Defining the physical gate of a mechanosensitive channel, MscL, by engineering metal-binding sites.
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.
Iscla
Irene
I
Department of Physiology, University of Texas-Southwestern Medical Center, Dallas, Texas 75390-9040, USA.
Levin
Gal
G
Wray
Robin
R
Reynolds
Robert
R
Blount
Paul
P
D001665:Binding Sites; D004926:Escherichia coli / Q000187:drug effects / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007473:Ion Channels / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D040542:Mechanotransduction, Cellular / Q000187:drug effects* / Q000502:physiology*; D008670:Metals / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D011312:Pressure; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism; D013104:Spheroplasts / Q000187:drug effects / Q000502:physiology; D013314:Stress, Mechanical; D013329:Structure-Activity Relationship
D029968:Escherichia coli Proteins; D007473:Ion Channels; D008670:Metals; C086596:MscL protein, E coli; D011994:Recombinant Proteins