Potential flecainide binding or interaction sites on RyR2 that have been implicated in single channel lipid bilayer studies
| Binding site# . | Mechanism . | Voltage dependence . | Likely location . | Effective [flecainide] range given or IC50/AC50 . | Significant characteristics . | . |
|---|---|---|---|---|---|---|
| #1aa | Open state fast block Po↓, To↓, Tc↕ | +40 mV only | Cytoplasmic in the transmembrane domain | IC50 15–17 µM | IC50 with diastolic [Ca2+]s independent of Po | Watanabe et al. (2009), Mehra et al. (2014), Bannister et al. (2015) |
| #1b1 | Flecainide-induced substate | Described at +40 mV only | Cytoplasmic in the transmembrane domain | 40–500 µM (Mehra) or 1–50 µM (Bannister) | Appears to be mainly associated with fast block | Mehra et al. (2014), Bannister et al. (2015) |
| #2a | Slow block Tc↑ | +40 mV only | Cytoplasmic in the transmembrane domain | 50–100 µM | Most apparent in low Po channels | Mehra et al. (2014) |
| #31 | Burst inhibition Po↓, To↓, Tc↕ | +40 mV only | Cytoplasmic in the transmembrane domain | 20–50 µM | cis 2 mM ATP and 0.1 μM Ca2+ | Hilliard et al. (2010), Mehra et al. (2014) |
| #4a | Activation Po↑, To↕, Tc↕, Fo↑ | +40 and −40 mV | Cytoplasmic peripheral | 0.5–5.0 µM | Mainly in low activity RyR2 Po < 0.08 | Salvage et al. (2021a) |
| #4b1 | Flecainide-stabilized substate Po↑, To↑, Tc↓ | +40 and −40 mV | Cytoplasmic peripheral | 5.0–10 µM | Mainly in low activity RyR2 Po < 0.08 | Salvage et al. (2021a) Figs. 3, 5, and 6 in this manuscript |
| #5 | Inhibition Po↓, To↕, Tc↑, Fo↓ | +40 and −40 mV | Cytoplasmic peripheral | 10–100 µM | Mainly in high activity RyR2 Po > 0.08 | Salvage et al. (2021a) |
| Binding site# . | Mechanism . | Voltage dependence . | Likely location . | Effective [flecainide] range given or IC50/AC50 . | Significant characteristics . | . |
|---|---|---|---|---|---|---|
| #1aa | Open state fast block Po↓, To↓, Tc↕ | +40 mV only | Cytoplasmic in the transmembrane domain | IC50 15–17 µM | IC50 with diastolic [Ca2+]s independent of Po | Watanabe et al. (2009), Mehra et al. (2014), Bannister et al. (2015) |
| #1b1 | Flecainide-induced substate | Described at +40 mV only | Cytoplasmic in the transmembrane domain | 40–500 µM (Mehra) or 1–50 µM (Bannister) | Appears to be mainly associated with fast block | Mehra et al. (2014), Bannister et al. (2015) |
| #2a | Slow block Tc↑ | +40 mV only | Cytoplasmic in the transmembrane domain | 50–100 µM | Most apparent in low Po channels | Mehra et al. (2014) |
| #31 | Burst inhibition Po↓, To↓, Tc↕ | +40 mV only | Cytoplasmic in the transmembrane domain | 20–50 µM | cis 2 mM ATP and 0.1 μM Ca2+ | Hilliard et al. (2010), Mehra et al. (2014) |
| #4a | Activation Po↑, To↕, Tc↕, Fo↑ | +40 and −40 mV | Cytoplasmic peripheral | 0.5–5.0 µM | Mainly in low activity RyR2 Po < 0.08 | Salvage et al. (2021a) |
| #4b1 | Flecainide-stabilized substate Po↑, To↑, Tc↓ | +40 and −40 mV | Cytoplasmic peripheral | 5.0–10 µM | Mainly in low activity RyR2 Po < 0.08 | Salvage et al. (2021a) Figs. 3, 5, and 6 in this manuscript |
| #5 | Inhibition Po↓, To↕, Tc↑, Fo↓ | +40 and −40 mV | Cytoplasmic peripheral | 10–100 µM | Mainly in high activity RyR2 Po > 0.08 | Salvage et al. (2021a) |
Changes in channel gating parameters (Po, To, Tc, Fo) are given relative to control values before flecainide addition, where data is given in publications or inferred from publication figures. Increase indicated by ↑, decrease indicated by ↓, and no significant effect indicated by ↕. #1b1, The flecainide-induced substate appears to be a component of fast block and is suggested to be a function of number of flecainide molecules in the pore (Mehra et al., 2014), although the possibility that it is the result of flecainide binding to an additional binding site has not been excluded. #31, The effect of flecainide in reducing burst activity may be an independent mechanism and separate binding site in the pore (Mehra et al., 2014), or could be explained by a component of transitions between fast and slow block with 100 nM cytoplasmic Ca2+. #4b1, The flecainide-stabilized substate is tentatively attributed to the activation site. The substate must contribute to activation, but the relative contributions from maximal and substate openings have not been dissected. It remains possible that the stabilization is the result of flecainide binding to separate substate sensitive sites.
Fast and slow block are apparent in the same records. Slow block clearly increases the closed times, but To for slow block was not separated from the reduction in To as a result of fast block.