Molecular Determinants Of Ca2+ Release Termination In The Cardiac Ryanodine Receptor

A longstanding question in the field of excitation-contraction coupling in cardiac muscle is how Ca2+ release from the sarcoplasmic reticulum (SR) is terminated. Recent studies have suggested that SR Ca2+ release terminates as a result of luminal Ca2+ dependent inactivation of the Ca2+ release channel/ryanodine receptor (RyR2). However, the molecular basis of luminal Ca2+ dependent inactivation of RyR2 is unknown. We have previously shown that the pore region of RyR2 is critical for the initiation of spontaneous Ca2+ release or store overload induced Ca2+ release (SOICR). In the present study, we determined whether the pore region of RyR2 is also important for Ca2+ release termination. To this end, we mutated each residue within the inner helix and the helix bundle crossing, and generated stable, inducible HEK293 cell lines expressing theses mutants. Using the fluorescence resonance energy transfer (FRET)-based luminal Ca2+ sensing protein, D1ER, we monitored the luminal Ca2+ dynamics in HEK293 cells expressing RyR2 wt and mutants during Ca2+ overload. Interestingly, we found that the G4871R mutation significantly lowered the critical luminal Ca2+ level at which Ca2+ release is terminated (the termination threshold), but it had no effect on the critical luminal Ca2+ level at which spontaneous Ca2+ release or SOICR occurs (the SOICR threshold), as compared with wt. In contrast, the I4862A mutation markedly lowered the SOICR threshold with little impact on the termination threshold. On the other hand, the Q4876A mutation lowered both the SOICR and termination thresholds, whereas the E4872A mutation raised both thresholds. Taken together, our data demonstrate that the pore region of RyR2 is an important determinant of both activation and termination of Ca2+ release, and suggest that the pathways for Ca2+ release activation and termination are distinct but overlap.