Flecainide Specifically Targets the Monovalent Countercurrent Through the Cardiac Ryanodine Receptor, While a Dominant Opposing Ca2+/Ba2+ Current Is Present

Figures

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  Figure 1. Flecainide blocks the RyR2-mediated countercurrent. Representative current traces of RyR2 channels recorded at 0 mV when equal concentrations of Tris+ (10/10 mM) on both sides of the BLM were present, followed by perfusing the cis compartment with a solution containing 125 mM (A) or 250 mM Tris+ (D) to drive the Tris+ countercurrent in the cytosol-to-SR lumen direction. A dominant Ca2+/Ba2+ current in the SR lumen-to-cytosol direction flowed through the RyR2 channel; therefore, openings are shown as upward deflections from the marked zero level. Flecainide of 5, 50, and 200 µM was added to the cis compartment corresponding to the cytosol. No partial blocking events were observed in the all-point amplitude histograms shown alongside the current traces. The increase in blocking events with increasing flecainide concentration is depicted in the RyR2 G versus flecainide relationship and manifests as an increase in the RyR2 G (B,E). No significant changes in Po were observed (C,F). Data for (B,C,E,F) are shown as average ± SEM from n = 8 independent experiments. Statistical significance at the level * p < 0.05 and ** p < 0.01 compared to 0 µM flecainide and # p < 0.05 and ### p <…

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  Figure 2. Flecainide does not affect the RyR2 channel when the Tris+ countercurrent is not present. (A) Representative current traces of the RyR2 channel recorded at 0 mV when equal concentrations of Tris+ (10/10 mM) on both sides of the BLM were present. A sole Ca2+/Ba2+ current in the SR lumen-to-cytosol direction flowed through the RyR2 channel; therefore, openings are shown as upward deflections from the marked zero level. Flecainide of 5, 50, and 200 µM was added to the cis compartment corresponding to the cytosol. No partial blocking events were observed in the all-point amplitude histograms, shown alongside the current traces. No significant changes in RyR2 G (B) and Po were detected (C). Data for (B,C) are shown as average ± SEM from n = 8 independent experiments. NS indicates not significant.

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  Figure 3. Quantitative comparison of flecainide’s ability to block the RyR2-mediated countercurrent. (A) The flecainide dependence of RyR2 G for 10/10 mM (white circles), 125/10 mM (red circles), and 250/10 mM (blue circles) Tris+ gradients. Red and blue solid lines show the best fits to the Hill equation; the black line represents the best fit to a linear function. (B) The fitted values of G0, EC50 for flecainide, and Gmax obtained for 125/10 mM and 250/10 mM Tris+ gradients. A significant change was only observed in the fitted value of G0 corresponding to the G value at 0 µM flecainide. Statistical significance at the level * p < 0.05 compared to 125/10 mM Tris+ gradient. NS indicates not significant.

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  Figure 4. Flecainide does not target SR Cl− channels. Representative current traces of SR Cl− channels recorded at 0 mV when 450/150 mM Cl− (A) or opposite 150/450 mM Cl− gradient (D) was established. When a sole Cl− current flows in the cytosol-to-SR lumen, openings are shown as upward deflections from the marked zero-level (A). When a sole Cl− current flows in the opposite direction, openings are shown as downward deflections from the marked zero level (D). Flecainide of 5, 50, and 200 µM was added to the cis compartment corresponding to the cytosol. No partial blocking events were observed in the all-point amplitude histograms, shown alongside the current traces. No significant changes in G (B,E) and Po were observed (C,F). Data for (B,C,E,F) are shown as average ± SEM from n = 9 independent experiments. NS indicates not significant.

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  Figure 5. Flecainide significantly interacts with the lipid membrane supported on a SiO2 surface of the QCM sensor. (A) Representative response signal of the SiO2 QCM sensor when the lipid membrane was formed over the sensor surface, followed by injection of 0.2, 1, 2, 5, and 10 µM flecainide. Downward arrows indicate times of flecainide additions. The flecainide dependence of −ΔfS when flecainide (B) or caffeine (C) was tested. The data indicate a strong interaction between flecainide and the lipid membrane and a weak interaction with caffeine. (D) The −ΔfS plotted as a function of flecainide concentration. Red solid line shows the best fit to the Langmuir adsorption isotherm when flecainide is added; the black line represents the best fit to a linear function when caffeine is added. Data for (B,C) are shown as average ± SEM from n = 2–6 independent experiments. Statistical significance at the level * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to 0 µM flecainide. NS indicates not significant.