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Updated: Jul 16, 2026
07:47
A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters
Published on: April 20, 2015
Uncoupling and turnover in a Cl-/H+ exchange transporter
Michael Walden1, Alessio Accardi, Fang Wu
1Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, MA 02454, USA.
The Journal of General Physiology
|March 29, 2007
Summary
Mutations in the CLC-ec1 protein
Area of Science:
- Biochemistry
- Molecular Biology
- Membrane Transport
Background:
- The CLC-ec1 protein facilitates chloride (Cl-) and proton (H+) exchange.
- Its precise transport mechanism remains incompletely understood.
Purpose of the Study:
- Investigate the role of conserved tyrosine residue Y445 in CLC-ec1 function.
- Determine the impact of Y445 mutations on ion transport stoichiometry and mechanism.
Main Methods:
- Site-directed mutagenesis of the CLC-ec1 Y445 residue.
- Measurement of H+/Cl- transport ratios in mutated proteins.
- Analysis of proton pumping and chloride conductivity in reconstituted liposomes.
Main Results:
- Mutations at Y445 cause uncoupling of H+ and Cl- transport.
- Proton pumping is weakened in uncoupled mutants.
- Conductive, uncoupled Cl- transport (leak) is observed.
- Unitary Cl- transport rates remain high (~4,000 s-1) for wild-type and mutants.
Conclusions:
- Tyrosine 445 is critical for coupled H+/Cl- transport in CLC-ec1.
- Altering Y445 disrupts the stoichiometry, leading to uncoupled Cl- flux.
- The study reveals insights into the molecular basis of ion translocation in CLC transporters.
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