Luminal addition of non-permeant Eu 3+ interferes with luminal Ca 2+ regulation of the cardiac ryanodine receptor.

•Non-permeant luminal Eu3+ interacts with RYR2 Ca2+ sites accommodating luminal Ca2+.•Luminal Ca2+-mediated RYR2 response to caffeine is antagonized by luminal Eu3+.•Gating behavior of the RYR2 channel is insensitive to luminal Eu3+.•RYR2 responsiveness to caffeine is likely controlled by a luminal-facing Ca2+ site.•RYR2 gating behavior is presumably regulated inside the channel pore.