Figure 6. State-dependent flecainide block.

(A) Representative steady-state current traces of WT and E1784K before and after flecainide (10 and 100 μmol/l). Cells were depolarized by –20 mV from a holding potential of –150 mV. Currents were normalized by the peak current before flecainide and were superimposed. Calibration bars indicate 1 nA and 1 ms. (B) Concentration-response curve for flecainide-induced tonic block in WT (n = 5) and E1784K (n = 5). The normalized peak currents were fit to the Hill equation B(%) = 100/(1 + (D / IC₅₀)nH), where B is the percentage block at drug concentration D, and nH is the Hill coefficient. The IC₅₀ values, representing dissociation constants for resting state (K_R) were WT, 150.3 μmol/l and E1784K, 20.4 μmol/l. Thus the mutant channel was far more sensitive to tonic block by flecainide. (C) Dissociation constant for inactivated channels. Cells were depolarized by prepulses ranging from –150 mV to –60 mV for 500 ms from a holding potential of –150 mV to ensure steady-state inactivation, followed by a –20-mV test pulse in the presence or absence of 30 μmol/l flecainide. The pulse protocol cycle time was 60 s. Peak Na currents were normalized to the maximum Na current in the absence or presence of flecainide and were fit to the Boltzmann equation. The dissociation constant for the inactivated state (K_I) was calculated using Bean’s equation (27): V_1/2 – V_1/2c = k × ln([1 + D / K_R] / [1 + D / K_I]), where V_1/2 and V_1/2c are the midpoints of steady-state inactivation for flecainide and control, respectively, D is the flecainide concentration, and K_R is the dissociation constant for the resting state. The K_R values are equivalent to the IC₅₀ values measured at a holding potential of –150 mV (WT, 150.3 μmol/l; E1784K, 20.4 μmol/l) in B.