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

Mechanosensitive channels of large conductance (MscL) protect bacteria from hypo-osmotic stress by opening a large pore in response to membrane tension. The transmembrane TM1 and TM2 helices together with the N-terminal amphipathic helix function as the primary force-sensing structural domains, whereas the role of the periplasmic loop in the channel gating remains poorly understood. Using experimental and computational approaches, our study demonstrates that mutating the loop residues (A64, Q65, G66, D67) or inserting a four-glycine flexible hinge at D67 site modified the properties of the channel recorded in giant E. coli spheroplasts and liposomes composed of azolectin or negatively charged lipids. Extending the periplasmic loop disrupted mechanical force transmission, reducing channel sensitivity to applied tension. Q65R channel mutant exhibited increased sensitivity in azolectin liposomes that was reduced in negatively charged liposomes and spheroplast membranes. The Q65E mutant showed decreased sensitivity across all preparations tested and exhibited channel flickering in negatively charged liposomes. Molecular dynamics simulations revealed that Q65E produced larger but more structurally restricted conformational changes, whereas wild-type MscL and Q65R exhibited rapid and extensive pore opening over a shorter timeframe. Our findings establish the periplasmic loop as a structural domain fine-tuning MscL gating through electrostatic interactions with surrounding lipids.