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CARDIOVASCULAR

Novel, Potent Inhibitors of Human Kv1.5 K⁺ Channels and Ultrarapidly Activating Delayed Rectifier Potassium Current

Department of Pharmacology, Merck Research Laboratories, West Point, Pennsylvania

Received January 12, 2006; accepted March 3, 2006.

	Abstract

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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 (I_Kur) in human atrial myocytes. Selectivity of DPO versus other cardiac K⁺ channels was demonstrated in human atrial myocytes (I_Kur versus transient outward potassium current) and guinea pig ventricular myocytes [I_Kur versus rapidly activating delayed rectifier potassium current (I_Kr), slowly activating delayed rectifier potassium current (I_Ks) and inward rectifier potassium current (I_K1), 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 I_Kr blocker. The selectivity and kinetics of inhibition hKv1.5 and I_Kur by DPO and the resulting selective prolongation of atrial repolarization could provide an effective profile for treatment of supraventricular arrhythmias.

Antiarrhythmic drugs are commonly used for the treatment of AF, to restore sinus rhythm, to facilitate electrical cardioversion, to prevent AF recurrence, and to control ventricular rate (Choudury and Lip, 2004). Currently available class I (sodium channel blockers) and class III (action potential-prolonging) antiarrhythmic agents reduce the rate of recurrence of AF, but they are of limited use because of a variety of potentially adverse effects, including ventricular proarrhythmia (Camm and Savelieva, 2004; Choudhury and Lip, 2004). Agents such as sotalol and amiodarone possess interesting and effective class III properties, but they produce adverse effects that cause them to be contraindicated in some patients. Most clinically available class III agents that increase myocardial refractoriness via prolongation of the cardiac action potential duration (APD) block at least one of the major cardiac repolarizing K⁺ currents, rapid delayed rectifier K⁺ current (I_Kr) or I_Ks, which are expressed in both human atria and ventricles. Because these agents prolong ventricular refractoriness and the electrocardiographic QT interval, they possess a risk for ventricular proarrhythmia, which limits their safety and utility for treatment of atrial arrhythmias (Camm and Savelieva, 2004; Choudhury and Lip, 2004). Recent efforts to develop safer and more selective Class III atrial antiarrhythmics have focused on the ultra-rapidly activating delayed rectifier K⁺ current I_Kur, which has been recorded in human atria but not ventricle (Wang et al., 1993; Amos et al., 1996). Compounds that inhibit I_Kur and human Kv1.5 (hKv1.5), the cloned channel subunit underlying I_Kur, have been described previously (Brendel and Peukert, 2002; Camm and Savelieva, 2004; Pecini et al., 2005). Most of these agents also affect a variety of other cardiac K⁺ channels, including transient outward current (I_to) (Kv4.3), I_KAch, and even I_Kr and I_Ks (Seki et al., 2002; Gögelein et al., 2004) with affinities comparable with that of Kv1.5 or I_Kur.

This study describes diphenyl phosphine oxide (DPO) compounds that are potent and selective frequency-dependent inhibitors of hKv1.5 channels. DPO inhibited hKv1.5 with IC₅₀ values ranging between 0.16 and 0.76 µM when the current was elicited at a pulsing frequency of 0.1 Hz. Potency increased with pacing frequency; IC₅₀ values increased from 0.05 to 0.71 µM at 1 Hz to 0.03 to 0.6 µM at 3 Hz. DPO inhibited I_kur of the human atrium in a manner similar to expressed hKv1.5 current and was selective for I_Kur versus I_Kr, I_Ks, and I_to of cardiac myocytes. A selected DPO prolonged action potential duration in human atrial but not ventricular myocytes. These properties have potential important implications for the safe and effective treatment and prevention of supraventricular arrhythmias.

	Materials and Methods

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All reagents were purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Chemicals (Fair Lawn, NJ), unless noted otherwise. All procedures related to the use of animals in these studies were reviewed and approved by the Institutional Animal Care and Use Committee at Merck Research Laboratories (West Point, PA) and conform with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, 1996).

DPO Solution Preparation. All test compounds (DPO) were prepared initially as 10 mM stock solutions by dissolving each compound in 100% dimethyl sulfoxide and were diluted into aqueous bath solutions to achieve final concentrations in the nanomolar to micromolar range. Aqueous test solutions had 0.1% final dimethyl sulfoxide vehicle concentration, in which there was no effect on any of the currents studied (data not shown).

