MscL as a Triggered Nanovalve: New Modifications to Improve Design

MscL is a small homopentameric bacterial protein that has, among other characteristics, an incredible pore size greater than 30A and the ability to gate in response to mechanical tension in the membrane. Because of its amenability, E.coli MscL has been the most studied mechanosensitive channel, serving as a paradigm of how a protein can sense and transduce mechanical force. Early on in the study of the channel a critical domain for MscL gating was revealed by forward genetic experiments screening for mutations that led to a gain-of-function (slowed- or no-growth) phenotypes: mutations at residue G22, within the pore, led to severe gain-of-function phenotypes. This residue is thought to form part of a “hydrophobic lock” that stabilizes the closed state of the channel. If this hydrophobic lock is disrupted by the insertion of a charge, the transition energy barrier for MscL gating is destabilized and the channel gates even in the absence of membrane tension. using this observation, researchers have successfully changed the modality of MscL to be sensitive to stimuli such as light and pH simply by chemically modifying the G22 site within the MscL channel. Due to its ability to be triggered by different stimuli and the large pore size, MscL has been proposed as a triggered nanovalve for its use in nanodevices such as a liposome drug delivery system. Here, by utilizing in vivo, flux and patch clamp assays, we characterize other neighboring residue that also form part of the hydrophobic lock and we show that the G22 site may not be the best choice for all modifications that change channel modality.