Molecular Basis of Luminal Ca2+ Gating of the Cardiac Ryanodine Receptor

Abstract

It has long been known that Ca2+ release from the sarcoplasmic reticulum (SR) can occur spontaneously when the SR Ca2+ content reaches a critical threshold, and that this spontaneous SR Ca2+ release can lead to lethal cardiac arrhythmias. Although it is clear that spontaneous SR Ca2+ release results from the activation of the cardiac ryanodine receptor (RyR2) by luminal Ca2+, the molecular basis of luminal Ca2+ activation of RyR2 remains undefined and controversial. An increasing body of evidence indicates that the RyR2 channel contains a luminal Ca2+ sensor distinct from its cytosolic Ca2+ sensor. To localize this luminal Ca2+ sensor, we have systematically mutated each of the negatively charged residues in the predicted pore-forming region of RyR2 that is likely to be accessible to luminal Ca2+. We found that alanine-mutations within and near the COOH-terminal end of the TM10 transmembrane helix (the pore inner helix) abolish the luminal, but not the cytosolic, Ca2+ activation of single RyR2 channels. HEK293 cells expressing these alanine-mutants display caffeine-induced Ca2+ release, but show no store-overload-induced Ca2+ release. Interestingly, introducing histidines into this region creates a high affinity metal binding site. The histidine-mutant channels are blocked by Ni2+ (<1 uM), while 100 uM Ni2+ does not block the RyR2 wt channels. Molecular modeling of the RyR2 channel pore based on the three-dimensional structure of the KcsA potassium channel reveals a cluster of negatively charged residues located within the internal pore at the helix bundle crossing, which is thought to form the ion gate of the RyR2 channel. Collectively, our results suggest that luminal Ca2+ opens the RyR2 channel by binding to the intracellular gate.