Electrophysiological Techniques. The whole-cell patch-clamp technique (Hamill et al., 1981) was used to measure ionic currents in stably transfected cell lines, isolated myocytes, and membrane patches excised from Xenopus laevis oocytes. The microelectrode technique was used to measure action potentials in isolated myocytes (Salata et al., 1998). The two-electrode voltage-clamp (TEVC) technique was used to measure ionic currents in X. laevis oocytes injected with cRNAs (Salata et al., 1998). Whole-cell patch-clamp (voltage-clamp) experiments were conducted using an Axopatch 200A or 200B amplifier (Axon Instruments, Union City, CA). Action potentials were recorded using an Axoclamp 2A or 2B amplifier (Axon Instruments). TEVC recordings were conducted using a Dagan TEV200A amplifier (Dagan, Minneapolis, MN). Analog signals were digitized with an Axon Digidata 1200 A/B interface and were acquired and analyzed with pClamp6 software (Axon Instruments) on an IBM-compatible computer.

Recording of hKv1.5 Currents in Stably Transfected CHO Cell Lines. Whole-cell currents were measured in CHO-K1 cell lines (American Type Culture Collection, Manassas, VA) stably transfected with the human Kv1.5 cDNA (Tamkun et al., 1991; GenBank accession no. M60451 [GenBank] ) using pcDNA3.1neo transcription vector (Invitrogen, Carlsbad, CA). Cells were maintained in culture (37°C; 5% CO₂) in Ham's F-12 growth media containing Geneticin (G418) for selection of the Kv1.5 heterologously expressed cDNA. For electrophysiological recordings, the cells were dissociated with 0.05% trypsin and 0.53 mM EDTA (room temperature; 2 min) and suspended in growth media and maintained in suspension by gently rocking in a capped test tube for up to 8 h. Cells were added dropwise to a 100-µl glass-bottomed test chamber, allowed to settle, and then superfused at 2 ml/min with HEPES-buffered saline (HBS) solution containing 132 mM NaCl, 4 mM KCl, 1.8 mM CaCl₂, 1.2 mM MgCl₂, 10 mM HEPES, and 11.1 mM glucose, pH 7.2. Pipette solution contained 119 mM potassium gluconate, 15 mM KCl, 3.2 mM MgCl₂, 5 mM HEPES, 5 mM EGTA, and 5 mM K₂ATP, pH 7.35.

Recording of I_Kur, I_to, and Action Potentials in Isolated Human Cardiac Myocytes. Human atrial myocytes were obtained from specimens of right atrial appendage obtained from patients undergoing cardiopulmonary bypass. All atrial specimens were grossly normal at the time of excision. Human ventricular myocytes were obtained from patients undergoing cardiac transplantation. All cardiac tissues were obtained in accordance with Temple University School of Medicine or Lankenau Hospital Institutional guidelines. Cardiac tissue samples were quickly immersed in ice-cold (0–4°C) cardioplegia solution containing 50 mM KH₂PO₄, 8 mM MgSO₄, 10 mM NaHCO₃, 5 mM adenosine, 25 mM taurine, 140 mM glucose, and 100 mM mannitol, pH 7.4, and bubbled with 100% O₂. Tissue samples were minced into 0.5- to 1-mm cubes and transferred to a 50-ml conical tube containing an ultralow calcium wash solution containing 137 mM NaCl, 5 mM KH₂PO₄, 1 mM MgSO₄, 10 mM taurine, 10 mM glucose, 5 mM HEPES, and 0.1 mM EGTA, pH 7.4 (22–24°C). The tissue was gently agitated by continuous bubbling with 100% O₂ for 5 min. The tissue was then incubated in 5 ml of solution containing 137 mM NaCl, 5 mM KH₂PO₄, 1 mM MgSO₄, 10 mM taurine, 10 mM glucose, and 5 mM HEPES supplemented with 0.1% bovine albumin, 1.5 mg/ml collagenase CLS II (Worthington Biochemicals, Lakewood, NJ), and 1.0 mg/ml protease type XXIV, pH 7.4 (37°C), and bubbled continuously with 100% O₂. The supernatant was removed after 40 min and discarded. The tissue samples were then incubated in a solution of the same ionic composition but supplemented with only collagenase and 100 µM CaCl₂. Microscopic examination of the medium was performed every 10 to 20 min to determine the quantity and quality of isolated cells. When the yield seemed maximal, the tissue samples were suspended in a modified Kraftbrühe solution containing 25 mM KCl, 10 mM KH₂PO₄, 25 mM taurine, 0.5 mM EGTA, 22 mM glucose, 55 mM glutamic acid, and 0.1% bovine albumin, pH 7.3 (22–24°C), and gently triturated using a largebore Pasteur pipette. The supernatant was collected in a 50-ml centrifuge tube. The cell suspension was centrifuged for 2 min at 1000 rpm, and the resulting pellet was resuspended in HBS solution containing 132 mM NaCl, 4 mM KCl, 1.2 mM MgCl₂, 0.2 mM CaCl₂, 10 mM HEPES, and 11.1 mM glucose, pH 7.2. Cells were used within 2 to 24 h after isolation.

