Michael C. Sanguinetti

hERG potassium channels and cardiac arrhythmia

hERG potassium channels are essential for normal electrical activity in the heart. Inherited mutations in the HERG gene cause long QT syndrome, a disorder that predisposes individuals to life-threatening arrhythmias. Arrhythmia can also be induced by a blockage of hERG channels by a surprisingly diverse group of drugs. This side effect is a common reason for drug failure in preclinical safety trials. Insights gained from the crystal structures of other potassium channels have helped our understanding of the block of hERG channels and the mechanisms of gating.

Molecular and Cellular Mechanisms of Cardiac Arrhythmias

We thank I. Splawski for advice, D. Atkinson for help preparing figures, and L. Morelli for assistance preparing the manuscript.

Compound Mutations A Common Cause of Severe Long-QT Syndrome

Background—Long QT syndrome (LQTS) predisposes affected individuals to sudden death from cardiac arrhythmias. Although most LQTS individuals do not have cardiac events, significant phenotypic variability exists within families. Probands can be very symptomatic. The mechanism of this phenotypic variability is not understood. Methods and Results—Genetic analyses of KVLQT1, HERG, KCNE1, KCNE2, and SCN5A detected compound mutations in 20 of 252 LQTS probands (7.9%). Carriers of 2 mutations had longer QTc intervals (527±54 versus 489±44 ms; P <0.001); all had experienced cardiac events (20 of 20 [100%] versus 128 of 178 [72%]; P <0.01) and were 3.5-fold more likely to have cardiac arrest (9 of 16 [56%] versus 45 of 167 [27%]; P <0.01; OR, 3.5; 95% CI, 1.2 to 9.9) compared with probands with 1 or no identified mutation. Two-microelectrode voltage clamp of Xenopus oocytes was used to characterize the properties of variant slow delayed rectifier potassium (IKs) channels identified in 7 of the probands. When wild-type and variant subunits were coexpressed in appropriate ratios to mimic the genotype of the proband, the reduction in IKs density was equivalent to the additive effects of the single mutations. Conclusions—LQTS-associated compound mutations cause a severe phenotype and are more common than expected. Individuals with compound mutations need to be identified, and their management should be tailored to their increased risk for arrhythmias.

Isoproterenol antagonizes prolongation of refractory period by the class III antiarrhythmic agent E-4031 in guinea pig myocytes. Mechanism of action.

The mechanism by which isoproterenol (ISO) prevents the prolongation of action potential duration (APD) and refractory period (RP) by the class III antiarrhythmic agent E-4031 was studied. E-4031 (1 microM) increased RP by 50% with no effect on contractile force in papillary muscles isolated from guinea pig heart. ISO (1 microM) increased force of contraction more than fivefold and decreased RP by 25%. The prolongation of RP by E-4031 was prevented by pretreatment of muscles with ISO. The prolongation of APD in isolated guinea pig ventricular myocytes by 5 microM E-4031 also was antagonized by prior exposure of the cells to 1 microM ISO. Instantaneous currents and delayed rectifier K+ currents, IK, were measured in isolated myocytes using the suction microelectrode voltage-clamp technique. Currents were measured in response to 225-msec depolarizing pulses from a holding potential of -40 mV. Previous studies have demonstrated that IK in these cells results from activation of two distinct outward K+ currents, IKs and IKr (specifically blocked by E-4031). ISO doubled the magnitude of IKs without significant effect on IKr. The instantaneous current, putatively identified as a Cl- current, also was doubled by ISO but was unaffected by E-4031. The augmented conductance of IKs and instantaneous current by ISO results in a decrease in RP. The small effect of E-4031 on APD and RP in the presence of ISO results from the smaller contribution of IKr relative to the augmented repolarizing currents.

Role of external Ca2+ and K+ in gating of cardiac delayed rectifier K+ currents

We sought to determine whether extracellular Ca2+ (Cae2+) and K+ (Ke+) play essential roles in the normal functioning of cardiac K+ channels. Reports by others have shown that removal of Cae2+and Ke+alters the gating properties of neural delayed rectifier (IK) and A-type K+ currents, resulting in a loss of normal cation selectivity and voltage-dependent gating. We found that removal of Cae2+and Ke+from the solution bathing guinea pig ventricular myocytes often induced a leak conductance, but did not affect the ionic selectivity or time-dependent activation and deactivation properties of IK. The effect of [K+]e on the magnitude of the two components of cardiac IK was also examined. IK in guinea pig myocytes is comprised of two distinct types of currents: IKr (rapidly activating, rectifying) and IKs (slowly activating). The differential effect of Cae2+on the two components of IK (previously shown to shift the voltage dependence of activation of the two currents in opposite directions) was exploited to determine the role of Ke+on the magnitude of IKs and IKr. Lowering [K+]e from 4 to 0 mM increased IKs, as expected from the change in driving force for K+, but decreased IKr. The differential effect of [K+]e on the two components of cardiac IK may explain the reported discrepancies regarding modulation of cardiac IK conductance by this cation.

