Desensitization, inactivation, and the tension-proof safety mechanism of inactivated MscS

MscS is the main low-threshold tension-activated osmolyte release valve in bacteria. Working alone or together with high-threshold MscL, it regulates turgor and protects cells from mechanical rupture during osmotic down-shock. The channel exhibits complex adaptive behavior, including desensitization and full inactivation, both of which occur at relatively low sub-lytic tension. There is debate over whether the commonly observed non-conductive state of MscS with the tension-sensing helices splayed away from the gate corresponds to the closed or inactivated state. In this work, using specialized pressure protocols in patch-clamp electrophysiology, we highlight the difference between reversible adaptation (desensitization) and inactivation. We show that inactivated channels cannot be reactivated with high tension, up to the limit of patch stability. This aligns with cryo-EM studies by Zhang et al. (Nature, 2021, 590:509-5018), who applied extreme tension to the splayed nanodisc-reconstituted MscS (PDB 6VYK) by depleting lipids with cyclodextrin, and observed a new flattened but apparently non-conductive structure (PDB 6VYM). To characterize these two states, we performed a steered Molecular Dynamics simulation from the initial splayed structure to the flattened conformation, confirming that they are connected through a smooth conformational pathway and remain largely dehydrated and entirely non-conductive throughout the transition. The data show that the initial splayed conformation meets all the criteria of the inactivated state, distorting but not opening under extreme tension. By combining patch-clamp experiments with simulations based on cryo-EM data, we demonstrate that inactivated MscS resists activation, thereby maintaining the membrane barrier when tension exceeds the activation threshold. SIGNIFICANCEBacterial energetics, which relies on the electrochemical proton gradient across the inner membrane as an intermediary, conflicts with the presence of a dense population of highly conductive mechano-activated channels in the same cytoplasmic membrane, which must be proton-tight. Moreover, the tension activation threshold for the common MscS channel is low and can be easily exceeded by fluctuations in the concentrations of internal or external osmolytes. In this paper, we describe the important adaptive inactivation of MscS at tensions near its activation threshold, and specifically, the complete resistance of inactivated MscS to opening at any tension. The data show another layer of tight regulation of MscS residing in the energy-coupling membranes.