Halide ion effects on human Ether-à-go-go related gene potassium channel properties.

The human Ether-à-go-go related gene (hERG) potassium channel has been widely used to counter screen potential pharmaceuticals as a biomarker to predict clinical QT prolongation. Thus, higher throughput assays of hERG are valuable for early in vitro screening of drug candidates to minimize failure in later-stage drug development due to this potentially adverse cardiac risk. We have developed a novel method utilizing potassium fluoride to improve throughput of hERG counter screening with an automated patch clamp system, PatchXpress 7000A. In that method, ∼50% substitution of internal Cl(-) with F(-) greatly increases success rate without substantially altering the biophysical properties of the hERG channel or compromising data quality. However, effect of F(-) or other halide ions on hERG channel properties has not been studied in detail. In this study, we examined effects of complete replacement of Cl(-) in internal solution with halide ions, F(-), or Br(-). We found that (1) F(-) slightly shifts the voltage dependence of hERG channel activation to more positive voltages, while Br(-) shifts it to more negative voltages; (2) Br(-) shifts to more positive voltages both the inactivation-voltage relationship and the peak position of channel full activation of hERG; (3) F(-) slows hERG activation, while both F(-) and Br(-) make the channel close faster; (4) neither F(-) nor Br(-) have any effect on hERG inactivation kinetics. In conclusion, compared to Cl(-), F(-) has subtle effect on hERG activation, while Br(-) has distinct effects on certain, but not all biophysical properties of hERG channel.

The anesthetized guinea pig: an effective early cardiovascular derisking and lead optimization model.

INTRODUCTION: In recent years, the anesthetized guinea pig has been used increasingly to evaluate the cardiovascular effects of drug-candidate molecules during lead optimization prior to conducting longer, more resource intensive safety pharmacology and toxicology studies. The aim of these studies was to evaluate the correlations between pharmacologically-induced ECG changes in the anesthetized cardiovascular guinea pig (CVGP) with ECG changes in conscious non-rodent telemetry models, human clinical studies and effects on key cardiac ion channels. METHODS: We compared the effects of 38 agents on ion channel inhibition to their ECG effects in the CVGP. 26 of these agents were also evaluated in non-rodent telemetry and compared to the results in the CVGP. RESULTS: The CVGP was highly sensitive for detecting QTc, PR and QRS interval prolongation mediated by inhibition of hERG, hCav1.2 and hNav1.5, respectively. There were robust correlations between ion channel inhibitory potencies and the free plasma concentrations (Cu) producing prolongation of the QTc, PR or QRS interval. Further evaluation showed that ECG changes in the CVGP were predictive of their effects on the QTc, PR and QRS intervals in non-rodent telemetry models with 92%, 92% and 100% accuracy, respectively. The CVGP proved to be 100% specific and 88%, 75% and 100% sensitive for QTc, PR and QRS interval prolongation, respectively. Similarly, the Cu that prolonged the QTc, PR and QRS in CVGP and humans correlated well. DISCUSSION: The CVGP is a sensitive model for assessing QTc, PR and QRS prolongation elicited by effects on hERG, hCav1.2 and hNav1.5, respectively. ECG changes in the CVGP are predictive of changes in non-rodent telemetry models and in humans (QTc). ECG parameters can be reliably evaluated with the CVGP model which increases the efficiency of CV derisking. Importantly, the design and implementation of this model is consistent with the "3Rs" for animal research.

MK-0448, a specific Kv1.5 inhibitor: safety, pharmacokinetics, and pharmacodynamic electrophysiology in experimental animal models and humans.

