Intracellular proton-transfer mutants in a CLC Cl-/H+ exchanger.

The Journal of General Physiology
Hyun-Ho Lim, Christopher Miller

Abstract

CLC-ec1, a bacterial homologue of the CLC family's transporter subclass, catalyzes transmembrane exchange of Cl(-) and H(+). Mutational analysis based on the known structure reveals several key residues required for coupling H(+) to the stoichiometric countermovement of Cl(-). E148 (Glu(ex)) transfers protons between extracellular water and the protein interior, and E203 (Glu(in)) is thought to function analogously on the intracellular face of the protein. Mutation of either residue eliminates H(+) transport while preserving Cl(-) transport. We tested the role of Glu(in) by examining structural and functional properties of mutants at this position. Certain dissociable side chains (E, D, H, K, R, but not C and Y) retain H(+)/Cl(-) exchanger activity to varying degrees, while other mutations (V, I, or C) abolish H(+) coupling and severely inhibit Cl(-) flux. Transporters substituted with other nonprotonatable side chains (Q, S, and A) show highly impaired H(+) transport with substantial Cl(-) transport. Influence on H(+) transport of side chain length and acidity was assessed using a single-cysteine mutant to introduce non-natural side chains. Crystal structures of both coupled (E203H) and uncoupled (E203V) mutants are similar to...Continue Reading

References

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Citations

Mar 6, 2010·The Journal of Membrane Biology·Richard J Naftalin
Jan 6, 2010·Pflügers Archiv : European journal of physiology·Leigh WellhauserChristine E Bear
Nov 10, 2009·Nature Structural & Molecular Biology·Alessandra PicolloAlessio Accardi
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Mar 13, 2013·The Journal of General Physiology·Juan Garcia-CelmaRaimund Dutzler
Dec 15, 2012·PLoS Biology·Hyun-Ho LimChristopher Miller
Aug 25, 2011·The Journal of Chemical Physics·Alexander M BerezhkovskiiHuan-Xiang Zhou
Sep 17, 2013·Nature Chemical Biology·Hyun-Ho LimChristopher Miller
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Feb 18, 2016·Journal of the American Chemical Society·Tao JiangEmad Tajkhorshid
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Dec 7, 2016·Pflügers Archiv : European journal of physiology·Ju Yong SeongInsuk So
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Sep 15, 2020·Science Advances·Hyuk-Joon LeeJae-Sung Woo
Aug 15, 2019·Proceedings of the National Academy of Sciences of the United States of America·Kunwoong ParkHyun-Ho Lim
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Nov 14, 2018·Journal of the American Chemical Society·Zhi WangGregory A Voth
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Methods Mentioned

BETA
PCR
gel filtration
X-ray

Software Mentioned

pClamp
COOT
REFMAC5
PHENIX
SciFinderScholar
PHASER

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