Disparate molecular mechanisms in cardiac ryanodine receptor channelopathies.

Aims: Mutations in the cardiac ryanodine receptor (RyR2) are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). This study investigates the underlying molecular mechanisms for CPVT mutations within the RyR2 N-terminus domain (NTD).

Methods and results: We consulted the high-resolution RyR2 structure in both open and closed configuration to identify mutations G357S/R407I and A77T, which lie within the NTD intra- and inter-subunit interface with the Core Solenoid (CSol), respectively. Their structural and functional roles were compared to R169L, a mutation that lies within the NTD-NTD inter-subunit interface. Using chemical cross-linking and co-immunoprecipitation assays, we show that R169L disrupts NTD tetramerization, while it does not alter the NTD-CSol interaction. Single cell Ca²⁺ imaging revealed that R169L increases the number of spontaneous Ca²⁺ transients and the proportion of oscillating cells, while reducing the Ca²⁺ store content. G357S and R407I do not affect NTD tetramerization, but they also do not alter the NTD-CSol interaction. Functionally, RyR2^G357S-expressing cells have Ca²⁺ handling properties similar to RyR2^WT. A77T enhances the NTD-CSol interaction, while it does not affect NTD tetramerization. Like R169L, A77T also increases the number of spontaneous Ca²⁺ transients and the proportion of oscillating cells, and it reduces the Ca²⁺ store content. However, unlike R169L that displays Ca²⁺ transients of normal amplitude and shorter duration, Ca²⁺ transients for A77T are of smaller amplitude and normal duration.

Conclusion: The NTD-CSol inter-subunit interface variant, A77T, produces a hyperactive channel by altering a different structure-function parameter to other CPVT mutations within the RyR2 NTD. Reduced NTD-NTD inter-subunit interaction and reinforced NTD inter-subunit interaction with CSol are distinct molecular mechanisms for gain-of-function RyR2 arrhythmogenic mutations.