BACKGROUND: We evaluated the viability of I(Kur) as a target for maintenance of sinus rhythm in patients with a history of atrial fibrillation through the testing of MK-0448, a novel I(Kur) inhibitor. METHODS AND RESULTS: In vitro MK-0448 studies demonstrated strong inhibition of I(Kur) with minimal off-target activity. In vivo MK-0448 studies in normal anesthetized dogs demonstrated significant prolongation of the atrial refractory period compared with vehicle controls without affecting the ventricular refractory period. In studies of a conscious dog heart failure model, sustained atrial fibrillation was terminated with bolus intravenous MK-0448 doses of 0.03 and 0.1 mg/kg. These data led to a 2-part first-in-human study: Part I evaluated safety and pharmacokinetics, and part II was an invasive electrophysiological study in healthy subjects. MK-0448 was well-tolerated with mild adverse experiences, most commonly irritation at the injection site. During the electrophysiological study, ascending doses of MK-0448 were administered, but no increases in atrial or ventricular refractoriness were detected, despite achieving plasma concentrations in excess of 2 µmol/L. Follow-up studies in normal anesthetized dogs designed to assess the influence of autonomic tone demonstrated that prolongation of atrial refractoriness with MK-0448 was markedly attenuated in the presence of vagal nerve simulation, suggesting that the effects of I(Kur) blockade on atrial repolarization may be negated by enhanced parasympathetic neural tone. CONCLUSIONS: The contribution of I(Kur) to human atrial electrophysiology is less prominent than in preclinical models and therefore is likely to be of limited therapeutic value for the prevention of atrial fibrillation.

Assessing use-dependent inhibition of the cardiac Na(+/-) current (I(Na)) in the PatchXpress automated patch clamp.

INTRODUCTION: The cardiac Na+ current (I(Na)) underlies the rapid depolarization of the cardiac myocyte, and block of the current slows cardiac conduction and increases the risk of ventricular arrhythmia. A feature of Na+ channel block termed use-dependence is important to the assessment of blocking potency. We developed a robust automated patch clamp assay to rapidly and routinely assess the use-dependent block of I(Na) by drug candidates. The assay clarifies whether drug candidates block more potently at increased heart rates and provides a quantitative score of use-dependence. METHODS: A use-dependent cardiac I(Na) assay was implemented on the PatchXpress 7000A, an automated whole-cell patch clamp device, using a HEK cell line stably expressing the human cardiac Na+ channel, Na(V)1.5. Stable recordings lasting up to 30 minutes were achieved by selection of holding potential (-100 mV) as well as an appropriate osmotic gradient to prevent time-dependent loss of cell capacitance and current. The final protocol allows evaluation of I(Na) inhibition at three pulsing rates at three test concentrations for each recorded cell. RESULTS: IC(50) values obtained for three standard I(Na) blockers lidocaine, mexiletine, and flecainide, at pulsing frequencies of 0.2 Hz, 1 Hz, and 3 Hz, were compared to IC(50) values obtained with conventional pipette patch clamp of the Na(V)1.5 cell line and of guinea pig cardiac myocytes using matched voltage protocols and pulsing rates. Absolute potencies were well correlated only under conditions of matched holding potential and fell within an approximately three-fold window. While absolute potencies could vary widely with holding potential, the fold increases in potency with increases in pulsing rates were less prone to variation of the holding potential. DISCUSSION: Use-dependence of cardiac Na+ channel block can be rapidly assessed in the PatchXpress platform and quantified at early stages of drug development to guide lead optimization.

Discovery of triarylethanolamine inhibitors of the Kv1.5 potassium channel.

A series of triarylethanolamine inhibitors of the Kv1.5 potassium channel have been prepared and evaluated for their effects in vitro and in vivo. The structure-activity relationship (SAR) studies described herein led to the development of potent, selective and orally active inhibitors of Kv1.5.

Optimization of Ca(v)1.2 screening with an automated planar patch clamp platform.

