Interactions of Anionic Phospholipids and Phosphatidylethanolamine with the Potassium Channel KcsA

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Description/Abstract

Fluorescence quenching methods have been used to study interactions of anionic phospholipids with the
potassium channel KcsA from Streptomyces lividans. Quenching of the Trp fluorescence of KcsA reconstituted into mixtures of
dioleoylphosphatidylcholine (DOPC) and an anionic phospholipid with dibromostearoyl chains is more marked at low mole
fractions of the brominated anionic phospholipid than is quenching in mixtures of dibromostearoylphosphatidylcholine and
nonbrominated anionic lipid. The quenching data are consistent with two classes of binding site for lipid on KcsA, one set
corresponding to annular binding sites around KcsA to which DOPC and two-chain anionic phospholipids bind with similar
affinities, the other set (non-annular sites) corresponding to sites at which anionic phospholipids can bind but from which DOPC
is either excluded or binds with very low affinity. The binding constant for tetraoleoylcardiolipin at the annular sites is significantly
less than that for DOPC, being comparable to that for dioleoylphosphatidylethanolamine. Tetraoleoylcardiolipin binds with
highest affinity to the non-annular sites, the affinity for dioleoylphosphatidylglycerol being the lowest. The affinity for
dioleoylphosphatidylserine decreases at high ionic strength, suggesting that electrostatic interactions between the anionic
phospholipid headgroup and positively charged residues on KcsA are important for binding at the non-annular site. The effect of
ionic strength on the binding of phosphatidic acid is less marked than on phosphatidylserine. The value of the binding constant
for the non-annular site depends on the extent of Trp fluorescence quenching following from binding at the non-annular site. It is
suggested that the non-annular site to which binding is detected in the fluorescence quenching experiments corresponds to the
binding site for phosphatidylglycerol detected at monomer-monomer interfaces in x-ray diffraction studies.