FIGURE 4.

Dissociation of the complex between Ca²⁺-saturated CaM and the CaM-binding domain of RyR1 by SPC, revealed by the fluorescence of dansyl-labeled CaM and the Trp of the RyR peptide. A, spectra of 0.2 μm Ca²⁺-saturated dansyl-CaM (○), 0.2 μm Ca²⁺-saturated dansyl-CaM with 0.5 μm RyR peptide (●), 0.2 μm Ca²⁺-saturated dansyl-CaM in the presence of 100 μm SPC (△), and 0.2 μm Ca²⁺-saturated dansyl-CaM in the presence of 0.5 μm RyR peptide and 100 μm SPC (▲). B, spectra of 1 μm RyR peptide (□), 1 μm RyR peptide with 1 μm Ca²⁺-saturated CaM (■), 1 μm RyR peptide in the presence of 100 μm SPC (▿), and 1 μm RyR peptide in the presence of 1 μm Ca²⁺-saturated CaM and 100 μm SPC (▼). Spectra in panels A and B are averaged from three independent measurements. C, effect of related lysophospholipids on the interaction between Ca²⁺-saturated CaM and the CaM-binding domain of RyR1. Bars depict the fluorescence intensity of 0.2 μm Ca²⁺-saturated dansyl-CaM in the presence of 0.5 μm RyR peptide and 100 μm lipids. D, the same as in panel C, but measuring the Trp fluorescence of the RyR peptide. Bars depict the fluorescence intensity of 1 μm RyR peptide in the presence of 1 μm Ca²⁺-saturated CaM and 100 μm lipids. Mean ± S.E. values in panels C and D were calculated from three independent experiments. The addition of SPC and LT-SPC brought forth a significant decrease in intensity (p < 0.05, see asterisks), based on Student's t test. E, concentration dependence of the complex dissociating ability of SPC. Ca²⁺-saturated dansyl-CaM and RyR peptide concentrations were 0.2 and 0.5 μm, respectively. F, the same as in panel E, but measuring the Trp fluorescence of the RyR peptide. RyR peptide and Ca²⁺-saturated CaM concentrations were both 1 μm. Data points in panels E and F represent the mean ± S.E. values of three independent determinations. Fitting a sigmoidal dose-response function yielded an EC₅₀ value of 19.4 ± 1.4 μm with a cooperativity coefficient of 2.6 ± 0.4 for dansyl-CaM, and an EC₅₀ value of 19.3 ± 3.4 μm with a cooperativity coefficient of 2.2 ± 0.8 for Trp fluorescence of the peptide.