For measurement of K⁺ currents, the bath solution contained 132 mM N-methyl-D-glucamine gluconate, 4 mM KCl, 1.2 mM MgCl₂, 10 mM HEPES, 11.1 mM glucose, and 0.4 µM nisoldipine. Pipette solution contained 110 mM K-aspartate, 20 mM KCl, 5 mM MgATP, 5 mM EGTA, and 10 mM HEPES, pH 7.2. All voltage-clamp experiments were performed at room temperature.

For recording action potentials, the bath solution was HBS solution containing 132 mM NaCl, 4 mM KCl, 1.2 mM MgCl₂, 1.8 mM CaCl₂, 10 mM HEPES, and 11.1 mM glucose, pH 7.2, maintained at 35 ± 0.5°C. Myocytes were impaled with sharp, high-resistance (40 M) microelectrodes containing 3 M KCl. Brief stimuli (5 ms) at 1.5 times the current threshold were used to trigger action potentials. A constant hyperpolarizing current of tailored amplitude for each myocyte was used to set the membrane potential uniformly at approximately –80 mV for myocyte action potential recordings (Table 3).

Recording of Kv1.5 and Kv3.1 Currents in X. laevis Oocytes. Kv currents heterologously expressed in X. laevis oocytes were recorded using the TEVC or inside-out patch-clamp technique, as described above. Plasmids encoding channel-forming subunit rat cDNAs were linearized with appropriate restriction enzymes, and cRNA was synthesized by standard procedures. cRNA was injected into X. laevis oocytes: 0.92 ng of rKv1.5 (Swanson et al., 1990; cDNA GenBank accession no. M27158 [GenBank] ) or rKv3.1 (Luneau et al., 1991; cDNA GenBank accession no. M6880); 250 ng of hKv1.5 (Tamkun et al., 1991; GenBank accession no. M60451 [GenBank] ). Oocytes were maintained at 18°C in ND-96, containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES, pH 7.5, supplemented with 550 mg/l sodium pyruvate and 100 µg/ml gentamicin. Macroscopic Kv currents were recorded with ND-96 bath solution and microelectrodes filled with 3 M KCl. In experiments testing the effect of millimolar TEA, TEA-Cl was added to ND-96 with equimolar substitution for NaCl. For hKv1.5 current recording on inside-out excised patches, the vitelline membrane was removed with forceps from the oocytes just before the experiments, and patches were excised from the animal pole. Patch recording was conducted under symmetrical ionic conditions. The pipette (extracellular) solution and the bath (intracellular) solution contained 116 mM potassium gluconate, 4 mM KCl, and 10 mM HEPES, pH 7.25; 30 µM CaCl₂ was added to the pipette solution to facilitate seal formation. Macroscopic Kv1.5 currents in the nonoampere range were routinely recorded from these patches.

Recording of Potassium Currents in Isolated Guinea Pig Ventricular Myocytes. Guinea pig ventricular myocytes were isolated following established protocols (Salata et al., 1998). Experiments were performed at 35°C. Currents were recorded using nominally Ca²⁺-free HBS bath solution containing 0.4 µM nisoldipine to block L-type Ca²⁺ current The intracellular pipette solution contained 500 mM potassium gluconate, 25 mM KCl, and 5 mM K₂ATP. Cells were voltage-clamped at a holding potential (V_h) of –50 mV to inactivate cardiac sodium current. A voltage-clamp protocol consisting of two sequential sweeps, each composed of a ramp and a step, were used to elicit the cardiac K⁺ currents I_K1, I_Kr, and I_Ks. Both protocols began with a 500-ms ramp from –80 to –50 mV, and the background inward rectifier K⁺ current (I_K1) was quantified as the peak outward current during each ramp. After the first ramp, a 500-ms step from –50 mV to a test potential (V_t) of –10 mV was applied to activate primarily I_Kr current, which was quantified as the peak of the tail current upon repolarization to the V_h of –50 mV. After the second ramp, a 1-s step from –50 mV to a V_t of +50 mV was applied to activate primarily I_Ks, which was quantified as the maximum steady-state current at the end of the 1-s activating step.

Determination of Potency and Frequency Dependence. Drug effects on each current were expressed as a fraction of control. The fractional inhibition values were averaged, and mean ± S.E.M. were plotted with SigmaPlot (Systat Software, Richmond, CA), and data were fitted with a Hill equation to calculate IC₅₀ as a measure of potency.

where n_H is the Hill coefficient.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Chemical structures of DPO compounds that are identified throughout this article by the numbers listed.

Determination of Kinetic Parameters of Inhibition. The time courses of the onset of inhibition during current activation by 500-ms depolarizing test pulses to a V_t of +40 mV at and varying concentrations of test agents were fitted to a single exponential decay function of the form I = I₀ e^–t/ + C, where I is the current amplitude measured at time t, I₀ is the current amplitude at time 0, is the time constant of decay, and C is a constant to adjust for any offset.

