The EF-hand Ca2+ Binding Domain Is Not Required for Cytosolic Ca2+ Activation of the Cardiac Ryanodine Receptor

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Activation of the cardiac ryanodine receptor (RyR2) by elevating cytosolic Ca2+ is a central step in the process of Ca2+-induced Ca2+ release, but the molecular basis of RyR2 activation by cytosolic Ca2+ is poorly defined. It has been proposed recently that the putative Ca2+ binding domain encompassing a pair of EF-hand motifs (EF1 and EF2) in the skeletal muscle ryanodine receptor (RyR1) functions as a Ca2+ sensor that regulates the gating of RyR1. Although the role of the EF-hand domain in RyR1 function has been studied extensively, little is known about the functional significance of the corresponding EF-hand domain in RyR2. Here we investigate the effect of mutations in the EF-hand motifs on the Ca2+ activation of RyR2. We found that mutations in the EF-hand motifs or deletion of the entire EF-hand domain did not affect the Ca2+-dependent activation of [3H]ryanodine binding or the cytosolic Ca2+ activation of RyR2. On the other hand, deletion of the EF-hand domain markedly suppressed the luminal Ca2+ activation of RyR2 and spontaneous Ca2+ release in HEK293 cells during store Ca2+ overload or store overload-induced Ca2+ release (SOICR). Furthermore, mutations in the EF2 motif, but not EF1 motif, of RyR2 raised the threshold for SOICR termination, whereas deletion of the EF-hand domain of RyR2 increased both the activation and termination thresholds for SOICR. These results indicate that, although the EF-hand domain is not required for RyR2 activation by cytosolic Ca2+, it plays an important role in luminal Ca2+ activation and SOICR.