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Biophys. J. BioFAST: First Published December 30, 2005. doi:10.1529/biophysj.105.075481
© 2005 by the Biophysical Society.

A more recent version of this article appeared on March 15, 2006.
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CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING

C-terminal charged cluster of MscL, RKKEE, functions as a pH sensor

Anna Kloda 1, Alexandre Ghazi 2 and Boris Martinac 1*

1 The University of Queensland
2 CNRS Universite Paris-Sud

* To whom correspondence should be addressed. E-mail: b.martinac{at}uq.edu.au.

Submitted on October 3, 2005
Revised on November 4, 2005
Accepted on 5 December 2005


  Abstract
The RKKEE cluster of charged residues located within the cytoplasmic helix of the bacterial mechanosensitive (MS) channel, MscL is essential for the channel function. The structure of MscL determined by X-ray crystallography and electron paramagnetic resonance (EPR) spectroscopy has revealed discrepancies toward the C-terminus suggesting that the structure of the C-terminal helical bundle differs depending on the pH of the cytoplasm. In the present study we examined the effect of pH as well as charge reversal and residue substitution within the RKKEE cluster, on the mechanosensitivity of E. coli MscL reconstituted into liposomes using the patch-clamp technique. Protonation of either positively or negatively charged residues within the cluster, achieved by changing the experimental pH or residue substitution within the RKKEE cluster, significantly increased the free energy of activation for the MscL channel due to an increase in activation pressure. Our data suggest that the orientation of the C-terminal helices relative to the aqueous medium is pH dependent indicating that the RKKEE cluster functions as a proton sensor by adjusting the channel sensitivity to membrane tension in a pH-dependent fashion. A possible implication of our results for the physiology of bacterial cells is briefly discussed.

Key Words: MS channels, lipid bilayer, membranes and transport, patch clamp, protein folding




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Copyright © 2005 by the Biophysical Society.