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 inhibi-tion 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.SIGNIFICANCE Answering the pathologically relevant, long-standing question of how the cardiac-and skeletal muscle -type ryanodine receptors are regulated by the Ca2+ content of the sarcoplasmic reticulum lumen is complicated by the technical issue that Ca2+ flowing through the channel interferes with its cytoplasmic Ca2+ regulation. An impermeable Ca2+ binding site agonist (Eu3+) allowed us to selectively study the luminal Ca2+ regulation of the channels. Our electrophysiological measurements gained new information about the function and location of the intrinsic luminal Ca2+ binding sites in both ryanodine receptor isoforms.