[³H]Ryanodine binding stimulation by calcins. (A) Dose-dependent activation of RyR1 by calcins, with boundaries set at 1 pM to 20 µM (n = 3–5). Heavy SR from rabbit skeletal muscle was incubated with 5 nM [³H]ryanodine in the absence (control) and the presence of the indicated concentrations of calcins. Binding conditions were specified in Materials and methods. The K_d was determined with the formula B = (B_max)/[1 + (K_d/[calcin])^nH), where B is specific binding of [³H]ryanodine, B_max is the maximum binding stimulated by calcin, and nH is the Hill coefficient. (B) Effect of calcins (100 nM each; n = 3–5) on the Ca²⁺-dependent [³H]ryanodine binding curve. Ca²⁺-dependent activation and inactivation were fitted with the formula B = ((B_max·[Ca²⁺])/(K_a + [Ca²⁺]))·(K_i/(K_i + [Ca²⁺])), where B is the specific binding of [³H]ryanodine, B_max is the maximum binding stimulated by calcin, K_a is the activation constant, and K_i is the inactivation constant. The specific [³H]ryanodine binding was standardized with the value of the control at pCa5 as 100%. (C–E) Radar charts illustrating the effect of calcins on K_a, K_i, and maximal amplitude (A_max; percentage of stimulation with respect to control). The black line corresponds to control (no calcin) and is included in all charts for reference. Caffeine (C, dashed line) moves K_a but has little effect on A_max and no effect on K_i. This effect contrasts with that of calcins (C–E, color lines as indicated), which affect K_a, K_i, and A_max.