Figure 3.
Ca2+ sensitivity of GCAP1 is affected by myristoylation.(A–D), Ca2+ binding isotherms obtained using fluorescent indicator dye BAPTA-2 titration protocol (Peshenko and Dizhoor, 2006). (A) Ca2+ binding by myristoylated D6S GCAP1 (◦), non-myristoylated WT GCAP1 (•), and non-myristoylated G2A GCAP1 (♦). (B) Comparison of the experimental data for Ca2+ binding by G2A GCAP1 (♦) with the theoretical curve for three-center binding model calculated using previously reported macroscopic association constants, 6.3 × 107, 5.0 × 106, and 2.0 × 103 M-1 (Dell'Orco et al., 2010) (- - -); the corresponding dissociation constants are shown next to each trace. (C,D) Change of the binding stoichiometry in non-myristoylated GCAP1 with one (D144N/D148G, C) or two (D100N/D102G/D144N/D148G, D) EF-hands inactivated. The data were fitted using two different models: panels (A,B)—by three-center binding model, Cabound/GCAP = (K1Caf + 2K1K2Ca2f + 3K1K2K3Ca3f)/(1 + K1Caf + K1K2Ca2f + K1K2K3Ca3f), where K1, K2, and K3 are macroscopic equilibrium constants; panels (C,D)—by simplified hyperbolic saturation function, (Cabound/GCAP) = Bmax × Cafree/(Cafree + Kd), where Cabound is the concentration of Ca2+ bound to GCAP1, calculated as Cabound = Catotal – Cafree, Bmax is mol of Ca2+ bound per mol of GCAP1 at saturation, Kd is the apparent dissociation constant. The data shown are representative from 3 to 5 independent experiments producing virtually identical results. (E) Normalized activity of the recombinant RetGC1 expressed in HEK293 cells reconstituted with 10 μM purified myristoylated GCAP1 (◦) or G2A GCAP1 (•) at different free Ca2+ concentrations and 1 mm free Mg2+. The activities in each series were normalized by the maximal activity in the corresponding series. The data were fitted by the equation, A = Amax+(Amax−Amin)/(1 + (Cafree/Ca1/2)n), where A is RetGC activity, Ca1/2 is the free Ca2+ concentration producing 50% effect and n is the Hill coefficient. For other conditions of the assay see Materials and Methods.