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Biophys. J. BioFAST: First Published August 31, 2004. doi:10.1529/biophysj.104.049833
© 2004 by the Biophysical Society.

A more recent version of this article appeared on November 1, 2004.
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CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING

Defining the physical gate of a mechanosensitive channel, MscL, by engineering metal binding sites

Irene Iscla 1, Gal Levin 1, Robin Wray 1, Robert Reynolds 1 and Paul Blount 2*

1 U. T. Southwestern Med. Ctr.
2 University of Texas Southwestern Med. Center

* To whom correspondence should be addressed. E-mail: paul.blount{at}utsouthwestern.edu.

Submitted on July 15, 2004
Revised on August 5, 2004
Accepted on 23 August 2004


  Abstract
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 orthologue 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 Ni++, Cd++ or Zn++ 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 Cd++ but stimulated by Ni++, 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 TM1 occurs early in the gating process

Key Words: ion channel gating, mechanosensation, metal binding, osmoregulation




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Copyright © 2004 by the Biophysical Society.