Design and characterization of a constitutively open KcsA

Cuello, Luis G.; Jogini, Vishwanath; Cortes, D. Marien; Sompornpisut, Amornrat; Purdy, Michael D.; Wiener, Michael C.; Perozo, Eduardo

doi:10.1016/j.febslet.2010.02.015

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Volume 584, Issue 6 , Pages 1133-1138, 19 March 2010

Design and characterization of a constitutively open KcsA

Edited by Julian Schroeder

Luis G. Cuello
- Department of Biochemistry and Molecular Biology, The University of Chicago, 929 E 57th Street, Chicago, IL 60637, USA
Vishwanath Jogini
- Department of Biochemistry and Molecular Biology, The University of Chicago, 929 E 57th Street, Chicago, IL 60637, USA
D. Marien Cortes
- Department of Biochemistry and Molecular Biology, The University of Chicago, 929 E 57th Street, Chicago, IL 60637, USA
Amornrat Sompornpisut
- Department of Biochemistry and Molecular Biology, The University of Chicago, 929 E 57th Street, Chicago, IL 60637, USA
Michael D. Purdy
- Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22906, USA
Michael C. Wiener
- Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22906, USA
Eduardo Perozo
- Department of Biochemistry and Molecular Biology, The University of Chicago, 929 E 57th Street, Chicago, IL 60637, USA
- Institute for Biophysical Dynamics, The University of Chicago, 929 E 57th Street, Chicago, IL 60637, USA
- Corresponding author. Address: Department of Biochemistry and Molecular Biology, The University of Chicago, 929 E 57th Street, Chicago, IL 60637, USA. Fax: +1 773 834 4632.

Received 13 September 2009; received in revised form 1 February 2010; accepted 1 February 2010. published online 11 February 2010.

Abstract

The molecular nature of the structure responsible for proton sensitivity in KcsA has been identified as a charge cluster that surrounds the inner helical bundle gate. Here, we show that this proton sensor can be modified to engineer a constitutively open form of KcsA, amenable to functional, spectroscopic and structural analyses. By combining charge neutralizations for all acidic and basic residues in the cluster at positions 25, 117–122 and 124 (but not E118), a mutant KcsA is generated that displays constitutively open channel activity up to pH 9. The structure of this mutant revealed that full opening appears to be inhibited by lattice forces since the activation gate seems to be only on the early stages of opening.

Keywords: K⁺ Channel, KcsA, pH sensor, Activation gate, Crystal structure