Recovery from inhibition at repolarizing potentials was measured with a series of paired pulses separated by a varying coupling interval. Each episode in a series consisted of a 500-ms conditioning pulse to +40 mV, followed by a 50-ms test pulse to +40 mV from a V_h of –80 mV. The coupling interval between the paired pulses was incrementally increased in each successive episode from 0.25 to 10 s with an interepisode interval of 60 s to allow complete current recovery before the next conditioning pulse. The time course of recovery of inhibition block was determined by plotting the fractional peak current (test pulse/conditioning pulse) as a function of coupling interval. This time course was fitted with a single exponential function of the form I = I₀ (1 – e^–t/) + C. In a model assuming a bimolecular interaction between the test agent (inhibitor) and the channel only in the open state, derived time constants () for the kinetics of block in the open state (i.e., depolarized membrane potentials) are related to the on and off kinetic rates by the expression 1/ = k_on (conc) + k_off, where k_on and k_off are expressed in units of µM^–1s^–1 and s^–1, respectively. Likewise, derived for the kinetics of recovery from block in the closed state (i.e., repolarized membrane potentials) are related to the off kinetic rate by the expression 1/ = k_off.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Concentration and frequency dependence of DPO-1 inhibition of hKv1.5 current. A, example of the concentration-dependent effect of DPO-1 on hKv1.5 current. Superimposed traces are shown for control and 0.03, 0.1, and 0.3 µM DPO-1. Open-channel block is evident from the concentration-related rate of decline in the current during the activating step depolarization to +40 mV and the slowing of the kinetics of deactivation during repolarization to –30 mV. Inset, control and 0.03 µM deactivating (*) tail currents at an expanded scale to highlight the "crossover" in the presence of DPO-1. B, frequency dependence of inhibition is illustrated by superimposition of currents evoked by the first and last (**) pulses in trains at 1 versus 3 Hz. C, frequency dependence is plotted as the amplitude of current at the end of activating pulses as a function of time in the presence of 0.3 µM DPO-1 expressed as a fraction of control. D, concentration-response relations at pulsing frequencies of 0.1, 1, and 3 Hz expressed as a fraction of control current (n = 3–4).

	Results

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Diphenyl Phosphine Oxides Inhibit hKv1.5 Preferentially in the Open State. In this study, five representative DPO compounds, with chemical structures shown in Fig. 1, were characterized and initially tested on CHO cells stably transfected with the human Kv1.5 channel pore-forming subunit. The potency for inhibition of hKv1.5 current for each of these compounds was determined with the voltage-clamp protocols illustrated in Fig. 2, which also exemplifies the effects of DPO-1. All five DPO inhibited hKv1.5 currents potently with submicromolar affinity as summarized in Table 1. Initially, hKv1.5 current was monitored during voltage-clamp test pulses delivered at 0.1 Hz during control and after application of increasing concentrations of each DPO (Fig. 2A, DPO-1). Thus, a steady-state level of inhibition was established for each concentration of a particular DPO. These agents seemed to inhibit the hKv1.5 channel preferentially in the open state. In the absence of pulsing, little or no inhibition of the Kv1.5 current was detected (data not shown); rather, after activation (opening) of the channels with a step depolarization to +40 mV, the current declined as a function of time and concentration during the test pulse (also see studies in excised patches, below). Furthermore, the kinetics of channel deactivation upon membrane repolarization was slowed by DPO with a "crossover" of the control tail current (Fig. 2A, inset).

Consistent with an open-channel blocking mechanism, DPO inhibition of Kv1.5 current was frequency-dependent to varying degrees for the different DPO. For example, the frequency dependence of DPO-1 is illustrated in Fig. 2, B to D. Conditions to assess frequency dependence were chosen to span physiologically relevant time courses and frequencies, with 150-ms depolarizations delivered at 1 and 3 Hz. Under control conditions, Kv1.5 currents during a train of pulses slowly developed small but significant cumulative inactivation that was more prominent at 3 Hz versus 1 Hz (Fig. 2B, top). In contrast, in the presence of DPO-1 there was more substantial concentration- and frequency-dependent inhibition of the hKv1.5 current as evident at a concentration of 0.03 µM (Fig. 2B, bottom). The level of inhibition was normalized as a fraction of the control current amplitude for each condition. The degree of fractional inhibition was determined isochronally (Fig. 2C) and was not necessarily an absolute "steady state" for each condition and test frequency. Nevertheless, DPO-1 inhibition of hKv1.5 current was clearly greater at 3 Hz than at 1 Hz. Overall the fractional current inhibition at each frequency was plotted as a function of the concentration of DPO-1, and each was fitted with a Hill equation (Fig. 2D). Table 1 lists the potencies of each compound (i.e., IC₅₀) at 0.1-, 1-, and 3-Hz pulsing frequencies, and the slope of the regression line fit to the log-log plots of IC₅₀ versus pulsing frequency provided an index of the degree of frequency dependence for each DPO (see Materials and Methods for details). For example, DPO-1 with the greatest slope displayed the greatest frequency dependence of inhibition.

