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.