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MscL, the highly conserved bacterial mechanosensitive channel of large conductance, is one of the best studied mechanosensors. It is a homopentameric channel that serves as a biological emergency release valve that prevents cell lysis from acute osmotic stress. We previously showed that the periplasmic region of the protein, particularly a single residue located at the TM1/periplasmic loop interface, F47 of Staphylococcus aureus and I49 of Escherichia coli MscL, plays a major role in both the open dwell time and mechanosensitivity of the channel. Here, we introduced cysteine mutations at these sites and found they formed disulfide bridges that decreased the channel open dwell time. By scanning a likely interacting domain, we also found that these sites could be disulfide trapped by addition of cysteine mutations in other locations within the periplasmic loop of MscL, and this also led to rapid channel kinetics. Together, the data suggest structural rearrangements and protein-protein interactions that occur within this region upon normal gating, and further suggest that locking portions of the channel into a transition state decreases the stability of the open state.

MscL, the highly conserved bacterial mechanosensitive channel of large conductance, is a homopentameric channel in the plasma membrane that serves as a biological emergency release valve that prevents cell lysis from acute osmotic stress. It is one of the best studied mechanosensors and serves as a paradigm of how a protein can sense and respond to membrane tension. While all orthologues tested thus far encode channel activity, many show functional differences. For example, E coli MscL has more stable openings and is more mechanosensitive than S. aureus MscL. Here we have utilized a chimera approach to correlate structural features with functional differences. Surprisingly, the data indicate that the periplasmic region of the protein, particularly a single residue located at the TM1/periplasmic loop interface, plays a major role in both the open dwell time and mechanosensitivity of the channel for both orthologues. The features of this single residue and interactions of this site were studied by employing site directed mutagenesis, cysteine trapping, and single channel patch clamp recordings. In sum, the data suggest that this single residue acts as a multi-switch, interacting differentially with various regions of the pentameric complex as the channel gates.