Gating of RYR2 channels from the arrhythmic RYR2-P2328S mouse heart and some unexpected actions of flecainide

The P2328S mutation in mice is associated with arrhythmia and spontaneous diastolic calcium release in atrial and ventricular myocytes and there is a corresponding leftward shift in the Ca2+-activation curve for mutant RYR2 channels from homozygous mouse hearts (Salvage et al. 2019. J Cell Sci. https://doi.org/10.1242/jcs.229039). P2328 is located in helical domain 1 (HD1) of RYR2. Local structural changes likely result when structurally active proline residues are replaced by structurally inert serine residues. We speculate that local structural changes in HD1 lead to sequential intradomain and interdomain stearic changes through the protein to the distant channel gate, which favor the open pore conformation. The drug flecainide prevents arrhythmia in humans and mouse models of CPVT by blocking NaV1.5 and RYR2 channels. Conventionally, flecainide blocks RYR2 channels in a voltage-dependent manner. We did not observe voltage-dependent pore block. This was possibly because, in contrast to previous studies, the only channel modulators that we used to produce end-diastolic control channel activity were 1 µM cytoplasmic Ca2+ and 1 mM luminal Ca2+. We observed previously unreported, voltage-independent increases in WT and P2328S channel activity at low flecainide concentrations, followed by a decline in activity at higher concentrations. The increase in activity dominated the effect of flecainide on P2328S channels. These effects suggested high-affinity flecainide binding to an activation site and lower-affinity binding to an inhibition site, both distant from the channel pore (Salvage et al. 2021. Cells. https://doi.org/10.3390/cells10082101). Unlike channel block by flecainide, the drug under our conditions stabilized intrinsic sub-conductance activity at +40 mV and −40 mV. Since flecainide effectively reduces CPVT arrythmia clinically and in animal models, we conclude that voltage-independent inhibition and voltage-dependent channel block prevail under cellular conditions. However, channel activation is important to note as it may be unmasked in other circumstances such as acquired cardiac disorders, mutations, or additional drug applications.