The characteristics of block and manner of access to the channel by DPO were studied in more detail in excised inside-out membrane patches from X. laevis oocytes expressing hKv1.5 currents (Fig. 3). DPO-1 was added to the bath (intracellular) solution at a saturating concentration of 1 µM, i.e., well above its IC₅₀. When the compound was applied with channels in the resting or closed state (i.e., while the membranes were kept at a holding potential of –80 mV), for as long as 3 min (Fig. 3), no block developed, as substantiated by the maintained amplitude of the initial peak current elicited with the first depolarization. However, with channel opening, block developed quickly as demonstrated by the decline of the current during the first depolarization and a rapid further decline in the current with subsequent depolarizations, and complete inhibition of the activating current occurred rapidly within a few pulses. This inhibition was reversible with washout of the compound (Fig. 3, A and B). In contrast, when the compound was applied during a train of depolarizing steps, the current was reduced in a manner similar to that observed in the stably transfected CHO cells, with more slowly developing cumulative block attributed to diffusion and equilibration of the compound in the bath and the on and off rates of block during the depolarization and repolarization steps (Fig. 3A, c and d). DPO-1 was similarly effective when applied intracellularly or extracellularly to the inside-out patches.

View larger version (23K): [in this window] [in a new window]   Fig. 3. Further characterization of open-channel block of hKv1.5 current by DPO-1. Experiments were conducted on excised inside-out patches from X. laevis oocytes expressing hKv1.5 currents. A, control currents elicited with application of depolarizing step pulses from a holding potential of –80 mV to a test potential of +40 mV were very stable (a). DPO-1 was then applied at a concentration of 1 µM to the bath (intracellular aspect of the patch) for 1 min in the absence of depolarization (horizontal bar labeled –80 mV). With reapplication of test pulses, the initial peak activating current level equaled that during control, but thereafter the onset of block developed during the first postrest pulse and increased and accumulated rapidly with each successive pulse (b). This result suggested that there was little or no closed-state block; rather, block developed only with activation or opening of channels upon depolarization. The inhibitory effect of DPO-1 was slowly and nearly fully reversible with washout (c). Upon re-exposure to DPO-1, but with continuous application depolarizing steps, block developed but over a more gradual time course (d), indicating components of wash-in equilibration and development of open-channel block. B, separate experiment similar to that in A during which control currents were very stable (a), and where after 1 µM DPO-1 was applied to the bath (intracellular aspect of the patch) but for 3 min in the absence of depolarization (horizontal bar labeled –80 mV). Likewise, the initial peak activating current level equaled that during control, but the onset of block during the first activating step was more rapid and complete (b), with little or no further block during subsequent pulses and near complete recovery from block with washout.

For each DPO examined in hKv1.5 currents expressed in CHO cells, the onset of block during a train of depolarizing steps exhibited a unique pattern, adequately modeled as a simple open-channel block, with parameters of kinetic on and off rates of block during the depolarization step, and concentration-independent off rate during repolarization. Current inhibition at depolarized potentials and the recovery from current inhibition upon repolarization were well fitted with the exponential rates predicted by this simple open-state model. Figures 4 and 5 illustrate this analysis for DPO-2. Table 1 presents a summary of kinetic parameters for all DPO examined in this study.

View larger version (13K): [in this window] [in a new window]   Fig. 4. Determination of kinetics of open-state block of hKv1.5 current. The rates of decline in activating currents were fitted with a single exponential decay function, and the time constants () were determined for each of the increasing concentrations of DPO-2 in this example (see Materials and Methods for details). Exponential fits are shown superimposed on the current traces. Reciprocal values of were plotted as a function of the concentration of DPO-2 (0.1, 0.3, 1, and 3 µM tested), and this relationship was fitted with a linear function (n = 4). For a simple channel open-state block model, the derived values of the slope and the y-axis intercept for the fitted line corresponds to the open-state on-rate (kon) and off-rate (koff) constants, respectively, the ratio of which corresponds to the dissociation constant, Kd (Kd = koff/kon).

View larger version (14K): [in this window] [in a new window]   Fig. 5. Determination of kinetics of recovery from block of hKv1.5 current. A, the rate of recovery from block of hKv1.5 channels in the closed state was assessed with a paired pulse protocol. Long (500-ms) followed by short (50-ms) depolarizing paired pulses were applied at incremental interpulse intervals to track the time course of recovery from inhibition in the absence (control) and presence of 1 µM DPO-2 (see Materials and Methods for details; n = 4). B, during control (), the ratios of the current amplitudes for the first versus the second pulse were essentially equal to 1 for all interpulse intervals. In the presence of DPO-2 (), the ratios of the peak current amplitudes elicited with the second short versus the first long pulse were plotted as function interpulse intervals. This time course was well fitted with a single exponential rising function. For a single-site binding model, the derived time constant corresponds to the reciprocal of the closed-state off-rate (koff).

