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Estimating the pKa values of basic and acidic side chains in ion channels using electrophysiological recordings: A robust approach to an elusive problem

Authors

  • Gisela D. Cymes,

    1. Department of Molecular and Integrative Physiology, Center for Biophysics and Computational Biology, and Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801
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  • Claudio Grosman

    Corresponding author
    1. Department of Molecular and Integrative Physiology, Center for Biophysics and Computational Biology, and Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801
    • 407 S. Goodwin Ave., 524 Burrill Hall, Urbana, IL 61801
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    • Ph.: +217-244-1736; Fax: +217-333-1133.


  • The authors state no conflict of interest.

Abstract

As a step toward gaining a better understanding of the physicochemical bases of pKa-value shifts in ion channels, we have previously proposed a method for estimating the proton affinities of systematically engineered ionizable side chains from the kinetic analysis of single-channel current recordings. We reported that the open-channel current flowing through mutants of the (cation-selective) muscle nicotinic acetylcholine receptor (AChR) engineered to bear single basic residues in the transmembrane portion of the pore domain fluctuates between two levels of conductance. Our observations were consistent with the idea that these fluctuations track directly the alternate protonation–deprotonation of basic side chains: protonation of the introduced basic group would attenuate the single-channel conductance, whereas its deprotonation would restore the wild-type-like level. Thus, analysis of the kinetics of these transitions was interpreted to yield the pKa values of the substituted side chains. However, other mechanisms can be postulated that would also be consistent with some of our findings but according to which the kinetic analysis of the fluctuations would not yield true pKas. Such mechanisms include the pH-dependent interconversion between two conformations of the channel that, while both ion permeable, would support different cation-conduction rates. In this article, we present experimental evidence for the notion that the fluctuations of the open-channel current observed for the muscle AChR result from the electrostatic interaction between fixed charges and the passing cations rather than from a change in conformation. Hence, we conclude that bona fide pKa values can be obtained from single-channel recordings. Proteins 2011; © 2011 Wiley-Liss, Inc.

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