title: Cysteine scanning of MscL transmembrane domains reveals residues critical for mechanosensitive channel gating.
abstract: 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.
authors.affiliation: Department of Physiology, University of Texas-Southwestern Medical Center, Dallas, Texas 75390-9040, USA.
authors.forename: Gal
authors.initials: G
authors.lastname: Levin
authors.forename: Paul
authors.initials: P
authors.lastname: Blount
mesh_terms: D015153:Blotting, Western; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides; D004594:Electrophysiology; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry*; D007473:Ion Channels / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000378:metabolism; D009995:Osmosis; D010641:Phenotype; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
chemical_list: D004220:Disulfides; D029968:Escherichia coli Proteins; D007473:Ion Channels; C086596:MscL protein, E coli; D003545:Cysteine