Interactions of anionic phospholipids and phosphatidylethanolamine with the potassium channel KcsA

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.

FIGURE 1

The non-annular binding site for anionic phospholipid on KcsA. The lipid molecule, modeled as diacylglycerol (DAG) in the crystal structure of KcsA, is bound at the interface between monomers 1 and 2, whose surfaces are shaded light and dark gray, respectively. The partial lipid molecule modeled as nonan-1-ol (FA) is also shown (PDF file 1K4C).

FIGURE 2

Fluorescence emission spectra for KcsA. Spectra are shown for KcsA reconstituted into bilayers of DOPC (solid line), DOPG (long dash), and DOPA (short dash). Wavelengths of maximum emission are listed in Table 1. The concentration of KcsA was 0.24 μM and the molar ratio of lipid to KcsA was 100:1. The buffer was 20 mM HEPES and 1 mM EGTA at pH 7.2.

FIGURE 3

Quenching of KcsA fluorescence in mixtures with phosphatidylserine. KcsA was reconstituted into bilayers containing mixtures of BrPC and DOPS (□), BrPS and DOPC (○), and DOPC and DOPS (▵). Fluorescence intensities are expressed as F/F_o where F_o is the fluorescence intensity in the nonbrominated lipid. In A, the solid lines for the BrPC/DOPS and BrPS/DOPC experiments show fits to Eq. 2 giving the values for relative binding constants listed in Table 2. The broken line shows an attempt to fit the data for the BrPS/DOPC experiment to the binding constant determined from the BrPC/DOPS experiment, as described in the text. In B, the solid line for the BrPC/DOPS experiment shows a fit to Eq. 2 giving the annular binding constant and the lines for the BrPS/DOPC experiments show fits to the annular/non-annular binding site model, assuming (solid line) that all the fluorescence can be quenched from the non-annular sites (Eq. 5), and (broken line) that only 60% of the fluorescence can be quenched from the non-annular sites (Eq. 6). Values for the binding constants are listed in Table 3. The buffer was 20 mM HEPES and 1 mM EGTA at pH 7.2.

FIGURE 4

Quenching of KcsA fluorescence in mixtures with phosphatidic acid. KcsA was reconstituted into bilayers containing mixtures of BrPC and DOPA (□,▪), BrPA and DOPC (○,•), and DOPC and DOPA (▵), in 20 mM HEPES, and 1 mM EGTA, at pH 7.2, in the absence of K⁺ (unfilled symbols) or in the presence of 500 mM KCl (solid symbols). Fluorescence intensities are expressed as F/F_o where F_o is the fluorescence intensity in the nonbrominated lipid. In A, the solid lines for the BrPC/DOPA and BrPA/DOPC experiments in the absence of K⁺ show fits to Eq. 2 giving the values for relative binding constants listed in Table 2. In B, the solid line for the BrPC/DOPA experiment in the absence of K⁺ shows a fit to Eq. 2 giving the annular binding constant and the lines for the BrPA/DOPC experiments in the absence of K⁺ show fits to the annular/non-annular binding site model, assuming (solid line) that all the fluorescence can be quenched from the non-annular sites (Eq. 5) and (broken line) that only 60% of the fluorescence can be quenched from the non-annular sites (Eq. 6). Fits of the data in 500 mM KCl to Eqs. 2 and 6 are shown by the dotted lines. Values for the binding constants are listed in Table 3.

FIGURE 5

Quenching of KcsA fluorescence by brominated phospholipids. KcsA was reconstituted into bilayers containing mixtures of nonbrominated lipid and the corresponding brominated lipid at pH 7.2. Fluorescence intensities are expressed as a fraction of the fluorescence for KcsA reconstituted in the nonbrominated lipid. Phospholipids were as follows: (○), phosphatidylserine; (□), phosphatidic acid; and (▵), cardiolipin. The lines show fits to Eq. 1 giving the values for n listed in Table 1. The buffer was 20 mM HEPES and 1 mM EGTA at pH 7.2.

FIGURE 6

Quenching of KcsA fluorescence in mixtures with phosphatidylserine at high ionic strength. The buffer was 20 mM HEPES, 1 mM EGTA at pH 7.2, and 500 mM KCl. KcsA was reconstituted into bilayers containing mixtures of BrPC and DOPS (□), and BrPS and DOPC (○). Fluorescence intensities are expressed as F/F_o where F_o is the fluorescence intensity in the nonbrominated lipid. In A, the solid lines show fits to Eq. 2 giving the values for relative binding constants listed in Table 2. In B, the solid line for the BrPC/DOPS experiment shows a fit to Eq. 2 giving the annular binding constant and the lines for the BrPS/DOPC experiments show fits to the annular/non-annular binding site model, assuming (solid line) that all the fluorescence can be quenched from the non-annular sites (Eq. 5) and (broken line) that only 60% of the fluorescence can be quenched from the non-annular sites (Eq. 6). Values for the binding constants are listed in Table 3.

FIGURE 7

Quenching of KcsA fluorescence in mixtures with phosphatidylethanolamine. KcsA was reconstituted into bilayers containing mixtures of BrPC and DOPE (□) and BrPE and DOPC (○). Fluorescence intensities are expressed as F/F_o where F_o is the fluorescence intensity in the nonbrominated lipid. The solid lines show fits to Eq. 2 giving the values for relative binding constants listed in Table 2.

FIGURE 8

Quenching of KcsA fluorescence in mixtures with phosphatidylglycerol. KcsA was reconstituted into bilayers containing mixtures of BrPC and DOPG (□) and BrPG and DOPC (○). Fluorescence intensities are expressed as F/F_o where F_o is the fluorescence intensity in the nonbrominated lipid. The solid line for the BrPC/DOPG experiment shows a fit to Eq. 2 giving the annular binding constant and the lines for the BrPG/DOPC experiments show fits to the annular/non-annular binding site model, assuming (solid line) that all the fluorescence can be quenched from the non-annular sites (Eq. 5) and (broken line) that only 60% of the fluorescence can be quenched from the non-annular sites (Eq. 6). Values for the binding constants are listed in Table 3. The buffer was 20 mM HEPES and 1 mM EGTA at pH 7.2.

FIGURE 9

Quenching of KcsA fluorescence in mixtures with cardiolipin. KcsA was reconstituted into bilayers containing mixtures of BrPC and TOCL (□) and BrCL and DOPC (○). Fluorescence intensities are expressed as F/F_o where F_o is the fluorescence intensity in the nonbrominated lipid. The solid line for the BrPC/TOCL experiment shows a fit to Eq. 2 giving the annular binding constant and the lines for the BrCL/DOPC experiments show fits to the annular/non-annular binding site model, assuming (solid line) that all the fluorescence can be quenched from the non-annular sites (Eq. 5) and (broken line) that only 60% of the fluorescence can be quenched from the non-annular sites (Eq. 6). Values for the binding constants are listed in Table 3. The buffer was 20 mM HEPES and 1 mM EGTA at pH 7.2. The mole fraction of cardiolipin was calculated on a chain basis to account for the fact that cardiolipin contains four fatty acyl chains and phosphatidylcholine, two.