BackgroundIvabradine is a specific bradycardic agent used in coronary artery disease and heart failure, lowering heart rate through inhibition of sinoatrial nodal HCN‐channels. This study investigated the propensity of ivabradine to interact with KCNH2‐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 ResultsPatch clamp recordings of hERG current (IhERG) were made from hERG expressing cells at 37°C. IhERG was inhibited with an IC50 of 2.07 μmol/L for the hERG 1a isoform and 3.31 μmol/L for coexpressed hERG 1a/1b. The voltage and time‐dependent characteristics of IhERG block 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 of hERG, 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 the MAPD90 restitution curve.ConclusionsIvabradine prolongs ventricular repolarization and alters electrical restitution properties at concentrations relevant to the upper therapeutic range. In absolute terms ivabradine does not discriminate between hERG and HCN channels: it inhibits IhERG with similar potency to that reported for native If and HCN channels, with S6 binding determinants resembling those observed for HCN4. These findings may have important implications both clinically and for future bradycardic drug design.

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 1.49 μM 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.

Ion channel behavior can depend strongly on temperature, with faster kinetics at physiological temperatures leading to considerable changes in currents relative to room temperature. These temperature-dependent changes in voltage-dependent ion channel kinetics (rates of opening, closing, inactivating, and recovery) are commonly represented with Q 10 coefficients or an Eyring relationship. In this article, we assess the validity of these representations by characterizing channel kinetics at multiple temperatures. We focus on the human Ether-à-go-go-Related Gene (hERG) channel, which is important in drug safety assessment and commonly screened at room temperature so that results require extrapolation to physiological temperature. In Part I of this study, we established a reliable method for high-throughput characterization of hERG1a (Kv11.1) kinetics, using a 15-second information-rich optimized protocol. In this Part II, we use this protocol to study the temperature dependence of hERG kinetics using Chinese hamster ovary cells overexpressing hERG1a on the Nanion SyncroPatch 384PE, a 384-well automated patch-clamp platform, with temperature control. We characterize the temperature dependence of hERG gating by fitting the parameters of a mathematical model of hERG kinetics to data obtained at five distinct temperatures between 25 and 37°C and validate the models using different protocols. Our models reveal that activation is far more temperature sensitive than inactivation, and we observe that the temperature dependency of the kinetic parameters is not represented well by Q 10 coefficients; it broadly follows a generalized, but not the standardly-used, Eyring relationship. We also demonstrate that experimental estimations of Q 10 coefficients are protocol dependent. Our results show that a direct fit using our 15-s protocol best represents hERG kinetics at any given temperature and suggests that using the Generalized Eyring theory is preferable if no experimental data are available to derive model parameters at a given temperature.

BackgroundThe familial Short QT Syndrome (SQTS) is associated with an increased risk of cardiac arrhythmia and sudden death. Gain-of-function mutations in the hERG K+ channel protein have been linked to variant 1 of the SQTS. A hERG channel pore (T618I) mutation has recently been identified in families with heritable SQTS. This study aimed to determine effects of the T618I-hERG mutation on (i) hERG current (IhERG) elicited by ventricular action potentials; (ii) the sensitivity of IhERG to inhibition by four clinically used antiarrhythmic drugs.MethodsElectrophysiological recordings of IhERG were made at 37°C from HEK 293 cells expressing wild-type (WT) or T618I hERG. Whole-cell patch clamp recording was performed using both conventional voltage clamp and ventricular action potential (AP) clamp methods.ResultsUnder conventional voltage-clamp, WT IhERG peaked at 0-+10 mV, whilst for T618I IhERG maximal current was right-ward shifted to ∼ +40 mV. Voltage-dependent activation and inactivation of T618I IhERG were positively shifted (respectively by +15 and ∼ +25 mV) compared to WT IhERG. The IhERG ‘window’ was increased for T618I compared to WT hERG. Under ventricular AP clamp, maximal repolarising WT IhERG occurred at ∼ -30 mV, whilst for T618I hERG peak IhERG occurred earlier during AP repolarisation, at ∼ +5 mV. Under conventional voltage clamp, half-maximal inhibitory concentrations (IC50) for inhibition of IhERG tails by quinidine, disopyramide, D-sotalol and flecainide for T618I hERG ranged between 1.4 and 3.2 fold that for WT hERG. Under action potential voltage clamp, T618I IC50s ranged from 1.2 to 2.0 fold the corresponding IC50 values for WT hERG.ConclusionsThe T618I mutation produces a more modest effect on repolarising IhERG than reported previously for the N588K-hERG variant 1 SQTS mutation. All drugs studied here appear substantially to retain their ability to inhibit IhERG in the setting of the SQTS-linked T618I mutation.

Background: Membrane-associated guanylate kinase (MAGUK) proteins function as adaptor proteins to mediate the recruitment and scaffolding of ion channels in the plasma membrane in various cell types. In the heart, the protein CASK (Calcium/CAlmodulin-dependent Serine protein Kinase) negatively regulates the main cardiac sodium channel, NaV1.5, which carries the sodium current (INa) by preventing its anterograde trafficking. CASK is also a new member of the dystrophin-glycoprotein complex, and like syntrophin, binds to the C-terminal domain of the channel. Objective: To unravel the mechanisms of CASK-mediated negative INa regulation, and interaction with the dystrophin-glycoprotein complex in cardiac myocytes. Methods: CASK adenoviral truncated constructs with sequential single functional domain deletions were designed for overexpression in cardiac myocytes: CASK CAMKII , CASK L27A , CASK L27B , CASK PDZ , CASK SH3 , CASK HOOK , and CASK GUK. Combination of whole-cell patch-clamp recording, total internal reflection fluorescence microscopy (TIRFm) and biochemistry experiments were conducted in cardiac myocytes to study the functional consequences of domain deletions. Results: We show that both L27B and GUK domains are required for the negative regulatory effect of CASK on INa and NaV1.5 surface expression and that the HOOK domain is essential for interaction with the cell adhesion dystrophin-glycoprotein complex. Conclusions: This study demonstrates that the multi-modular structure of CASK confers an ability to simultaneously interact with several targets within cardiomyocytes. Through its L27B, GUK, and HOOK domains, CASK potentially provides the ability to control channel delivery at adhesion points in cardiomyocyte.