INTRODUCTION: Ca(v)1.2 channels play an important role in shaping the cardiac action potential. Screening pharmaceutical compounds for Ca(v)1.2 block is very important in developing drugs without cardiac liability. Ca(v)1.2 screening has been traditionally done using fluorescence assays, but these assays have some limitations. Patch clamping is considered the gold standard for ion channel studies, but is very labor intensive. The purpose of this study was to develop a robust medium throughput Ca(v)1.2 screening assay in PatchXpress 7000A by optimizing cell isolation conditions, recording solutions and experimental parameters. Under the conditions established, structurally different standard Ca(v)1.2 antagonists and an agonist were tested. METHODS: HEK-293 cells stably transfected with hCa(v)1.2 L-type Ca channel were used. For experiments, cells were isolated using 0.05% Trypsin. Currents were recorded in the presence of 30 mM extracellular Ba2+ and low magnesium intracellular recording solution to minimize rundown. Ca(v)1.2 currents were elicited from a holding potential of -60 mV at 0.05 Hz to increase pharmacological sensitivity and minimize rundown. Test compounds were applied at increasing concentrations for 5 min followed by a brief washout. RESULTS: Averaged peak Ca(v)1.2 current amplitudes were increased from 10 pA/pF to 15 pA/pF by shortening cell incubation and trypsin exposure time from 2.5 min at 37 degrees C to 1 min at room temperature and adding 0.2 mM cAMP to the intracellular solution. Rundown was minimized from 2%/min to 0.5%/min by reducing the intracellular free Mg2+ from 2.7 mM to 0.2 mM and adding 100 nM Ca2+. Under the established conditions, we tested 8 structurally different antagonists and an agonist. The IC(50) values obtained ranked well against published values and results obtained using traditional clamp experiments performed in parallel using the expressed cell line and native myocytes. DISCUSSION: This assay can be used as a reliable pharmacological screening tool for Ca(v)1.2 block to assess compounds for cardiac liability during lead optimization.

The hERG channel and risk of drug-acquired cardiac arrhythmia: an overview.

This review summarizes current knowledge of the cardiac rapidly activating delayed rectifier potassium current (I(Kr)), and its connection to drug-acquired QT prolongation and the associated risk of ventricular arrhythmia and fibrillation. The molecular characterization of hERG as the structural correlate of I(Kr) and the link between inherited long QT and the KCNH2 gene (hERG), have facilitated mechanistic studies of drug-acquired QT prolongation. The development of high throughput assays to evaluate drug effects on hERG has provided an avenue to determine structure activity relations (SAR) within chemical series. More than 10 years of collective data and structural considerations support the notion that hERG is an unusually promiscuous target among potassium channels, but that defining SAR within a chemical series is a viable strategy to reduce or eliminate hERG activity. Despite a critical need to minimize drug effects on hERG, one should always keep in mind that hERG is not the only structural correlate of QT prolongation, and that QT prolongation is a sub-optimal biomarker for ventricular arrhythmia and fibrillation.

Modeling of the adrenergic response of the human IKs current (hKCNQ1/hKCNE1) stably expressed in HEK-293 cells.

Stable coexpression of human (h)KCNQ1 and hKCNE1 in human embryonic kidney (HEK)-293 cells reconstitutes a nativelike slowly activating delayed rectifier K+ current (HEK-I(Ks)), allowing beta-adrenergic modulation of the current by stimulation of endogenous receptors in the host cell line. HEK-I(Ks) was enhanced two- to fourfold by isoproterenol (EC50 = 13 nM), forskolin (10 microM), or 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (50 microM), indicating an intact cAMP-dependent ion channel-regulating pathway analogous to the PKA-dependent regulation observed in native cardiac myocytes. Activation kinetics of HEK-I(Ks) were accurately fit with a novel modified second-order Hodgkin-Huxley (H-H) gating model incorporating a fast and a slow gate, each independent of each other in scale and adrenergic response, or a "heterodimer" model. Macroscopically, beta-adrenergic enhancement shifted the current activation threshold to more negative potentials and accelerated activation kinetics while leaving deactivation kinetics relatively unaffected. Modeling of the current response using the H-H model indicated that observed changes in gating could be explained by modulation of the opening rate of the fast gate. Under control conditions at nearly physiological temperatures (35 degrees C), rate-dependent accumulation of HEK-I(Ks) was observed only at pulse frequencies exceeding 3 Hz. Rate-dependent accumulation of I(Ks) at high pulsing rate had two phases, an initial staircaselike effect followed by a slower, incremental accumulation phase. These phases are readily interpreted in the context of a heterodimeric H-H model with two independent gates with differing closing rates. In the presence of isoproterenol after normalizing for its tonic effects, rate-dependent accumulation of HEK-I(Ks) appeared at lower pulse frequencies and was slightly enhanced (approximately 25%) over control.

