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

Dysregulation of the cardiac ryanodine receptor (RYR2) by luminal Ca has been implicated in a life-threatening, stress-induced arrhythmogenic disease. The mechanism of luminal Ca-mediated RYR2 regulation is under debate, and it has been attributed to Ca binding on the cytosolic face (the Ca feedthrough mechanism) and/or the luminal face of the RYR2 channel (the true luminal mechanism). The molecular nature and location of the luminal Ca site is unclear. At the single-channel level, we directly probed the RYR2 luminal face by Eu, considering the non-permeant nature of trivalent cations and their high binding affinities for Ca sites. Without affecting essential determinants of the Ca feedthrough mechanism, we found that luminal Eu competitively antagonized the activation effect of luminal Ca on RYR2 responsiveness to cytosolic caffeine, and no appreciable effect was observed for luminal Ba (mimicking the absence of luminal Ca). Importantly, luminal Eu caused no changes in RYR2 gating. Our results indicate that two distinct Ca sites (available for luminal Ca even when the channel is closed) are likely involved in the true luminal mechanism. One site facing the lumen regulates channel responsiveness to caffeine, while the other site, presumably positioned in the channel pore, governs the gating behavior.