Interaction between gallopamil and cardiac ryanodine receptors

1 In a sarcoplasmic reticulum fraction obtained from rat hearts, the analysis of equilibrium [ 3 H]‐ryanodine binding showed high and low affinity sites ( K D = 1.3 n m and 2.8 μ m , B max = 2.2 pmol mg −1 and 27.8 pmol mg −1 ). The dissociation rate constant increased at 1 μ m VS 4 n m [ 3 H]‐ryanodine concentration, and micromolar ryanodine slowed the dissociation of nanomolar ryanodine. 2 The binding of 4 n m [ 3 H]‐ryanodine was not affected by gallopamil, while the binding of 100 n m to 18 μ m [ 3 H]‐ryanodine was partly displaced. Data analysis suggested that gallopamil inhibited low affinity [ 3 H]‐ryanodine binding, with IC 50 in the micromolar range. 3 Gallopamil decreased the dissociation rate constant of 1 μ m [ 3 H]‐ryanodine. While gallopamil alone did not affect the dissociation of 4 n m [ 3 H]‐ryanodine, gallopamil and micromolar ryanodine slowed it to a greater extent than micromolar ryanodine alone. 4 Our results are consistent with the hypothesis that the ryanodine receptor is a negatively cooperative oligomer, which undergoes a sequential alteration after ryanodine binding. Gallopamil has complex actions: it inhibits ryanodine binding to its low affinity site(s), and probably modulates the cooperativity of ryanodine binding and/or the transition to a receptor state characterized by slow ryanodine dissociation. These molecular actions could account for the previously reported effect of gallopamil on the sarcoplasmic reticulum calcium release channel.