Scientific review and recommendations on preclinical cardiovascular safety evaluation of biologics.

Biological therapeutic agents (biologicals), such as monoclonal antibodies (mAbs), are increasingly important in the treatment of human disease, and many types of biologicals are in clinical development. During preclinical drug development, cardiovascular safety pharmacology studies are performed to assess cardiac safety in accord with the ICH S7A and S7B regulations that guide these studies. The question arises, however, whether or not it is appropriate to apply these guidelines, which were devised primarily to standardize small molecule drug testing, to the cardiovascular evaluation of biologicals. We examined the scientific literature and formed a consensus of scientific opinion to determine if there is a rational basis for conducting an in vitro hERG assay as part of routine preclinical cardiovascular safety testing for biologicals. We conclude that mAb therapeutics have very low potential to interact with the extracellular or intracellular (pore) domains on hERG channel and, therefore, are highly unlikely to inhibit hERG channel activity based on their targeted, specific binding properties. Furthermore, mAb are large molecules (>140,000 Da) that cannot cross plasma membranes and therefore would be unable to access and block the promiscuous inner pore of the hERG channel, in contrast with typical small molecule drugs. Consequently, we recommend that it is not appropriate to conduct an in vitro hERG assay as part of a preclinical strategy for assessing the heart rate corrected QT interval (QTc) prolongation risk of mAbs and other types of biologicals. It is more appropriate to assess QTc risk by integrating cardiovascular endpoints into repeat-dose general toxicology studies performed in an appropriate non-rodent species. These recommendations should help shape future regulatory strategy and discussions for the cardiovascular safety pharmacology testing of mAbs as well as other biologicals and provide guidance for the preclinical cardiovascular evaluation of such agents.

Improved throughput of PatchXpress hERG assay using intracellular potassium fluoride.

Blockade of the human ether-a-go-go-related gene (hERG) potassium channel, with a consequent possibility of QT prolongation and increased susceptibility to a characteristic polymorphic ventricular arrhythmia, torsade de pointes, is an important cause of withdrawal of drugs from the market. In the aftermath of recent drug withdrawals, regulatory agencies now require in vitro hERG screening of all pharmaceutical compounds that are targeted for human use. To minimize the potential for failure in later-stage drug development, many pharmaceutical and biotechnology companies have begun to use automated patch clamp systems with higher throughput than conventional manual patch-clamp techniques to conduct routine functional hERG screening during drug discovery and early development. We have optimized an automated patch-clamp hERG screening method for the PatchXpress 7000A system (Molecular Devices, Sunnyvale, CA) using potassium fluoride (KF) in the internal recording solution. In this study we show that (1) the biophysical and pharmacological properties of hERG current recorded with KF are similar to those with standard potassium chloride solutions, (2) use of KF significantly improves the success rate of hERG screening using PatchXpress without compromising data quality, and (3) utilization of KF can significantly increase the throughput of hERG screening with PatchXpress.

Application of PatchXpress planar patch clamp technology to the screening of new drug candidates for cardiac KCNQ1/KCNE1 (I Ks) activity.

