MscL, a mechanosensitive channel found in many bacteria, protects cells from hypotonic shock by reducing intracellular pressure through release of cytoplasmic osmolytes. First isolated from Escherichia coli, this protein has served as a model for how a protein senses and responds to membrane tension. Recently the structure of a functionally uncharacterized MscL homologue from Mycobacterium tuberculosis was solved by x-ray diffraction to a resolution of 3.5 Å. Here we demonstrate that the protein forms a functional MscL-like mechanosensitive channel in E. coli membranes and azolectin proteoliposomes. Furthermore, we show that M. tuberculosis MscL crystals, when re-solubilized and reconstituted, yield wild-type channel currents in patch clamp, demonstrating that the protein does not irreversibly change conformation upon crystallization. Finally, we apply functional clues acquired from the E. coli MscL to the M. tuberculosis channel and show a mechanistic correlation between these channels. However, the inability of the M. tuberculosis channel to gate at physiological membrane tensions, demonstrated by in vivo E. coli expression and in vitro reconstitution, suggests that the membrane environment or other additional factors influence the gating of this channel.Mechanosensation, the process of detecting a mechanical stimulus, is integral to a vast array of sensory systems. These sensory systems encompass a span from hearing to proprioception to osmoregulation, yet are among the least understood at the molecular level. The advent of patch clamp analysis revolutionized the study of mechanosensitive systems. Electrophysiological evidence quickly implicated mechanosensitive (MS) 1 channels as the primary transducing element in these sensory cascades (1, 2). Soon, MS channel activities had been detected in more than 30 cell types (3) including embryonic chick skeletal muscle cells and frog muscle, where they were first discovered (4, 5).These systems have remained veiled because of the very low abundance of the channels or the tissues that bear them. In addition, the lack of tangible ligands for MS channels has hampered efforts to enrich a population by biochemical means. This veil began to lift when MS channel activities were detected in bacteria. Early studies in Escherichia coli giant spheroplasts (6) showed MS conductances, and soon similar activities were detected in giant protoplasts of Bacillus subtilis (7) and Streptococcus faecalis (8). At least three MS activities are now recognized in E. coli: MscL (mechanosensitive channel large conductance), MscS (Msc small), and MscM (Msc mini) (9). Several studies support a role for these bacterial MS channels in osmoregulation, serving as "emergency relief valves" in response to acute hypotonic shock (10 -12).Previously it was demonstrated that a 136-amino acid protein from E. coli formed a homo-multimeric membrane-bound complex that correlated with the large membrane conductance (13-15). This protein, MscL, became the first MS channel to be cloned and subjected to molecul...

The mechanosensitive channel of large conductance (MscL), a bacterial channel, is perhaps the best characterized mechanosensitive protein. A structure of the Mycobacterium tuberculosis ortholog has been solved by x-ray crystallography, but details of how the channel gates remain obscure. Here, cysteine scanning was used to identify residues within the transmembrane domains of Escherichia coli MscL that are crucial for normal function. Utilizing genetic screens, we identified several mutations that induced gain-of-function or loss-of-function phenotypes in vivo. Mutants that exhibited the most severe phenotypes were further characterized using electrophysiological techniques and chemical modifications of the substituted cysteines. Our results verify the importance of residues in the putative primary gate in the first transmembrane domain, corroborate other residues previously noted as critical for normal function, and identify new ones. In addition, evaluation of disulfide bridging in native membranes suggests alterations of existing structural models for the "fully closed" state of the channel.

MscL is a mechanosensitive channel of large conductance that functions as an ''emergency release valve,'' allowing bacteria to survive acute hypoosmotic stress. Although Escherichia coli MscL is the best-studied mechanosensitive channel, structural rearrangements occurring during gating remain disputed. Introduction of a charged residue into the pore of MscL was shown to result in a reduced-viability phenotype. Here, we probe for residues in the transmembrane domains that are exposed to the aqueous environment in the presence and absence of hypoosmotic shock by reacting a charged sulfhydryl reagent with substituted cysteines. Subsequent analysis of cell viability allows for an assessment of residues exposed in the closed and opening states in vivo. The results suggest that the crystal structure of MscL derived from the Mycobacterium tuberculosis orthologue may reflect a nearly closed rather than fully closed state and support a clockwise rotation of the pore-forming first transmembrane domain on gating.

The six-transmembrane channels are thought to be composed of two modules: pore and sensor. Whereas the modular design of the pore has been established, the modularity of the sensor remains hypothetical. As a first step toward establishing the modularity of this region, we searched for genes where the sensor is found independent of the pore and have identified new members of the sensor superfamily. Analysis of these sensors reveals a motif shared among not only these newly discovered members and voltage-gated, transient receptor potential, and polycystin channel sensors, but also MscL, a bacterial mechanosensitive channel. Mutational analyses presented here and in previous studies demonstrate that highly conserved residues within this motif are required for normal channel activity; mutations of residues within this motif in different subfamilies lead to consistent channel phenotypes. Previous studies have demonstrated that peptides containing this motif and the adjacent conserved transmembrane domain elicit channel activities when reconstituted into lipid membranes. These data provide evidence for the modularity of the sensor, imply a model for its evolution, suggest a common origin for mechano- and voltage-sensing, and may offer a glimpse of the properties of the first sensor/channel.

The mechanosensitive channel of large conductance, MscL, of Escherichia coli is one of the best-studied mechanosensitive proteins. Although the structure of the closed or "nearly-closed" state of the Mycobacterium tuberculosis ortholog has been solved and mechanisms of gating have been proposed, the transition from the closed to the open states remains controversial. Here, we probe the relative position of specific residues predicted to line the pore of MscL in either the closed state or during the closed-to-open transition by engineering single-site histidine substitutions and assessing the ability of Ni2+, Cd2+ or Zn2+ ions to affect channel activity. All residues predicted to be within the pore led to a change in channel threshold pressure, although the direction and extent of this change were dependent upon the mutation and metal used. One of the MscL mutants, L19H, exhibited gating that was inhibited by Cd2+ but stimulated by Ni2+, suggesting that these metals bind to and influence different states of the channel. Together, the results derived from this study support the hypotheses that the crystal structure depicts a "nearly closed" rather than a "fully closed" state of MscL, and that a clockwise rotation of transmembrane domain 1 occurs early in the gating process.