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(Circulation Research. 1995;77:1151-1155.)
© 1995 American Heart Association, Inc.

Articles

Cloned Human Inward Rectifier K⁺ Channel as a Target for Class III Methanesulfonanilides

J. Kiehn, B. Wible, E. Ficker, M. Taglialatela, A.M. Brown

From the Rammelkamp Center for Research, MetroHealth Campus, Case Western Reserve University, Cleveland, Ohio; the Department of Physiology, Baylor College of Medicine, Houston, Tex; and the Department of Neuroscience Section of Pharmacology (M.T.), Second School of Medicine, University of Naples, Italy.

Correspondence to A.M. Brown, Rammelkamp Center, 2500 MetroHealth Drive, Cleveland, OH 44109-1998.

	Abstract

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Abstract Methanesulfonanilide derivatives such as dofetilide are members of the widely used Class III group of cardiac antiarrhythmic drugs. A methanesulfonanilide-sensitive cardiac current has been identified as I_Kr, the rapidly activating component of the repolarizing outward cardiac K⁺ current, I_K. I_Kr may be encoded by the human ether-related gene (hERG), which belongs to the family of voltage-dependent K⁺ (K_v) channels having six putative transmembrane segments. The hERG also expresses an inwardly rectifying, methanesulfonanilide-sensitive K⁺ current. Here we show that hIRK, a member of the two-transmembrane-segment family of inward K⁺ rectifiers that we have cloned from human heart, is a target for dofetilide. hIRK currents, expressed heterologously in Xenopus oocytes, are blocked by dofetilide at submicromolar concentrations (IC₅₀=533 nmol/L at 40 mV and 20°C). The drug has no significant blocking effect on the human cardiac K_v channels hK_v1.2, hK_v1.4, hK_v1.5, or hK_v2.1. The block is voltage dependent, use dependent, and shortens open times in a manner consistent with open-channel block. While steady state block is strongest at depolarized potentials, recovery from block is very slow even at hyperpolarized potentials (tau=1.17 seconds at -80 mV). Thus, block of hIRK may persist during diastole and might thereby affect cardiac excitability.

Key Words: human inward rectifier K⁺ channel • Class III antiarrhythmic drugs • dofetilide • I_Kr

	Introduction

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Ventricular tachycardia and fibrillation are the major causes of sudden cardiac death. For patients at risk, preventive therapy makes use of Class III antiarrhythmic drugs, which block cardiac K⁺ currents.¹ The rationale is that block of I_K during the CAP prolongs refractoriness, making the heart less susceptible to reentrant arrhythmias. One type of Class III drug, the methanesulfonanilides (dofetilide, E-4031, and D-sotalol), has been shown to block I_Kr and to prolong the CAP.^{2 3 4} Block of other K⁺ channels by methanesulfonanilides is not precluded.

Until recently, the molecular identity of I_Kr was unknown, but a link between I_Kr, the human "ether-a-go-go"–related gene hERG, and the hereditary long QT syndrome has been established.⁵ Thus, hERG injected into Xenopus oocytes produced currents very similar to I_Kr recorded from cardiomyocytes. In another interpretation, the hERG is reported to express an inwardly rectifying, methanesulfonanilide-sensitive K⁺ current.⁶

It is also important to examine drugs for their potential effect on other cloned ion channels to clarify and investigate additional effects. Therefore, we have examined K_v channels having six putative transmembrane segments and IRK channels having two putative transmembrane segments. K_v channels contribute to outward plateau and repolarizing currents; IRKs contribute to terminal repolarization of the CAP and to the resting membrane potential. We found that dofetilide at submicromolar concentrations produces significant block of an IRK channel, hIRK, that we have recently cloned from human heart⁷ and produces no significant block of K_v channels.

	Materials and Methods

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Electrophysiology
Whole-cell and single-channel currents were recorded from oocytes with conventional two-microelectrode voltage-clamp and patch-clamp techniques, respectively.⁸ Macropatch currents were measured by using pipettes made from borosilicate glass, with tip openings of 10 to 15 µm. After achieving a gigaseal, macropatches were excised and positioned in the stream of a large pipette (diameter, 2 mm) to wash out the naturally occurring blocking particles, Mg²⁺ and polyamines, from the oocyte.⁹ Dofetilide was applied to the cytoplasmic surface of the inside-out patches by changing the solution flowing through the application pipette.

Solutions and Drug Administration
Voltage-clamp measurements were performed in a bath (MES solution) containing (in mmol/L) 2.5 KOH, 120 N-methyl-D-glucamine, 122.5 MES, 2 Mg(OH)_2, and 10 HEPES (pH 7.3). In all macropatch and single-channel measurements the bath (EDTA-K⁺ solution) contained (in mmol/L) 100 KCl, 10 EDTA, and 10 HEPES (pH 7.3). In the measurements with Mg²⁺ in the bath, EDTA was replaced by EGTA. The extracellular "low-K⁺" pipette solution contained (in mmol/L) 5 KCl, 100 NaCl, 1.5 CaCl₂, 2 MgCl_2, and 10 HEPES (pH 7.3). The "high-K⁺" pipette solution contained (in mmol/L) 100 KCl, 2 MgCl_2, and 10 HEPES (pH 7.3).