A cardiac safety concern for QT prolongation and potential for pro-arrhythmia exists due to inhibition of the cardiac slowly activating delayed rectifier potassium current, I(Ks). Selective inhibitors of I Ks have been shown to prolong the QT interval in animal models. On the other hand, I Ks has been considered as a target for anti-arrhythmic therapy due to certain biophysical and pharmacological properties and its expression pattern in the heart. Consequently, we have developed a method utilizing a human embryonic kidney (HEK)-293 cell line expressing KCNQ1/KCNE1 (genes that encode for the I Ks channel) as a model for screening of new compounds for I Ks activity. This study was designed (1) to establish and optimize the experimental conditions for measurement of I Ks using PatchXpress() 7000A (Molecular Devices Corporation, Sunnyvale, CA) and (2) to test the effects of I Ks inhibitors and compare the 50% inhibitory concentration (IC50) values determined with PatchXpress versus conventional patch clamp in order to validate the PatchXpress approach for higher-throughput I Ks screening. Biophysical properties of HEK/I Ks recorded with PatchXpress were similar to those recorded with conventional patch-clamp and reported in the literature. The IC50 values for I Ks block determined with PatchXpress correlated well with conventional patch-clamp values from HEK-293 cells as well as from native cardiac myocytes for the majority of compounds tested. Electrophysiological recording of I Ks expressed in HEK-293 cells with the PatchXpress is of acceptable quality for screening purposes. This approach can be utilized for functional prescreening of development compounds for I Ks inhibition either for optimizing lead anti-arrhythmic or other therapeutic candidates or to exclude compounds with the potential to prolong QT.

Kinesin spindle protein (KSP) inhibitors. Part 6: Design and synthesis of 3,5-diaryl-4,5-dihydropyrazole amides as potent inhibitors of the mitotic kinesin KSP.

3,5-diaryl-4,5-dihydropyrazoles were discovered to be potent KSP inhibitors with excellent in vivo potency. These enzyme inhibitors possess desirable physical properties that can be readily modified by incorporation of a weakly basic amine. Careful adjustment of amine basicity was essential for preserving cellular potency in a multidrug resistant cell line while maintaining good aqueous solubility.

Kinesin spindle protein (KSP) inhibitors. Part 7: Design and synthesis of 3,3-disubstituted dihydropyrazolobenzoxazines as potent inhibitors of the mitotic kinesin KSP.

Observations from two structurally related series of KSP inhibitors led to the proposal and discovery of dihydropyrazolobenzoxazines that possess ideal properties for cancer drug development. The synthesis and characterization of this class of inhibitors along with relevant pharmacokinetic and in vivo data are presented. The synthesis is highlighted by a key [3+2] cycloaddition to form the pyrazolobenzoxazine core followed by diastereospecific installation of a quaternary center.

Novel, potent inhibitors of human Kv1.5 K+ channels and ultrarapidly activating delayed rectifier potassium current.

We have identified a series of diphenyl phosphine oxide (DPO) compounds that are potent frequency-dependent inhibitors of cloned human Kv1.5 (hKv1.5) channels. DPO inhibited hKv1.5 expressed in Chinese hamster ovary cells in a concentration-dependent manner preferentially during channel activation and slowed the deactivating tail current, consistent with a predominant open-channel blocking mechanism. Varying kinetics of DPO interaction with Kv1.5 channels resulted in differing potencies and frequency dependencies of inhibition that were comparable for both expressed hKv1.5 current and native ultrarapidly activating delayed rectifier potassium current (IKur) in human atrial myocytes. Selectivity of DPO versus other cardiac K+ channels was demonstrated in human atrial myocytes (IKur versus transient outward potassium current) and guinea pig ventricular myocytes [IKur versus rapidly activating delayed rectifier potassium current (IKr), slowly activating delayed rectifier potassium current (IKs) and inward rectifier potassium current (IK1), and one compound (DPO-1) was shown to be 15-fold more selective for Kv1.5 versus Kv3.1 channels expressed in Xenopus oocytes. DPO-1 also prolonged action potentials of isolated human atrial but not ventricular myocytes, in contrast to the effect of a selective IKr blocker. The selectivity and kinetics of inhibition hKv1.5 and IKur by DPO and the resulting selective prolongation of atrial repolarization could provide an effective profile for treatment of supraventricular arrhythmias.