View larger version (25K): [in this window] [in a new window]   Fig. 6. Concentration and frequency dependence of DPO-1 inhibition of IKur. A, example of the concentration-dependent effect of DPO-1 on native IKur of a human atrial myocyte. Superimposed current traces are shown for control and 0.03 and 0.3 µM DPO-1. B, frequency dependence of inhibition is illustrated by superimposition of currents evoked by the first and last (**) pulses in trains at 1 versus 3 Hz. C, frequency dependence of IKur inhibition is plotted as the amplitude of current at the end of activating pulses as a function of time in the presence of 0.3 µM DPO-1 expressed as a fraction of control. D, concentration-response relations at pulsing frequencies of 0.1, 1, and 3 Hz expressed as a fraction of control current (n = 2–4).

View larger version (15K): [in this window] [in a new window]   Fig. 7. Selectivity of DPO-1 for IKur versus Ito. A, currents were elicited with depolarizing step to +40 mV from a holding potential of –80 mV, and deactivating tail currents were recorded during a repolarizing step to –50 mV during control and in the presence of DPO-1 (*). Ito was evident as a relatively rapidly activating and deactivating current during the depolarizing step. In the presence of DPO-1, the current seemed to be shifted downward with little change in its time course. B, drug-sensitive difference current was a relatively rapidly activating current with little or no inactivation, indicating a lack of effect of DPO on Ito. C, with application of a depolarizing prepulse to inactivate Ito, the remaining current was characteristic of IKur, a rapidly activating, delayed rectifier with deactivating tail current upon repolarization that was largely inhibited by 1 µM DPO-1. In human atrial myocytes, there was a DPO-insensitive sustained outward current (Isus) that seemed to be instantaneously activating and deactivating (**).

Further selectivity testing of DPO was conducted on repolarizing cardiac outward K⁺ currents of guinea pig ventricular myocytes. At a concentration of 3 µM, DPO-1 and DPO-2 had had no significant effect on I_Kr and significant (P < 0.05) but small (<25%) effects on I_K1 and I_Ks (Table 2).

DPO also show a high degree of selectivity for Kv1.5 versus Kv3.1. In experiments conducted on rat Kv1.5 and rat Kv3.1 currents heterologously expressed in Xenopus oocytes, DPO-1 inhibited Kv1.5 and Kv3.1 currents with IC₅₀ values of 0.31 and 4.7 µM, respectively. TEA, a well characterized inhibitor of Kv currents, was shown to inhibit Kv1.5 and Kv3.1 currents predictably, with IC₅₀ values of 140 and 0.13 µM, respectively (data not shown). DPO, not unexpectedly, showed little or no selectivity for Kv1.5 over other Kv1.x currents (data not shown).

DPO Prolongs Repolarization of Human Atrial but Not Ventricular Myocytes. DPOs were used to assess the contribution of I_Kur to human atrial repolarization, and inhibition of I_Kur with DPO-1 produced significant prolongation of human atrial APD. At 0.1, 0.2, and 1 µM, and a pacing rate of 1 Hz, DPO-1 increased time when action potential repolarizes to 50% of peak amplitude (APD₅₀) by 25, 118, 137, and 115%, respectively, indicating a concentration-dependent but apparently self-limiting effect (Table 3). It is also worth noting that the effect of DPO-1 on APD₅₀ is larger than the effect on time when the action potential repolarizes to 90% of peak amplitude (APD₉₀). The analysis of action potential parameters is summarized in Table 3. APD₅₀, APD₉₀, and action potential amplitude measured at 50 ms to capture effects on early repolarization were significantly increased (P < 0.05) at concentrations of 0.2 and 1 µM DPO-1, compared with control. In addition, DPO-1 displayed a modest frequency-dependent prolongation of human atrial APD (Fig. 8A). In contrast, DPO-1 had no detectable effect on the APD of human ventricular myocytes (Fig. 8B), whereas the well known I_Kr blocker MK-499 substantially prolonged APD. A constant hyperpolarizing current injection was applied to the isolated human atrial myocytes to standardize the "resting" membrane potentials (RMPs) (Table 3) at approximately –80 mV (see Materials and Methods).