Molecular determinants of voltage-dependent human ether-a-go-go related gene (HERG) K+ channel block.

The structural determinants for the voltage-dependent block of ion channels are poorly understood. Here we investigate the voltage-dependent block of wild-type and mutant human ether-a-go-go related gene (HERG) K+ channels by the antimalarial compound chloroquine. The block of wild-type HERG channels expressed in Xenopusoocytes was enhanced as the membrane potential was progressively depolarized. The IC50 was 8.4 ± 0.9 μmwhen assessed during 4-s voltage clamp pulses to 0 mV. Chloroquine also slowed the apparent rate of HERG deactivation, reflecting the inability of drug-bound channels to close. Mutation to alanine of aromatic residues (Tyr-652 or Phe-656) located in the S6 domain of HERG greatly reduced the potency of channel block by chloroquine (IC50 > 1 mm at 0 mV). However, mutation of Tyr-652 also altered the voltage dependence of the block. In contrast to wild-type HERG, block of Y652A HERG channels was diminished by progressive membrane depolarization, and complete relief from block was observed at +40 mV. HERG channel block was voltage-independent when the hydroxyl group of Tyr-652 was removed by mutating the residue to Phe. Together these findings indicate a critical role for Tyr-652 in voltage-dependent block of HERG channels. Molecular modeling was used to define energy-minimized dockings of chloroquine to the central cavity of HERG. Our experimental findings and modeling suggest that chloroquine preferentially blocks open HERG channels by cation-π and π-stacking interactions with Tyr-652 and Phe-656 of multiple subunits.

Influence of ATP-sensitive potassium channel modulators on ischemia-induced fibrillation in isolated rat hearts

We have confirmed the findings of Kantor and colleagues that ischemia-induced fibrillation in isolated Langendorff-perfused rat hearts can be prevented by glyburide, a blocker of ATP-dependent K channels. These data suggest that block of ATP-dependent K current [IK(ATP)] is a novel antiarrhythmic mechanism. This hypothesis was further tested by evaluating the effects of another sulfonylurea IK(ATP) blocker, tolbutamide (1 mM) and two agents known to activate these channels in cardiac tissue, BRL 34915 (10 microM) and pinacidil (30 microM). Similar to glyburide, tolbutamide was also effective in preventing fibrillation in this isolated rat heart model. The IK(ATP) activators enhanced the rate of tachycardia and shortened the time required for the hearts to develop fibrillation. Coadministration of glyburide with either IK(ATP) activator prevented their effects. It is concluded that activation of IK(ATP) during global ischemia contributes to the development of fibrillation in the perfused rat heart model.

Molecular Determinants of hERG Channel Block

Drug-induced block of cardiac hERG K+ 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 -160 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, (+)- N -[1′ -(6-cyano-1,2,3,4-tetrahydro-2(R)-naphthalenyl)-3,4-dihydro-4(R)-hydroxyspiro(2H -1-benzopyran-2,4′ -piperidin)-6-yl]-methanesulfonamide] monohydrochloride (MK-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.

Modulation of potassium channels by antiarrhythmic and antihypertensive drugs.

Agents that modulate cardiac and smooth muscle K+ channels have stimulated considerable interest in recent years because of their therapeutic potential in a number of cardiovascular diseases. Foremost among these drugs are the so-called Class III antiarrhythmic agents, which act by prolonging cardiac action potentials, and K+ channel openers, which hyperpolarize and thereby relax smooth muscle cells. Many of the newly developed Class III antiarrhythmic agents probably act by specific block of one subtype of delayed rectifier K+ current, IKr, whereas other agents block more than one type of cardiac K+ current. Much controversy exists over the specific type of K+ channel (or channels) in smooth muscle that are activated by the K+ channel openers. Both groups of K+ channel modulators have great therapeutic promise, but the Class III antiarrhythmic agents may suffer from a side-effect that is directly linked to their specific mechanism of action.

Antiarrhythmic Drug Target Choices and Screening

Abstract— Most antiarrhythmic drugs are ion channel blockers, and to date, those tested in large randomized placebo-controlled clinical trials have shown no decrease in mortality outcome. This apparent lack of survival benefit may result from the significant liabilities associated with these agents that offset any long-term benefit. Despite the current success of implantable defibrillators and the future promise of gene therapy, there is still a pressing need for new antiarrhythmic drugs. An improved understanding of cardiac ion channels and novel approaches to target selection and compound screening will provide new opportunities for drug discovery in the near future. Here, we briefly review the multiple mechanisms of arrhythmia, the history of drug failures, and the possibilities that evolving technologies may provide in the search for more efficacious and safer antiarrhythmic drugs.