Analysis of a protein region involved in permeation and gating of the voltage-gated Torpedo chloride channel ClC-0

• Published in: The Journal of physiology Vol. 498; no. Pt 3; pp. 691 - 702
• Main Authors: Ludewig, U, Jentsch, T J, Pusch, M
• Format: Journal Article
• Language: English
• Published: England The Physiological Society 01-02-1997
• Subjects: Animals; Cell Membrane Permeability - genetics; Cell Membrane Permeability - physiology; Chloride Channels - genetics; Chloride Channels - metabolism; Chloride Channels - physiology; Electric Organ - innervation; Electric Organ - metabolism; Electric Stimulation; Electrophysiology; Ion Channel Gating - genetics; Ion Channel Gating - physiology; Membrane Potentials - physiology; Mutagenesis; Mutation; Patch-Clamp Techniques; Proteins - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Torpedo - physiology; Xenopus - metabolism
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1113/jphysiol.1997.sp021893

1. The chloride channel from the Torpedo electric organ, ClC-0, is controlled by two distinct ('fast' and 'slow') voltage-dependent
gates. Here we investigate the effects of mutations in a region after putative transmembrane domain D12. A mutation in this
region has previously been shown to change fast gating and permeation. 2. We used a combination of site-directed mutagenesis
with two-electrode voltage-clamp and patch-clamp measurements. 3. Most conservative substitutions have minor effects, while
more drastic mutations change kinetics and voltage dependence of fast gating, as well as ion selectivity and rectification.
4. While ClC-0 wild-type (WT) channels deactivate fully in two-electrode voltage clamp at negative voltages, channels do not
close completely in patch-clamp experiments. Open probability is increased by intracellular chloride in a concentration- but
not voltage-dependent manner. 5. In several mutants, including K519R, the minimal macroscopic open probability of fast gating
is larger than in WT. Mutant channels fluctuate at negative potentials between open and closed conformations. Open probability
is much more effectively increased by intracellular chloride than in WT. The observations support the idea that permeating
ions inside the pore stabilize the open state. 6. Besides effects on permeation and gating of single protopores, some mutations
affect 'slow' gating. In summary, the region after D12 participates in fast as well as in slow gating; mutations additionally
influence permeation properties.