View larger version (17K): [in this window] [in a new window]   Fig. 8. Effects of DPO-1 on human atrial versus ventricular action potentials. A, DPO-1 at a concentration of 0.2 µM increased APD of a human atrial myocyte stimulated at 1 and 3 Hz. Although APD during control decreased with an increase in stimulus frequency from 1 to 3 Hz as expected, the DPO-induced increase in APD was greater at 3 Hz than at 1 Hz, thus showing a frequency-dependent APD prolongation. For each condition, 50 individual action potentials were averaged, and the resting membrane potential was set at –80 mV by transmembrane application of a constant hyperpolarizing current of 160 pA in this example. B, in contrast to the effect on human atrial myocytes, DPO-1 up to 1 µM had no effect on APD of human ventricular myocyte, whereas 100 nM MK-499, a selective inhibitor of IKr, produced a large increase in ventricular APD. No current was injected to set the resting membrane potential in this ventricular myocyte.

	Discussion

Top Abstract Materials and Methods Results Discussion References

Overall, similar inhibitory effects were noted on hKv1.5 overexpressed in CHO cells and on I_Kur current in human atrial myocytes, in terms of potency, frequency, and rate dependence. Whether the native channel requires modulatory subunits or whether these subunits are present at the correct stoichiometry in the CHO cell line overexpressing Kv1.5 subunits remains to be determined.

The assertion that DPO compounds act mechanistically as Kv1.5 open-channel blockers is based on several lines of evidence. First, DPO accelerated the rate of current decay and slowed current deactivation (tail current crossover; Fig. 2). Second, the onset of block, after diffusion and equilibration of the blocker, could only be established upon depolarization (Fig. 3). Third, potency increased as a direct function of stimulation frequency. Finally, the time course of block during depolarization, and the time course of recovery from block during repolarization were well fitted with kinetic on and off rates in a simple model of open channel block (Figs. 4 and 5). None of the compounds studied decreased in potency as a function of increasing stimulation frequency, and one of them, DPO-1, showed a 5-fold increase when the pulse interval was increased from 0.1 to 3 Hz.

The measured parameters of block summarized in Table 1 provide a good qualitative correlation between predicted and measured effects of frequency (0.1, 1, and 3 Hz) on the potencies of block. For example, a connection between slow k_off values for the closed state (e.g., DPO-1) or fast k_on values for the open state (e.g., DPO-4) and compound potency could be established. Likewise, for DPO-1, the k_off value for the closed state was much slower than that for any of the other compounds and might play a role in its steeper frequency dependence.

A number of compounds have been studied with a kinetic analysis similar to that used in this study, including zatebradine (Valenzuela et al., 1996), loratadine (Delpon et al., 1997), and bisindolylmaleimide (Choi et al., 2000), and they have been characterized as open-channel blockers of Kv1.5 channels. However, at least one compound, bertosamil, an analog of tedisamil, with obvious open-channel blocking properties, has been proposed to also modulate the rate of channel inactivation (Godreau et al., 2002). At present, we cannot exclude the possibility that some of the effects described here might also involve mechanisms such as acceleration of C-type inactivation and reduced recovery from the inactivated state.

The selectivity profile for DPO described in this study is substantially different from that of other agents with reported atrial antiarrhythmic activity. Some of these, developed as Kv1.5 channel inhibitors, also block I_to or I_KAch channels with similar affinities. For example, the benzopyran derivative NIP-141/142 (Seki et al., 2002) and the biphenyl AVE0018 (Gögelein et al., 2004) are relatively nonselective, whereas Kv1.5 selectivity has been reported for the biphenyl S9947 (Bachmann et al., 2001). The phenethoxycyclohexane RSD1235 (Cardiome) has been reported to block Kv1.5, I_to, and other potassium currents as well as sodium channels (Fedida et al., 2005), although Kv1.5 selectivity has been reported for C9356 (Fedida et al., 2003). Amiodarone and its derivatives dronedarone and SSR149774C inhibit a number of ion channels, including Kv1.5, at therapeutic concentrations, and they also exert antiadrenergic and antiangiotensin II effects (Doggrell and Hancox, 2004; Gautier et al., 2004). Compounds such as irbesartan, developed as an angiotensin II type 1 receptor antagonist, block Kv1.5 and Kv4.3 with micromolar affinity (Moreno et al., 2003).

The action of DPO to prolong human atrial but not ventricular action potential duration supports a selective therapeutic role for Kv1.5 inhibition in AF. In this context, DPO-1 has been shown to increase myocardial refractoriness in rat and in African green monkey, as anticipated by the cardiac distribution of I_Kur channels in these species. DPO-1 prolongs refractory periods of both atria and ventricle in rats, but produces atrial-selective increased refractoriness in primate (Regan et al., 2006). Likewise, the antiarrhythmic efficacy of DPO-1 in anesthetized dogs was compared with MK-499 and ibutilide, blockers of I_Kr and I_to, respectively, and with propafenone, a sodium current blocker. Although the effect of DPO-1 in this study was atrial-selective, the other drugs, which nonselectively block repolarizing currents expressed in both atria and ventricle, produced additional cardiac effects (Stump et al., 2005).

