Inactivation of the Bacterial Mechanosensitive Channel MscL Involves Flexible Transmembrane Helices and a ‘Dry’ Gate

Abstract

MscL, a mechanosensitive channel of large conductance, is an emergency release valve residing in the cytoplasmic membrane of E. coli. Under osmotic shock, when membrane tension approaches the lytic limit of ∼10 mN/m, MscL opens a 3 nm pore relieving osmotic stress. The conformational transition in the pentameric complex was previously envisioned as a tilting iris-like motion of tightly coupled pairs of the centrally located TM1 and peripheral TM2 helices. Wetting the hydrophobic constriction formed by the rings of L19 and V23 was identified as is the rate-limiting step over a barrier of more than 50 kT. While adaptation of MscL was reported, the channel was generally considered non-inactivating. Special pressure protocols involving prolonged conditioning steps and short saturating test pulses revealed that after a 30 s exposure to half-saturating pressure (p0.5) in spheroplast patches, about 20% of MscL population reversibly inactivates. The channels return to the resting state within 1 s upon pressure release. Introduction of a flexible double glycine motif (A91G/I92G) in TM2 dramatically increased the rate of inactivation resulting in a 90% silent channel population after a 10 s step to p0.5. Single-channel traces revealed a split of concerted 70 pA opening transitions into a staircase of irregular ∼7 pA substates in the double glycine mutant. The additional hydrophilic substitution in the constriction (V23T/A91G/I92G) pre-hydrates the pore, reduces p0.5 by ∼55% and, while generating multiple substates, completely abolishes inactivation. In extrapolated-motion simulations TM2 kinks at A91G/I92G and its C-terminal end separates from TM1. We propose that in WT MscL TM1s and TM2s are tightly coupled, whereas in the double glycine mutant the unsupported TM1s may reform the tight hydrophobic seal independent of the positions of TM2s thus creating a tension-insensitive non-conductive state.