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 cryoelectron microscopy (cryo-EM) studies by Zhang et al., 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.