The Journal of General Physiology
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Published online March 26, 2007
doi:10.1085/jgp.200709756
The Journal of General Physiology, Vol. 129, No. 4, 317-329
The Rockefeller University Press, 0022-1295 $30.00
© 2007 Walden et al.
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ARTICLE

 Uncoupling and Turnover in a Cl–/H+ Exchange Transporter



Michael Walden, Alessio Accardi, Fang Wu, Chen Xu, Carole Williams, and Christopher Miller

Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, MA 02454

Correspondence to Christopher Miller: cmiller{at}brandeis.edu

The CLC-family protein CLC-ec1, a bacterial homologue of known structure, stoichiometrically exchanges two Cl– for one H+ via an unknown membrane transport mechanism. This study examines mutations at a conserved tyrosine residue, Y445, that directly coordinates a Cl– ion located near the center of the membrane. Mutations at this position lead to "uncoupling," such that the H+/Cl– transport ratio decreases roughly with the volume of the substituted side chain. The uncoupled proteins are still able to pump protons uphill when driven by a Cl– gradient, but the extent and rate of this H+ pumping is weaker in the more uncoupled variants. Uncoupling is accompanied by conductive Cl– transport that is not linked to counter-movement of H+, i.e., a "leak." The unitary Cl– transport rate, measured in reconstituted liposomes by both a conventional initial-velocity method and a novel Poisson dilution approach, is ~4,000 s–1 for wild-type protein, and the uncoupled mutants transport Cl– at similar rates.

Abbreviations used in this paper: Vln, valinomycin.


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