Antiarrhythmic strategies to treat AF have long pursued the idea that prolonging action potential duration in the heart can be of therapeutic benefit, but only recently have these strategies focused on atrial-specific effects (Brendel and Peukert, 2002; Nattel, 2002; Camm and Savelieva, 2004; Choudhury and Lip, 2004; Greenlee and Vidaillet, 2004; Pecini et al., 2005). Many of the agents specifically developed as I_Kur blockers, such as AVE0118 and related biphenyls S9947 and S20951 [GenBank] (Knobloch et al., 2004), anthranilic amides (Peukert et al., 2004), and the benzopyran NIP-141/142 (Seki et al., 2002), display mixed ion channel activity, as discussed above. In light of results on animal models, the argument has been presented that some mixed ion channel inhibition, such as I_KAch or Kv4.3 block, as opposed to I_K1, human ether-a-go-go-related gene, or sodium channel block, can be of benefit, or at least not be detrimental (Brendel and Peukert, 2003). Likewise, it has been argued that effects of inhibitors of I_Kur on remodeled atria are more pronounced, despite evidence that I_Kur current is down-regulated in remodeled atria (Blaauw et al., 2004). Recently, the notion that I_Kur block in atria can prolong action potential duration, but only after AF remodeling, has been put forward, based on the observed effect of 4-aminopyridine and AVE0118 on atrial trabeculae from either sinus rhythm or AF patients (Wettwer et al., 2004). Clearly, the specific pharmacological properties of cardiac channel inhibitors will help to determine the therapeutic benefit of selective versus nonselective I_Kur inhibition in maintaining sinus rhythm and in preventing or converting AF. In this context, the DPO compounds presented in this study, with submicromolar potency on Kv1.5, intriguing selectivity profiles and frequency-dependent effects, and clear action potential-prolonging effects on non-AF myocytes, could be useful tools to help assess the role played by I_Kur in normal versus AF atrial myocytes.

	Acknowledgements

 
We thank Lori Mixson for invaluable expertise in performing the statistical analyses.

	Footnotes

 
doi:10.1124/jpet.106.101162.

ABBREVIATIONS: AF, atrial fibrillation; I_Kur, ultrarapidly activating delayed rectifier potassium current; hKv1.5, human Kv1.5; APD, action potential duration; I_to, transient outward potassium current; I_KAch, G-protein regulated, muscarinic-activated potassium current; I_Kr, rapidly activating delayed rectifier potassium current; I_Ks, slowly activating delayed rectifier potassium current; DPO, diphenyl phosphine oxide; DPO-1, (2-isopropyl-5-methylcyclohexyl) diphenylphosphine oxide; DPO-2, 2-(diphenylphosphinylmethyl)-4'-fluoro-3,5,3'-trimethylbiphenyl; DPO-3, 1,2-diphenylvinyl diphenylphosphinate; DPO-4, 2-methyl-1-(3-nitrophenyl)-1-propenyl diphenylphosphinate; DPO-5, octyl diphenylphosphinate; TEVC, two-electrode voltage-clamp; CHO, Chinese hamster ovary; HBS, HEPES-buffered saline; TEA, tetraethylammonium; I_K1, inward rectifier potassium current; MK-499, 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; RMP, resting membrane potential; NIP-141/142, (3R*,4S*)-4-cyclopropylamino-3,4-dihydro-2,2-dimethyl-6-(4-methoxy-phenylacethylamino)-7-nitro-2H-1-benzopyran-3-ol; AVE0118, 2'-{[2-(4-methoxyphenyl)-acetylamino]-methyl}-biphenyl-2-carboxylic acid (2-pyridin-3-yl-ethyl)-amide; S9947, [2'-(2-pyridin-2-yl-ethylcarbamoyl)-biphenyl-2-ylmethyl]-carbamic acid benzyl ester; S20951 [GenBank] , 2'-{[2-(4-methoxy-phenyl)-acetylamino]-methyl}-biphenyl-2-carboxylic acid 2,4-difluorobenzylamide; RSD1235, (1R,2R)-2-[(3R)-hydroxypyrrolidinyl]-1-(3,4-dimethoxy-phenethoxy-cyclohexane; SSR149774C, 2-butyl-3-{4-[3-(dibutylamino)propyl]benzoyl}-1-benzofuran-5-carboxylate isopropyl fumarate.

¹ Current affiliation: Department of Preclinical Strategy and Safety Evaluation, Merck Research Laboratories, West Point, PA 19486.

² Current affiliation: Department of Pain Pharmacology, Merck Research Laboratories, West Point, PA 19486.

³ Current affiliation: Pfizer Global Research and Development, Groton, CT 06340.

Address correspondence to: Dr. Joseph J. Salata, Merck Research Laboratories, Cellular Electrophysiology-Preclinical Strategy and Safety Evaluation, WP81-218, P.O. Box 4, 770 Sumneytown Pike, West Point, PA 19486. E-mail: joseph_salata{at}merck.com

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