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Location and orientation of minK within the I(Ks) potassium channel complex
1Department of Pharmacology, Division of Genetic Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6304, USA.
Abstract:
The slowly activating cardiac potassium current (I(Ks)) is generated by a heteromultimeric potassium channel complex consisting of pore-forming (KvLQT1) and accessory (minK) subunits belonging to the KCNQ and KCNE gene families, respectively. Evidence indicating that minK residues line the I(Ks) pore originates from the observation that two minK cysteine mutants (G55C and F54C) render I(Ks) Cd2+-sensitive. We have identified a single cysteine residue in the KvLQT1 S6 segment (Cys-331) that contributes to Cd2+ coordination in conjunction with cysteine residues engineered into the minK transmembrane domain. This observation indicates that minK resides in close proximity to S6 in the I(Ks) channel complex. On the basis of homology modeling that compares the KvLQT1 S6 segment with the structure of the bacterial potassium channel KcsA, we predict that the sulfhydryl side chain of Cys-331 projects away from the central axis of the KvLQT1 pore and suggest that minK resides outside of the permeation pathway. A preliminary model illustrating the orientation of minK with S6 was validated by successful prediction of a novel Cd2+ binding site created within the I(Ks) channel complex by engineering additional cysteine residues into both subunits. Our results indicate the location and orientation of minK within the I(Ks) channel complex and further suggest that Cd2+ exerts its effect on I(Ks) through an allosteric mechanism rather than direct pore blockade.
Insights
The accessory minK subunit is near the KvLQT1 S6 segment in cardiac potassium channels (I(Ks)). This proximity suggests minK influences I(Ks) function allosterically, not by direct pore blockade.
Area of Science:
- Molecular Biology
- Cardiovascular Physiology
- Ion Channel Function
Background:
- The slowly activating cardiac potassium current (I(Ks)) is crucial for heart rhythm.
- I(Ks) is formed by KvLQT1 and minK subunits, with previous studies suggesting minK lines the pore.
Purpose of the Study:
- To determine the precise location and orientation of the minK subunit within the I(Ks) channel complex.
- To elucidate the mechanism by which cadmium ions (Cd2+) affect I(Ks) function.
Main Methods:
- Site-directed mutagenesis to introduce cysteine residues into KvLQT1 and minK subunits.
- Cadmium sensitivity assays to probe subunit proximity and pore accessibility.
- Homology modeling based on KcsA bacterial potassium channel structure.
Main Results:
- A cysteine residue in KvLQT1 S6 (Cys-331) was identified to coordinate with engineered minK cysteines.
- Homology modeling predicted KvLQT1 Cys-331 faces away from the pore, suggesting minK is not in the permeation pathway.
- A novel Cd2+ binding site was engineered, validating a model of minK's proximity to KvLQT1 S6.
Conclusions:
- minK is located in close proximity to the KvLQT1 S6 segment within the I(Ks) channel complex.
- Cadmium ions likely affect I(Ks) function via an allosteric mechanism, not direct pore blockade, due to the positioning of minK.