The Interfacial Electrostatics Defines MscS Gating and Inactivation

Abstract

The membrane tension that drives transitions in mechanosensitive proteins is transmitted primarily through the polar/apolar interfaces of the lipid bilayer. The mechanosensitive channel of small conductance (MscS) of E. coli has two prominent arginines, R46 and R74, in each subunit which anchor the TM1 and TM2 helices to the cytoplasmic boundary of the membrane. Alignments of 171 MscS homologs show that the presence of positive charges (R, K or H) in these two locations is highly conserved. A distinctive property of WT MscS is that it inactivates under moderate tension, thus properly terminating an osmotic permeability response. Under depolarizing voltages, MscS inactivation is accelerated; hyperpolarization, on the contrary, slows down inactivation and accelerates recovery. Therefore, MscS utilizes normal membrane potential to return back to the resting state. We generated a variety of double mutants at these positions to investigate the interactions with the lipid head groups and examine the voltage dependence of inactivation. We found that N or T substitutions for R46 and R74 produce functional channels, even preserving the voltage sensitivity. The D, A or V mutations produce channels that do not rescue the osmotically sensitive MJF465 strain and do not open in full, demonstrating 10-30% of WT conductance. L, Y and W mutants support the gating and rescue, but remove the voltage dependence of inactivation and recovery. The data indicates that polar interactions between helix-anchoring residues and interfacial lipid phosphates are critical for gating. The transition to the open or inactivated state likely increases the number of favorable contacts with phosphates. Voltage dependence of inactivation can be related to the Lippmann's electrowetting effect, accumulation of co-ions or counter-ions in the double layer on the cytoplasmic surface, which changes the hydration of the TM2-TM3 interface that transmits tension to the gate.