Flunarizine is a highly potent inhibitor of cardiac hERG potassium current.

Flunarizine has been widely used for the management of a variety of disorders such as peripheral vascular diseases, migraine, and epilepsy. The majority of its beneficial effects have been attributed to its ability to inhibit voltage-gated Ca2+ channels in the low micromolar range, albeit non-selectively, as flunarizine has been shown to inhibit a variety of ion channels. We examined the effects of flunarizine on potassium currents through cardiac channels encoded by the human ether-a-go-go related gene (hERG) stably expressed in CHO cells. In this study, we have characterized the effect of flunarizine on biophysical properties of hERG potassium currents with standard whole-cell voltage-clamp techniques. Notably, flunarizine is a highly potent inhibitor of hERG current with an IC50 value of 5.7 nM. The effect of flunarizine on hERG potassium current is concentration and time dependent, and displays voltage dependence over the voltage range between -40 and 0 mV. At concentrations near or above the IC50, flunarizine causes a negative shift in the voltage dependence of hERG current activation and accelerates tail current deactivation. Flunarizine preferentially blocks the activated state of the channel and displays weak frequency dependence of inhibition. Flunarizine also inhibits KCNQ1/KCNE1 channel current with an IC50 of 0.76 microM.

In vivo canine cardiac electrophysiologic profile of 1,4-benzodiazepine IKs blockers.

Previous cardiac electrophysiologic studies of blockers of the slowly activating delayed rectifier (IKs) current have focused primarily on ventricular repolarization. This report summarizes an extensive in vivo cardiac electrophysiologic profile of four 1,4-benzodiazepine IKs blocker analogues (L-761334, L-763540, L-761710, and L-768673) in dogs. At 3.0 mg/kg intravenously, all four analogues elicited 14.5%-21.4% increases in ventricular refractoriness and 19.2%-22.6% increases in QTc interval. Concomitant 11.1%-13.5% increases in atrial refractoriness were noted with all four analogues. Decreases in sinus heart rate of 8.4%-17.3% were noted with all four compounds. No effects on atrial, His Purkinje, ventricular conduction or atrial and ventricular excitation were observed. One analogue, L-761710, significantly delayed atrioventricular (AV) nodal conduction (40.7+/-17.4% increase in atrial-to-His interval) and increased the AV conduction system functional refractory period 19.9+/-6.2%. The lack of effect of the other three 1,4-benzodiazepine IKs blockers on AV nodal function at dosages producing comparable effects on atrial and ventricular refractoriness suggest that the AV nodal effects of L-761710 were unrelated to IKs blockade. These findings indicate IKs plays important roles in both atrial and ventricular refractoriness as well as pacemaker function in the dog heart, suggesting potential utility for IKs blockers in the treatment of atrial and ventricular arrhythmias.

Saxitoxin is a gating modifier of HERG K+ channels.

Potassium (K+) channels mediate numerous electrical events in excitable cells, including cellular membrane potential repolarization. The hERG K+ channel plays an important role in myocardial repolarization, and inhibition of these K+ channels is associated with long QT syndromes that can cause fatal cardiac arrhythmias. In this study, we identify saxitoxin (STX) as a hERG channel modifier and investigate the mechanism using heterologous expression of the recombinant channel in HEK293 cells. In the presence of STX, channels opened slower during strong depolarizations, and they closed much faster upon repolarization, suggesting that toxin-bound channels can still open but are modified, and that STX does not simply block the ion conduction pore. STX decreased hERG K+ currents by stabilizing closed channel states visualized as shifts in the voltage dependence of channel opening to more depolarized membrane potentials. The concentration dependence for steady-state modification as well as the kinetics of onset and recovery indicate that multiple STX molecules bind to the channel. Rapid application of STX revealed an apparent "agonist-like" effect in which K+ currents were transiently increased. The mechanism of this effect was found to be an effect on the channel voltage-inactivation relationship. Because the kinetics of inactivation are rapid relative to activation for this channel, the increase in K+ current appeared quickly and could be subverted by a decrease in K+ currents due to the shift in the voltage-activation relationship at some membrane potentials. The results are consistent with a simple model in which STX binds to the hERG K+ channel at multiple sites and alters the energetics of channel gating by shifting both the voltage-inactivation and voltage-activation processes. The results suggest a novel extracellular mechanism for pharmacological manipulation of this channel through allosteric coupling to channel gating.

