While several studies on Eco- or Tb-MscL have suggested that the periplasmic region may play a role in MscL channel regulation (Ajouz et al., 2000
; Blount et al., 1996b
; Jeon and Voth, 2008
; Maurer et al., 2000
; Maurer and Dougherty, 2003
; Park et al., 2004
; Tsai et al., 2005
), no one had successfully identified a single sub-domain or residue that acted as a variable spring element, in either a consistent or opposing manner, across bacterial species. In this study, using a chimera approach we were able to identify such subdomains, and ultimately, a single residue.
We initially found that the periplasmic loop of Eco-MscL confers short channel openings and that of Sa-MscL defines long openings. In contrast, the relatively conserved TM1/periplasmic lipid interface (44–49) influenced channel kinetics in an opposite manner with that from Eco-MscL effecting long open dwell times, and that from Sa-MscL generating short open dwell times. Thus, these two periplasmic regions appear to work in concert within these two orthologues, and the engineered combination of the Eco-MscL TM1/periplasmic lipid interface and the Sa-MscL periplasmic loop leads to MscL channels with a stabilized open state and extremely long open dwell times.
The further dissection of the TM1/periplasmic loop interface led to the finding that a single residue at the lipid interface, I49 for Eco-MscL and the analogous site F47 for Sa-MscL, has profound effects on channel kinetics, thus suggesting an important role of protein-lipid interactions at this region. Furthermore, we generated one particular mutant by site directed mutagenesis, Sa-MscL F47L, which has an extreme channel phenotype in that it shows a severe hysteresis as well as delay in both opening and closing. These data demonstrate that not only can the energy barrier for gating, or the transition between closed and open states, be modified by a mutation at Sa-F47, but that the transition from closed to open can be drastically different from that for going from open to closed states. A recent molecular dynamic simulation study on Eco-MscL channel gating suggests that, during the gating process, membrane thinning induces a kink within the TM1/periplasmic loop interface, essentially in and around Eco-I49, which causes outward motion of the periplasmic loop away from the pore center (Deplazes et al., 2012
). We propose that the easing or resisting of some restraint, such as this proposed kink formation, may modulate the transition between open and closed states, like adjusting the spring on a clasp knife.
Studying additional channels altered by site-directed mutagenesis, we found consistent correlations between residue properties and their effects on channel function for Sa- as well as Eco-MscL; these data suggest a common functional role for this site between orthologues. As a general rule, hydrophobic residues led to longer open times, although streamlined hydrophobic residues appear to be more efficient in this aspect than those containing bulkier aromatic rings, presumably because of some sort of steric hindrance of the latter. The role of this residue in channel mechanosensation was also studied, and more hydrophobic substitutions were found to decrease mechanosensitivity. Therefore, increasing the hydrophobicity appears to increase the energy barrier required to achieve an open state, but once achieved, the open state is stabilized; increasing the hydrophilicity does the opposite, demonstrating that this spring element is adjusted by hydrophobicity. These findings are reminiscent of early studies suggesting that hydrophobic interactions, coined a “hydrophobic lock”, at the pore constriction keeps the channel closed; according to this theory, it is the exposure of these residues to the aqueous solution that is a major energy barrier for channel opening (Blount et al., 2007
; Blount and Moe, 1999
; Moe et al., 2000
; Yoshimura et al., 1999
). Note, however, that the “lock” is due to the bundling of hydrophobic residues from each of the subunits at the constriction of the pore, whereas here we found a residue at the lipid interface where no such bundling of residues is likely to occur.
Although we found that the hydrophobicity of this spring element at the TM1 periplasmic lipid interface is important in both channel kinetics and mechanosensation, it is important to note that there are differences in the two correlations. For example, aromatic amino acids fit the hydropathy correlation for mechanosensation but not open dwell time. Several studies emphasize the importance of aromatics in potential lipid interactions for MscL (Sawada et al., 2012
) as well as other mechanosensitive channels such as TRPY1 and other TRP channels (Su et al., 2007
; Zhou et al., 2007
) and belts of aromatics are seen in other membrane proteins, including KcsA (Doyle et al., 1998
). Thus, aromatic residue-lipid interactions may play more of a role in mechanosensation than kinetics. We also observed that there is no correlation between mechanosensitivity and open dwell time for the various substitutions. Hence, although the relative hydrophobicity of this spring element effects changes in both kinetics and mechanosensation, it does so by different underlying mechanisms.
In summary, by initially using a chimera approach, we identified a single amino acid serves as a “clasp knife spring” for the resistance and stability of channel opening and closing. The hydrophobicity and steric shape of this residue, complemented by the periplasmic loop, appears to define channel mechanosensitivity and kinetics in both Sa- and Eco-MscL.