Eu detects two functionally distinct luminal Ca binding sites in ryanodine receptors.

Ryanodine receptors (RyRs) are Ca release channels, gated by Ca 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 Ca 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 Eu, an impermeable agonist of Ca binding sites, as a probe to avoid this complication and to gain more specific information about the function of the luminal Ca 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 Ca, whereas RyR1 is inhibited. These results were qualitatively reproducible using Eu. The luminal regulation of RyR1 carrying a mutation associated with malignant hyperthermia was not different from that of the wild-type. RyR1 inhibition by Eu 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 Ca binding site sequences. We propose that RyR2 bears an activation site, which is missing in RyR1, but both isoforms share the same inhibitory Ca binding site near the channel gate.