Novel 5-cyclopropyl-1,4-benzodiazepin-2-ones as potent and selective I(Ks)-blocking class III antiarrhythmic agents.

Novel 5-cyclopropyl-1,4-benzodiazepin-2-ones having various N-l substituents were identified as potent and selective blockers of the slowly activating cardiac delayed rectifier potassium current (I(Ks)). Compound 11 is the most potent I(Ks) channel blocker reported to date.

Functional and pharmacological properties of canine ERG potassium channels.

We established HEK-293 cell lines that stably express functional canine ether-à-go-go-related gene (cERG) K(+) channels and examined their biophysical and pharmacological properties with whole cell patch clamp and (35)S-labeled MK-499 ([(35)S]MK-499) binding displacement. Functionally, cERG current had the hallmarks of cardiac delayed rectifier K(+) current (I(Kr)). Channel opening was time- and voltage dependent with threshold near -40 mV. The half-maximum activation voltage was -7.8 +/- 2.4 mV at 23 degrees C, shifting to -31.9 +/- 1.2 mV at 36 degrees C. Channels activated with a time constant of 13 +/- 1 ms at +20 mV, showed prominent inward rectification at depolarized potentials, were highly K(+) selective (Na(+)-to-K(+) permeability ratio = 0.007), and were potently inhibited by I(Kr) blockers. Astemizole, terfenadine, cisapride, and MK-499 inhibited cERG and human ERG (hERG) currents with IC(50) values of 1.3, 13, 19, and 15 nM and 1.2, 9, 14, and 21 nM, respectively, and competitively displaced [(35)S]MK-499 binding from cERG and hERG with IC(50) values of 0.4, 12, 35, and 0.6 nM and 0.8, 5, 47, and 0.7 nM, respectively. cERG channels had biophysical properties appropriate for canine action potential repolarization and were pharmacologically sensitive to agents known to prolong QT. A novel MK-499 binding assay provides a new tool to detect agents affecting ERG channels.

Increasing I(Ks) corrects abnormal repolarization in rabbit models of acquired LQT2 and ventricular hypertrophy.

Excessive action potential (AP) prolongation and early afterdepolarizations (EAD) are triggers of malignant ventricular arrhythmias. A slowly activating delayed rectifier K+ current (I(Ks)) is important for repolarization of ventricular AP. We examined the effects of I(Ks) activation by a new benzodiazepine (L3) on the AP of control, dofetilide-treated, and hypertrophied rabbit ventricular myocytes. In both control and hypertrophied myocytes, L3 activated I(Ks) via a negative shift in the voltage dependence of activation and a slowing of deactivation. L3 had no effect on L-type Ca(2+) current or other cardiac K+ currents tested. L3 shortened AP of control, dofetilide-treated, and hypertrophied myocytes more at 0.5 than 2 Hz. Selective activation of I(Ks) by L3 attenuates prolonged AP and eliminated EAD induced by rapidly activating delayed rectifier K+ current inhibition in control myocytes at 0.5 Hz and spontaneous EAD in hypertrophied myocytes at 0.2 Hz. Pharmacological activation of I(Ks) is a promising new strategy to suppress arrhythmias resulting from excessive AP prolongation in patients with certain forms of long QT syndrome or cardiac hypertrophy and failure.