Flecainide increases Kir2.1 currents by interacting with cysteine 311, decreasing the polyamine-induced rectification

Abstract

Both increase and decrease of cardiac inward rectifier current (I(K1)) are associated with severe cardiac arrhythmias. Flecainide, a widely used antiarrhythmic drug, exhibits ventricular proarrhythmic effects while effectively controlling ventricular arrhythmias associated with mutations in the gene encoding Kir2.1 channels that decrease I(K1) (Andersen syndrome). Here we characterize the electrophysiological and molecular basis of the flecainide-induced increase of the current generated by Kir2.1 channels (I(Kir2.1)) and I(K1) recorded in ventricular myocytes. Flecainide increases outward I(Kir2.1) generated by homotetrameric Kir2.1 channels by decreasing their affinity for intracellular polyamines, which reduces the inward rectification of the current. Flecainide interacts with the HI loop of the cytoplasmic domain of the channel, Cys311 being critical for the effect. This explains why flecainide does not increase I(Kir2.2) and I(Kir2.3), because Kir2.2 and Kir2.3 channels do not exhibit a Cys residue at the equivalent position. We further show that incubation with flecainide increases expression of functional Kir2.1 channels in the membrane, an effect also determined by Cys311. Indeed, flecainide pharmacologically rescues R67W, but not R218W, channel mutations found in Andersen syndrome patients. Moreover, our findings provide noteworthy clues about the structural determinants of the C terminus cytoplasmic domain of Kir2.1 channels involved in the control of gating and rectification.

Fig. 1.

Flecainide increases I_Kir2.1. (A) I_Kir2.1 traces recorded by applying the protocol shown in the absence and presence of flecainide. (B) I–V curves for currents measured at the end of the pulses. (Inset) Data at potentials positive to E_K in an expanded scale. *P < 0.05 and **P < 0.01 vs. control. (C) Flecainide-induced change on the current recorded at −50 mV in cells expressing homotetramers of Kir2.1, Kir2.2, or Kir2.3 channels or heterotetramers of Kir2.1 + Kir2.2 and Kir2.1 + Kir2.3. *P < 0.05 vs. Kir2.1. Each point/bar represents the mean ± SEM of five or more experiments.

Fig. 2.

Flecainide increases ventricular but not atrial I_K1. Voltage ramp (800 ms) from −100 to −10 mV (A) and I–V curves (B) for human atrial I_K1 in the absence and presence of flecainide. Representative I_K1 traces recorded in guinea-pig atrial (C) or ventricular (E) myocytes by applying the protocol shown. I–V curves for guinea-pig atrial (D) or ventricular (F) I_K1. (G) I–V of the Ba²⁺-sensitive and Ba²⁺-insensitive currents recorded in ventricular myocytes before and after application of flecainide. (H) mRNA expression level of Kir2.x channels in guinea-pig atrial and ventricular samples. First lane shows molecular weight marker (1,000–100 bp). Lanes 2–4 and 5–7 show Kir2.1 (325 bp), Kir2.2 (291 bp), and Kir2.3 (303 bp) mRNA expression in atrial and ventricular tissue, respectively. GAPDH gene was used as internal standard. Each point represents the mean ± SEM of four experiments in each group. *P < 0.05 vs. control.

Fig. 3.

Flecainide 1 μM increases mean open time, f_o, and P_o, of Kir2.1 channels. (A) Single-channel recordings under control conditions and after perfusion with flecainide. Closed- and open-channel levels are indicated by C and O, respectively. Unitary current amplitude (B), mean open time (C), f_o (D), and P_o (E) in the absence and presence of flecainide. (F) P_o–V in control conditions and in the presence of flecainide. Solid lines represent the fit of a Boltzmann function to the data. (G) Single-channel current-voltage relationships in the absence and presence of flecainide. Solid lines represent the fit of a linear function to the data. Each point/bar represents the mean ± SEM of six experiments. *P < 0.05 and **P < 0.01 vs. control.

Fig. 4.

Flecainide decreases polyamine blockade. (A) I_Kir2.1 normalized to the amplitudes at −120 mV at potentials positive to E_K in control conditions and in the presence of flecainide. (B) Mean relative Gc in control conditions and in the presence of flecainide. Solid lines represent the fit of a Boltzmann function to the data. (Inset) Mean I–V curve and the current predicted, assuming a linear unblocked current in control conditions. (C) Current traces recorded at +70 mV in excised inside-out patches from HEK-293 cells expressing Kir2.1 channels in control conditions and after cytoplasmic surface application of Spm in the absence and presence of flecainide. Dashed lines represent the zero current level. (D) Percentage of current inhibition at +70 mV in excised inside-out patches as a function of Spm concentrations in the absence and presence of flecainide. (E) I–V curves for E224A Kir2.1 channels in the absence and presence of flecainide. (F) Flecainide-induced change on the current recorded at −50 mV in CHO cells expressing WT, E224A, E299A, and D259A Kir2.1 channels. *P < 0.05 vs. control. Each point/bar represents the mean ± SEM of five or more experiments.

Fig. 5.

Cys311 is critical for the flecainide-induced increase of I_Kir2.1. The I–V curves for currents recorded in cells expressing C311A Kir2.1 (A) and A312C Kir2.2 (B) channels in the absence and presence of flecainide. (C) Current traces recorded in a cell expressing A312C Kir2.2 channels in control conditions and in the presence of flecainide. (D) Flecainide-induced change on the current recorded at 40 mV positive to E_K in cells expressing WT or mutant Kir2.x channels. *P < 0.05 vs. control. Each point/bar represents the mean ± SEM of five or more experiments.

Fig. 6.

Effects of flecainide on Kir2.1 channel density. Current traces recorded in a cell expressing R67W (A) and R218W (B) Kir2.1 in the absence and presence of flecainide. Current traces recorded in cells expressing R67W (C) and R218W (D) Kir2.1 channels incubated with flecainide for 24 h. The I–V curves for currents recorded in cells expressing R67W (E), L222I (F), WT (G), and C311A (H) Kir2.1 channels in control conditions and after incubation with flecainide. In G, squares represent acute effects of flecainide produced in cells incubated with flecainide. *P < 0.01 vs. control. ^ΦP < 0.05 vs. incubated cells. Each point represents the mean ± SEM of eight experiments.