Eu³⁺ detects two functionally distinct luminal Ca²⁺ binding sites in ryanodine receptors

Ryanodine receptors (RyRs) are Ca²⁺ release channels, gated by Ca²⁺ in the cytosol and the sarcoplasmic reticulum lumen. Their regulation is impaired in certain cardiac and muscle diseases. Although a lot of data is available on the luminal Ca²⁺ regulation of RyR, its interpretation is complicated by the possibility that the divalent ions used to probe the luminal binding sites may contaminate the cytoplasmic sites by crossing the channel pore. In this study, we used Eu³⁺, an impermeable agonist of Ca²⁺ binding sites, as a probe to avoid this complication and to gain more specific information about the function of the luminal Ca²⁺ sensor. Single-channel currents were measured from skeletal muscle and cardiac RyRs (RyR1 and RyR2) using the lipid bilayer technique. We show that RyR2 is activated by the luminal addition of Ca²⁺, whereas RyR1 is inhibited. These results were qualitatively reproducible using Eu³⁺. The luminal regulation of RyR1 carrying a mutation associated with malignant hyperthermia was not different from that of the wild-type. RyR1 inhibition by Eu³⁺ was extremely voltage dependent, whereas RyR2 activation did not depend on the membrane potential. These results suggest that the RyR1 inhibition site is in the membrane's electric field (channel pore), whereas the RyR2 activation site is outside. Using in silico analysis and previous results, we predicted putative Ca²⁺ binding site sequences. We propose that RyR2 bears an activation site, which is missing in RyR1, but both isoforms share the same inhibitory Ca²⁺ binding site near the channel gate.