Drug-induced block of cardiac hERG K ϩ channels causes acquired long QT syndrome. Here, we characterized the molecular mechanism of hERG block by two low-potency drugs (Nifekalant and bepridil) and two high-potency drugs 1-[2-(6-methyl-2pyridyl)ethyl]-4-(4-methylsulfonyl aminobenzoyl)piperidine (E-4031) and dofetilide). Channels were expressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage-clamp technique. All four drugs progressively reduced hERG current during a 20-s depolarization to 0 mV after a 10-min pulse-free period, consistent with the preferential block of open channels. Recovery from block in response to pulses to Ϫ160 mV was observed for D540K hERG channels but not for wild-type hERG channels, suggesting that all four drugs are trapped in the central cavity by closure of the activation gate. The molecular determinants of hERG channel block were defined by using a site-directed mutagenesis approach. Mutation to alanine of three residues near the pore helix (Thr623, Ser624, and Val625) and four residues in Ser6 (Gly648, Tyr652, Phe656, and Val659) reduced channel sensitivity to block by dofetilide and E-4031, effects identical with those reported previously for two other methanesulfonanilides, (ϩ)- -499) and ibutilide. The effect of nifekalant on mutant channels was similar, except that V659A retained normal sensitivity and I655A channels were less sensitive. Finally, mutation of the three residues near the pore helix and Phe656 in the Ser6 domain reduced channel block by bepridil. We conclude that the binding site is not identical for all drugs that preferentially block hERG in the open state.Class III antiarrhythmic drugs are defined by their ability to block potassium channels and prolong the action potential duration of cardiomyocytes. Some of the most potent class III drugs such as dofetilide, E-4031, and MK-499 are structural analogs of the methanesulfonanilide sotalol, a compound with low potency. All of these drugs prolong action potentials by a relatively specific block of the rapid delayed rectifier K ϩ current, I Kr (Sanguinetti and Jurkiewicz, 1990). Sotalol is the only class III methanesulfonanilide that has been shown in clinical trials to improve prognosis; however, the potency of I Kr block by sotalol is several hundred-fold weaker than dofetilide, E-4031, or MK-499. The discrepancy between the beneficial clinical profile and a low potency for I Kr block has long been reported for sotalol and may indicate the greater importance of the ␤-adrenergic receptor blocking activity compared with I Kr block.The human I Kr channel is encoded by HERG and mutations in this gene cause long QT syndrome , a disorder of cardiomyocyte repolarization that predisposes affected individuals to an increased risk of torsades de pointes and lethal ventricular fibrillation. The most common cause of prolonged QT interval is treatment with class III antiarrhythmic agents and side effects associated with treatment with certain noncardiac medications. For this re...

