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A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters
Published on: April 20, 2015
Amphiphilic blockers punch through a mutant CLC-0 pore
Xiao-Dong Zhang1, Tsung-Yu Chen
1Center for Neuroscience and Department of Neurology, University of California, Davis, CA 95618, USA.
The Journal of General Physiology
|December 18, 2008
Summary
Amphiphilic molecules like p-chlorophenoxy acetate (CPA) block CLC-0 channel pore mutants. Studies on the E166G mutant reveal blocker "punch-through" at negative potentials, indicating molecules can exit the pore.
Area of Science:
- Molecular biology
- Ion channel biophysics
- Membrane transport
Background:
- Amphiphilic molecules, including p-chlorophenoxy acetate (CPA) and octanoate, are known to inhibit CLC-0 channel function.
- Pore-open mutants of CLC-0 exhibit voltage-dependent block by these molecules.
Purpose of the Study:
- To investigate the phenomenon of blocker "punch-through" in the CLC-0 E166G pore-open mutant.
- To provide further evidence for CPA and octanoate punch-through by examining blocker dissociation kinetics.
Main Methods:
- Electrophysiological recordings of CLC-0 E166G mutant.
- Voltage-clamp experiments in symmetrical 140 mM Cl(-) solutions.
- Measurement of blocker dissociation rates under varying voltage and ionic conditions.
Main Results:
- The block of the E166G mutant by CPA showed multiphasic voltage dependence, with apparent affinity decreasing at potentials more negative than -80 mV.
- Dissociation rate of CPA from the E166G pore reached a minimum at -80 mV in symmetrical 140 mM Cl(-).
- CPA dissociation rate shifted with the Cl(-) reversal potential, supporting the punch-through hypothesis and suggesting asymmetric pore movement.
Conclusions:
- The findings provide strong evidence for the "punch-through" of CPA and octanoate in the CLC-0 E166G pore.
- The results suggest that blocker movement through the CLC-0 pore is voltage-dependent and exhibits asymmetry, with outward movement being more restricted.
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