain and the gate through association of the beta domain and the cytoplasmic end of the pore-lining TM3 helix. This association imposes kinks on TM3 that lead to closing or inactivation. Our analysis shows that mutations disrupting the TM3-beta association dramatically decrease closing rates, impede inactivation and make the channel insensitive to crowding agents and voltage. Conversely, mutations predicted to strengthen the TM3-beta interactions make the channel hard to open and inactivate silently under ramp stimulation. Models with straightened TM3s and separated beta domains representing the resting and open states are supported by an engineered salt bridge, which was not observed in the crystal structure and strongly destabilized the open state. Simulations suggest that in the resting state, with beta domains separated from TM3, the cage is prolate in shape, protruding more into the cytoplasm, whereas conversion to an oblate shape and approaching the membrane favors TM3b-beta association. We conclude that the shape changes make the cage an intracellular crowding sensor which functions through labile kink-stabilizing TM3-beta interactions. The cage is the first example of a hollow channel domain that can provide feedback on the degree of cytoplasm condensation (crowding) that disengages the gate and prevents efflux of osmolytes.1. Koprowski P, Grajkowski W, Isacoff EY and Kubalski A. 2011. J Biol Chem. 286:877-88.