Scanning MscL Channels with Targeted Post-Translational Modifications for Functional Alterations.

ABSTRACT

Mechanosensitive channels are present in all living organisms and are thought to underlie the senses of touch and hearing as well as various important physiological functions like osmoregulation and vasoregulation. The mechanosensitive channel of large conductance (MscL) from Escherichia coli was the first protein shown to encode mechanosensitive channel activity and serves as a paradigm for how a channel senses and responds to mechanical stimuli. MscL plays a role in osmoprotection in E. coli, acting as an emergency release valve that is activated by membrane tension due to cell swelling after an osmotic down-shock. Using an osmotically fragile strain in an osmotic down-shock assay, channel functionality can be directly determined in vivo. In addition, using thiol reagents and expressed MscL proteins with a single cysteine substitution, we have shown that targeted post-translational modifications can be performed, and that any alterations that lead to dysfunctional proteins can be identified by this in vivo assay. Here, we present the results of such a scan performed on 113 MscL cysteine mutants using five different sulfhydryl-reacting probes to confer different charges or hydrophobicity to each site. We assessed which of these targeted modifications affected channel function and the top candidates were further studied using patch clamp to directly determine how channel activity was affected. This comprehensive screen has identified many residues that are critical for channel function as well as highlighted MscL domains and residues that undergo the most drastic environmental changes upon gating.