Electrostatic Interactions Between the Transmembrane and Cytoplasmic Domains Critically Stabilize Tension-Sensitive States in MSCS

Abstract

The small mechanosensitive channel MscS is a bacterial osmolyte release valve with homologs found in all walled organisms. E. coli MscS readily responds to abrupt steps of tension in the cytoplasmic membrane, but under sustained stimulation it enters the tension-insensitive inactivated state. Upon tension release, MscS recovers within 2 s. In the crystal structure of WT MscS, the gate region (end of TM3a) is the only connecting element between the transmembrane (TM) and the cytoplasmic (cage) domains. It has been predicted that the two domains can make additional contacts through salt bridges between D62 on the TM1-TM2 loop and the R128-R131 cluster on the cage. Our experiments show that disrupting this salt bridge with D62R(N) substitutions does not affect desensitization, but instead, it drastically speeds up the process of inactivation and decreases the rate of recovery. The mutations also open a path for silent inactivation at sub-threshold tensions bypassing channel opening. Swapping the charges (D62R/R131D) restores the normal inactivation phenotype. Our new models suggest that the D62-R128/131 bridge critically stabilizes the positions of the lipid-facing TM1-TM2 helices along central TM3s and their association through the F68-L111-L115 hydrophobic cluster which transmits force from the membrane to the gate, in both closed and open states. Simulations suggested that not only the G113 flexible region on TM3 is necessary for inactivation, but the G76 hinge on TM2 might be needed, too. Experiments confirmed that G76A substitution abolishes inactivation. Analyzing combined mutations with opposing effects on inactivation (D62N/G113A, D62N/G76A) reveals a strong contribution of the loop-cage interactions to the stability of tension-sensitive states. The predicted hinge action of G76 suggests that twisting of TM1-TM2 may be the inactivation mechanism that disrupts the bridges while disengaging these helices from the gate.