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Abstract:
Interactions of fatty acids with the potassium channel KcsA were studied using Trp
fluorescence quenching and electron paramagnetic resonance (EPR) techniques. The brominated analogue of
oleic acid was shown to bind to annular sites on KcsA and to the nonannular sites at each protein
−
protein
interface in the homotetrameric structure with binding constants relative to dioleoylphosphatidylcholine of
0.67
±
0.04 and 0.87
±
0.08, respectively. Mutation of the two Arg residues close to the nonannular binding
sites had no effect on fatty acid binding. EPR studies with a spin-labeled analogue of stearic acid detected a
high-affinity binding site for the fatty acid with strong immobilization. Fluorescence quenching studies with the
spin-labeled analogue showed that the binding site detected in the EPR experiments could not be one of the annular or
nonannular binding sites. Instead, it is proposed that the EPR studies detect binding to the central hydrophobic cavity of the
channel, with a binding constant in the range of
∼
0.1
−
1
μ
M
