Background: Ivabradine is a specific bradycardic agent used in coronary artery disease and heart failure, lowering heart rate through inhibition of sinoatrial nodalHCN‐channels. This study investigated the propensity of ivabradine to interact withKCNH2‐encoded human Ether‐à‐go‐go–Related Gene (hERG) potassium channels, which strongly influence ventricular repolarization and susceptibility to torsades de pointes arrhythmia.Methods and Results: Patch clamp recordings ofhERGcurrent (I_hERG) were made fromhERGexpressing cells at 37°C. I_hERGwas inhibited with anIC₅₀of 2.07 μmol/L for thehERG1a isoform and 3.31 μmol/L for coexpressedhERG1a/1b. The voltage and time‐dependent characteristics of I_hERGblock were consistent with preferential gated‐state‐dependent channel block. Inhibition was partially attenuated by the N588K inactivation‐mutant and the S624A pore‐helix mutant and was strongly reduced by the Y652A and F656A S6 helix mutants. In docking simulations to a MthK‐based homology model ofhERG, the 2 aromatic rings of the drug could form multiple π‐π interactions with the aromatic side chains of both Y652 and F656. In monophasic action potential (MAP) recordings from guinea‐pig Langendorff‐perfused hearts, ivabradine delayed ventricular repolarization and produced a steepening of theMAPD₉₀restitution curve.Conclusions: Ivabradine prolongs ventricular repolarization and alters electrical restitution properties at concentrations relevant to the upper therapeutic range. In absolute terms ivabradine does not discriminate betweenhERGandHCNchannels: it inhibits I_hERGwith similar potency to that reported for native I_fandHCNchannels, with S6 binding determinants resembling those observed forHCN4. These findings may have important implications both clinically and for future bradycardic drug design.

One form of the short QT syndrome (SQT3) has been linked to the D172N gain-in-function mutation to Kir2.1, which preferentially increases outward current through channels responsible for inward rectifier K⁺ current (I_K1). This study investigated mechanisms by which the Kir2.1 D172N mutation facilitates and perpetuates ventricular arrhythmias. The ten Tusscher et al. model for human ventricular action potentials (APs) was modified to incorporate changes to I_K1 based on experimentally observed changes to Kir2.1 function: both heterozygous (WT-D172N) and homozygous (D172N) mutant scenarios were studied. Cell models were incorporated into heterogeneous one-dimensional (1D), 2D tissue, and 3D models to compute the restitution curves of AP duration (APD-R), effective refractory period (ERP-R), and conduction velocity (CV). Temporal and spatial vulnerability of ventricular tissue to re-entry was measured and dynamic behaviour of re-entrant excitation waves (lifespan and dominant frequency) in 2D and 3D models of the human ventricle was characterized. D172N ‘mutant’ I_K1 led to abbreviated APD and ERP, as well as steeper APD-R and ERP-R curves. It reduced tissue excitability at low excitation rates but increased it at high rates. It increased tissue temporal vulnerability for initiating re-entry, but reduced the minimal substrate size necessary to sustain re-entry. SQT3 ‘mutant’ I_K1 also stabilized and accelerated re-entrant excitation waves, leading to sustained rapid re-entry. Increased I_K1 due to the Kir2.1 D172N mutation increases arrhythmia risk due to increased tissue vulnerability, shortened ERP, and altered excitability, which in combination facilitate initiation and maintenance of re-entrant circuits.

