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pH-dependent gating in the Streptomyces lividans K+ channel
L G Cuello1, J G Romero, D M Cortes
1Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville.
Biochemistry
|April 16, 1998
Summary
The small K+ channel from Streptomyces lividans (SKC1) functions as a pH-gated channel, opening at acidic pH. This discovery enables a new method for studying potassium channel function using radiotracer influx.
Area of Science:
- Biophysics
- Molecular Biology
- Ion Channel Research
Background:
- The small K+ channel from Streptomyces lividans (SKC1) is a well-expressed, stable channel suitable for structural studies.
- Functional characterization of SKC1 is crucial for understanding its mechanism and potential applications.
Purpose of the Study:
- To develop a functional assay for purified and reconstituted SKC1 using radiotracer uptake.
- To investigate the gating properties and ion selectivity of SKC1.
Main Methods:
- Reconstitution of SKC1 into liposomes and planar lipid bilayers.
- Radiotracer (86Rb+) uptake experiments to measure channel activity.
- Single-channel electrophysiology in planar lipid bilayers.
- Limited proteolysis to determine channel orientation.
Main Results:
- SKC1-mediated 86Rb+ uptake was observed at acidic external pH (3.5) and high intravesicular pH (7.5), indicating pH-gated influx.
- The channel exhibited K+ selectivity (K+ > Rb+ > NH4+ >> Na+ > Li+) and was blocked by Ba2+.
- Single-channel recordings in planar bilayers showed activity primarily at acidic pH (<5.0), with a main conductance of 135 pS.
- Proteolysis indicated a right-side-out orientation, suggesting the gating site is extracellular.
Conclusions:
- SKC1 functions as a pH-gated potassium channel, likely regulated by protonation of extracellular residues.
- The findings support the use of radiotracer influx assays for functional characterization of reconstituted SKC1.
- This study reveals a potentially novel gating mechanism for SKC1.