Mechanosensitive (MS) channels are membrane proteins that respond to mechanical stimuli and they play critical roles in prokaryotes and eukaryotes. This study explores the contributions of the four periplasmic loop residues of the E. coli mechanosensitive channel of large conductance (MscL)—A64, Q65, G66, and D67—to channel gating. Using site-directed spin labelling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy, we demonstrated that these residues interact directly with the membrane lipid bilayer during the channel opening by membrane tension sensitivity of the channel. We further explored how mutations at these sites affect biophysical properties of MscL in giant E . coli spheroplasts and liposomes made of different lipids (soy azolectin and negatively charged lipids comprising of 70% phosphatidylcholine and 30% phosphatidylglycerol) using patch clamp fluorometry and molecular dynamics simulations. We found that mutation of Q65 to arginine (Q65R) increased the sensitivity of the channel, while substitution to glutamic acid (Q65E) decreased the sensitivity of the channel to membrane tension in giant spheroplasts as well as in negatively charged liposomes. Our molecular dynamic simulation data further revealed that under in-plane radial tension Q65E exhibits more pronounced early conformational changes under tension, whereas the rapid and extensive opening of the channel at later stages occurs within a shorter time frame primarily in the wild-type and Q65R mutant channel. Mutation of A64 and G66 to either E (A64E, G66E) or R (A64R) decreased the tension sensitivity of the channel in negatively charged lipids. Additionally, extending the periplasmic loop by adding four glycine residues resulted in decrease tension sensitivity of the channel. Our findings provide novel insights into the protein-lipid interactions influencing MscL opening kinetics and underscore the importance of the periplasmic loop in regulating mechanosensitivity of the channel.
Kingsley C. Duru (Sun,) studied this question.