Background-Recent clinical electrophysiology studies and successful results of radiofrequency catheter ablation therapy suggest that high-frequency focal activity in the pulmonary veins (PVs) plays important roles in the initiation and perpetuation of atrial fibrillation, but the mechanisms underlying the focal arrhythmogenic activity are not understood. Methods and Results-Extracellular potential mapping of rabbit right atrial preparations showed that ryanodine (2 mol/L) caused a shift of the leading pacemaker from the sinoatrial node to an ectopic focus near the right PV-atrium junction. The transmembrane potential recorded from the isolated myocardial sleeve of the right PV showed typical atrial-type action potentials with a stable resting potential under control conditions. Treatment with ryanodine (0.5 to 2 mol/L) resulted in a depolarization of the resting potential and a development of pacemaker depolarization. These changes were enhanced transiently after an increase in the pacing rate: a self-terminating burst of spontaneous action potentials (duration, 33.6Ϯ5.0 s; nϭ32) was induced by a train of rapid stimuli (3. A trial fibrillation (AF) is the most common of all sustained tachyarrhythmias and is one of the major causes of stroke. The most widely accepted mechanism of AF is multiple reentrant wavelets. 1 However, recent clinical studies have shown that paroxysmal AF is initiated by bursts of premature excitations originating primarily in the pulmonary veins (PVs), and radiofrequency ablation or electrical isolation of these foci can eliminate AF. 2,3 More recently, Haïs-saguerre et al 4 reported that in patients with drug-resistant chronic AF and structural heart disease, after electrical cardioversion, the PVs are also the dominant trigger reinitiating AF. Therefore, the PVs are important not only for the initiation of AF but also for its maintenance. 5 The myocardial fibers of the left atrium wrap around the PVs entering the left atrium to form "myocardial sleeves" (MSs), 6,7 and this structure is the origin of focal activity. 8 Previous studies have suggested that PVMSs show a variety of spontaneous activities such as sinoatrial (SA) node-type automaticity, rapid spontaneous activities via early or delayed afterdepolarizations, 8 -12 and microreentry based on a marked heterogeneous tissue structure. 12,13 However, the electrophysiological properties of PVMSs have not been fully characterized.In the present study, we investigated the electrophysiological properties of rabbit PVMSs. The results show that addition of 0.5 to 2 mol/L ryanodine to PVMSs uncovers pacing-induced spontaneous activity. Ryanodine at low concentrations locks the sarcoplasmic reticulum (SR) Ca 2ϩ release channel, the ryanodine receptor (RyR), in a subconductance state, causing a Ca 2ϩ -independent Ca 2ϩ release from the SR. 14 MethodsRabbits weighing 1.5 to 2.0 kg (Chubu-Kagaku-Shizai, Nagoya, Japan) were anesthetized with pentobarbital (30 to 40 mg/kg IV), and the heart was quickly excised. The right atrium, including the SA...

Abstract. Block of cardiac hERG K + channels by the antihistamine terfenadine and the prokinetic agent cisapride is associated with prolonged ventricular repolarization and an increased risk of ventricular arrhythmia. Here, we used a site-directed mutagenesis approach to determine the molecular determinants of hERG block by terfenadine and cisapride. Wild-type and mutant hERG channels were heterologously expressed in Xenopus laevis oocytes and characterized by measuring whole cell currents with two-microelectrode voltage clamp techniques. Mutation of T623, S624, Y652, or F656 to Ala reduced channel sensitivity to block by terfenadine. The same mutations reduced sensitivity to cisapride. These data confirm our previous findings that polar residues (T623, S624) located near the base of the pore helix and aromatic residues (Y652, F656) located in the S6 domain are key molecular determinants of the hERG drug binding site. Unlike methanesulfonanilides (dofetilide, MK-499, E-4031, ibutilide) or clofilium, mutation of V625, G648, or V659 did not alter the sensitivity of hERG channels to terfenadine or cisapride. As previously proposed by molecular modeling studies (Farid R, et al. Bioorg Med Chem. 2006;14:3160-3173), our findings suggest that different drugs can adopt distinct modes of binding to the central cavity of hERG.

The widespread distribution of HCN4 can explain the widespread location of the leading pacemaker site during sinus rhythm, the extensive region of tissue that has to be ablated to stop sinus rhythm, and the widespread distribution of ectopic foci responsible for atrial tachycardia.

