Opening threshold and kinetics of the MscL mechanosensitive channel are regulated by its periplasmic loop

Mechanosensitive (MS) channels are membrane proteins that respond to mechanical stimuli and are essential across prokaryotic and eukaryotic organisms. The E. coli mechanosensitive channel of large conductance (MscL) provides a powerful model for dissecting protein-lipid interactions underlying mechanotransduction. Here, we investigated the contribution of four periplasmic loop residues (A64, Q65, G66, D67) to MscL gating. Using site-directed spin labelling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy, we show that these residues interact directly with the lipid bilayer during the channel opening through membrane tension sensitivity of the channel. We further examined how mutations at these periplasmic loop residues affect biophysical properties of MscL in giant E. coli spheroplasts and liposomes composed of azolectin or negatively charged lipids (70% phosphatidylcholine and 30% phosphatidylglycerol) using patch clamp electrophysiology, patch fluorometry and molecular dynamics simulations. Substitution of Q65 residue with arginine (Q65R) increased channel sensitivity to membrane tension, whereas replacement with glutamic acid (Q65E) decreased the sensitivity across all systems tested. Molecular dynamics simulations revealed that under in-plane radial tension Q65E exhibited larger conformational changes under tension, whereas the wild-type (WT) and Q65R mutant channels showed rapid and extensive opening at later stages within a shorter time frame. Insertion of a four-glycine hinge at D67 also reduced tension sensitivity, consistent with impaired force transmission. Substitution of A64 and G66 to either glutamic acid (A64E, G66E) or arginine (A64R) decreased tension sensitivity of the channel only in liposomes of negatively charged lipids. Together, these findings deepen our understanding of protein-lipid interactions governing MscL opening kinetics and highlights the contribution of the periplasmic loop in regulating mechanosensitivity.