The antiarrhythmic drug amiodarone delays cardiac repolarisation through inhibition of hERG-encoded potassium channels responsible for the rapid delayed rectifier potassium current (IKr). This study aimed to elucidate molecular determinants of amiodarone binding to the hERG channel. Whole-cell patch-clamp recordings were made at 37°C of ionic current (IhERG) carried by wild-type (WT) or mutant hERG channels expressed in HEK293 cells. Alanine mutagenesis and ligand docking were used to investigate the roles of pore cavity amino-acid residues in amiodarone binding. Amiodarone inhibited WT outward IhERG tails with a half-maximal inhibitory concentration (IC50) of ∼45nM, whilst inward IhERG tails in a high K(+) external solution ([K(+)]e) of 94mM were blocked with an IC50 of 117.8nM. Amiodarone's inhibitory action was contingent upon channel gating. Alanine-mutagenesis identified multiple residues directly or indirectly involved in amiodarone binding. The IC50 for the S6 aromatic Y652A mutation was increased to ∼20-fold that of WT IhERG, similar to the pore helical mutant S624A (∼22-fold WT control). The IC50 for F656A mutant IhERG was ∼17-fold its corresponding WT control. Computational docking using a MthK-based hERG model differentiated residues likely to interact directly with drug and those whose Ala mutation may affect drug block allosterically. The requirements for amiodarone block of aromatic residues F656 and Y652 within the hERG pore cavity are smaller than for other high affinity IhERG inhibitors, with relative importance to amiodarone binding of the residues investigated being S624A∼Y652A>F656A>V659A>G648A>T623A.

The class Ic antiarrhythmic drug flecainide inhibits KCNH2-encoded "hERG" potassium channels at clinically relevant concentrations. The aim of this study was to elucidate the underlying molecular basis of this action. Patch clamp recordings of hERG current (IhERG) were made from hERG expressing cells at 37°C. Wild-type (WT) IhERG was inhibited with an IC50 of 1.49μM and this was not significantly altered by reversing the direction of K(+) flux or raising external [K(+)]. The use of charged and uncharged flecainide analogues showed that the charged form of the drug accesses the channel from the cell interior to produce block. Promotion of WT IhERG inactivation slowed recovery from inhibition, whilst the N588K and S631A attenuated-inactivation mutants exhibited IC50 values 4-5 fold that of WT IhERG. The use of pore-helix/selectivity filter (T623A, S624A V625A) and S6 helix (G648A, Y652A, F656A) mutations showed <10-fold shifts in IC50 for all but V625A and F656A, which respectively exhibited IC50s 27-fold and 142-fold their WT controls. Docking simulations using a MthK-based homology model suggested an allosteric effect of V625A, since in low energy conformations flecainide lay too low in the pore to interact directly with that residue. On the other hand, the molecule could readily form π-π stacking interactions with aromatic residues and particularly with F656. We conclude that flecainide accesses the hERG channel from the cell interior on channel gating, binding low in the inner cavity, with the S6 F656 residue acting as a principal binding determinant.

KCNQ1 (alias KvLQT1 or Kv7.1) and KCNE1 (alias IsK or minK) co-assemble to form the voltage-activated K⁺ channel responsible for I_Ks—a major repolarizing current in the human heart—and their dysfunction promotes cardiac arrhythmias. The channel is a component of larger macromolecular complexes containing known and undefined regulatory proteins. Thus, identification of proteins that modulate its biosynthesis, localization, activity, and/or degradation is of great interest from both a physiological and pathological point of view. Using a yeast two-hybrid screening, we detected a direct interaction between β-tubulin and the KCNQ1 N-terminus. The interaction was confirmed by co-immunoprecipitation of β-tubulin and KCNQ1 in transfected COS-7 cells and in guinea pig cardiomyocytes. Using immunocytochemistry, we also found that they co-localized in cardiomyocytes. We tested the effects of microtubule-disrupting and -stabilizing agents (colchicine and taxol, respectively) on the KCNQ1–KCNE1 channel activity in COS-7 cells by means of the permeabilized-patch configuration of the patch-clamp technique. None of these agents altered I_Ks. In addition, colchicine did not modify the current response to osmotic challenge. On the other hand, the I_Ks response to protein kinase A (PKA)-mediated stimulation depended on microtubule polymerization in COS-7 cells and in cardiomyocytes. Strikingly, KCNQ1 channel and Yotiao phosphorylation by PKA—detected by phospho-specific antibodies—was maintained, as was the association of the two partners. We propose that the KCNQ1–KCNE1 channel directly interacts with microtubules and that this interaction plays a major role in coupling PKA-dependent phosphorylation of KCNQ1 with I_Ks activation.