No abstract

Abstract-Recent work on isolated sinoatrial node cells from rabbit has suggested that sarcoplasmic reticulum Ca 2ϩ release plays a dominant role in the pacemaker potential, and ryanodine at a high concentration (30 mol/L blocks sarcoplasmic reticulum Ca 2ϩ release) abolishes pacemaking and at a lower concentration abolishes the chronotropic effect of ␤-adrenergic stimulation. The aim of the present study was to test this hypothesis in the intact sinoatrial node of the rabbit. Spontaneous activity and the pattern of activation were recorded using a grid of 120 pairs of extracellular electrodes. Ryanodine 30 mol/L did not abolish spontaneous activity or shift the position of the leading pacemaker site, although it slowed the spontaneous rate by 18.9Ϯ2.5% (nϭ6). After ryanodine treatment, ␤-adrenergic stimulation still resulted in a substantial chronotropic effect (0.3 mol/L isoproterenol increased spontaneous rate by 52.6Ϯ10.5%, nϭ5). In isolated sinoatrial node cells from rabbit, 30 mol/L ryanodine slowed spontaneous rate by 21.5Ϯ2.6% (nϭ13). It is concluded that sarcoplasmic reticulum Ca 2ϩ release does not play a dominating role in pacemaking in the sinoatrial node. The full text of this article is available at http://www.circresaha.org. release activates inward Na ϩ -Ca 2ϩ exchange current, and this helps to generate the pacemaker depolarization. Two studies by Lakatta and coinvestigators 1,2 published recently in Circulation Research have placed the spotlight on this mechanism: Bogdanov et al 1 suggested that SR Ca 2ϩ release may be obligatory for pacemaking, because they observed that a high concentration (30 mol/L) of ryanodine, which blocks the SR Ca 2ϩ release channel, abolishes the spontaneous activity of isolated SA node cells from rabbit. Vinogradova et al 2 boldly suggested that the positive chronotropic effect of ␤-adrenergic stimulation is the result of the increase in the Ca 2ϩ transient caused by ␤-adrenergic stimulation, because they observed that the chronotropic effect in isolated SA node cells from rabbit is abolished or greatly reduced after the suppression of the Ca 2ϩ transient by a submaximal concentration of ryanodine (3 mol/L). These recent reports are surprising. Previously, the role of SR Ca 2ϩ release was thought to be more minor, because in isolated SA node cells from rabbit and guinea pig, suppression of the Ca 2ϩ transient by a variety of interventions (including up to 10 mol/L ryanodine) did not abolish pacemaking and just decreased spontaneous rate by 21% to 37%. [3][4][5] In this scenario, it is assumed that multiple ionic currents (I Na , I Ca,L , I Ca,T , I K,r , I b,Na , and I f as well as I NaCa ) are involved in the generation of the pacemaker potential. 6 As highlighted by DiFrancesco and Robinson, 7 the conclusion that the chronotropic effect of ␤-adrenergic stimulation is the result of an increase in the Ca 2ϩ transient is also surprising, because the chronotropic effect of ␤-adrenergic stimulation has been previously attributed to actions on ionic currents such as I Ca,...

Regional differences in electrical activity in rabbit sinoatrial node have been investigated by recording action potentials throughout the intact node or from small balls of tissue from different regions. In the intact node, action potential duration was greatest at or close to the leading pacemaker and declined markedly in all directions from it, e.g., by 74 ± 4% (mean ± SE, n = 4) to the crista terminalis. Similar data were obtained from the small balls. The gradient is down the conduction pathway and will help prevent reentry. In the intact node, a zone of inexcitable tissue with small depolarizations of <25 mV or stable resting potentials was discovered in the inferior part of the node, and this will again help prevent reentry. The intrinsic pacemaker activity of the small balls was slower in tissue from more inferior (as well as more central) parts of the node [e.g., cycle length increased from 339 ± 13 ms ( n = 6) to 483 ± 13 ms ( n = 6) in transitional tissue from more superior and inferior sites], and this may help explain pacemaker shift.

Effects of block of the rapid delayed rectifier K+current ( I K,r) by E-4031 on the electrical activity of small ball-like tissue preparations from different regions of the rabbit sinoatrial node were measured. The effects of partial block of I K,r by 0.1 μM E-4031 varied in different regions of the node. In tissue from the center of the node spontaneous activity was generally abolished, whereas in tissue from the periphery spontaneous activity persisted, although the action potential was prolonged, the maximum diastolic potential was decreased, and the spontaneous activity slowed. After partial block of I K,r, the electrical activity of peripheral tissue was more like that of central tissue under normal conditions. One possible explanation of these findings is that the density of I K,r is greater in the periphery of the node; this would explain the greater resistance of peripheral tissue to I K,r block and help explain why, under normal conditions, the maximum diastolic potential is more negative, the action potential is shorter, and pacemaking is faster in the periphery.