Dofetilide (N-[4-(2-{[4-(methanesulfonamino)phenoxyl]-N-methylethylamino}ethyl)phenyl]methanesulfonamide, Pfizer Central Research) was dissolved in distilled water, acidified to pH 3.0 by addition of HCl in a stock solution to 10 mmol/L, and stored at -20°C. On the day of the experiments, the stock solution was diluted with the intracellular solution (EDTA-K⁺solution) to the desired concentration. All measurements were done at room temperature (20°C).

Data Analysis
Data were low-pass filtered at 1 to 2 kHz (-3 dB, 4-pole Bessel filter) before digitalization at 5 to 10 kHz. PCLAMP software (Axon Instruments) was used for the generation of the voltage-pulse protocols and for data acquisition. Statistical data are expressed as mean±SD.

Molecular Biology
Heterologous expression in Xenopus oocytes of complementary RNAs encoding hIRK, IRK1 (which is 70% identical to hIRK), and ROMK1 (which is 39% identical to hIRK) was performed as previously described.^{7 8 10} The origin of the K_v channel cDNAs is as follows: hK_v1.5 was previously cloned in our laboratory¹¹ and subcloned into A⁺-pCRII, a vector that we constructed to maximize expression in oocytes.⁸ hK_v1.4 was obtained by reverse-transcribed PCR of human heart total RNA with oligonucleotides derived from the published sequence,¹² sequenced, and subcloned into A⁺-pCRII. We cloned hK_v2.1 from a human brain cDNA library and found the predicted amino acid sequence to be consistent with the published sequence.¹³ hK_v1.2 was kindly provided by O. Pongs (Genbank accession No. L02752).

	Results

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Nature of Dofetilide Block
hIRK is clearly an inward rectifier, but whole-cell outward currents between -80 and -30 mV are significant (Fig 1A and 1B). For inside-out macropatches excised in a zero Mg²⁺–zero polyamine solution, dofetilide applied to the cytoplasmatic side blocked outward currents in a slow, time-dependent manner (Fig 1C). Consequently, the dofetilide-sensitive currents (Fig 1D) have rising phases and an activation threshold (Fig 1E). While these properties are similar to those described for I_Kr in guinea pig² and human atrial cardiomyocytes,¹⁴ the steady state current-voltage relationship (Fig 1E) and the tail currents (Fig 1D) are significantly different.

View larger version (31K): [in this window] [in a new window]   Figure 1. Currents (A, B) and steady state current-voltage relationships using two-microelectrode voltage-clamp recordings of hIRK currents expressed in Xenopus oocytes. B, Voltage-dependent block by intracellular polyamines causes the hump in the current-voltage relationship.9 20 25 Holding potential: -45 mV; test pulses: -105 to 5 mV in 10-mV steps of 300 milliseconds. Bath contained 2.5 mmol/L K+ (MES solution). C through H, Block by dofetilide of outward hIRK currents. C, Control currents from an inside-out macropatch and the effects of dofetilide. Holding potential: -80 mV; prepulse: -40 mV, 100 milliseconds; test pulse: 20 mV, 400 milliseconds; return pulse: -40 mV, 400 milliseconds. D, Digitally subtracted dofetilide-sensitive current over the voltage range -140 to 60 mV in 20-mV steps. E, Current-voltage relationship of the dofetilide-sensitive current, measured at the end of a 400-millisecond test pulse. F, Concentration dependence of dofetilide block of macropatch currents. G, Dose-response curves with the normalized values (Idrug/Icontrol) expressed as a function of the dofetilide concentration at different membrane potentials. Solid lines are fits to the binding equation Idrug/Icontrol=1/(1+X/Kd)n, where X is the dofetilide concentration and n is the Hill coefficient. Fitted values for n were 0.8 and displayed as 0.8 in the graph. Log Kd was displayed against membrane voltage at two different extracellular K+ concentrations (n=3). H, Fit of the points resulted in equal values (assuming z=1) for both K+concentrations, according to the following Woodhull equation: Kd(V)=Kd(0) exp (zVF/RT),16 where z is the effective valence and F, R, and T have their usual thermodynamic meaning. Bath: EDTA-K+ solution; pipette: low-K+ solution.

The steady state block was stronger at more depolarized potentials (Fig 1D and 1E), and the steady state current-voltage relationship for the dofetilide-sensitive hIRK current reached a plateau at about +40 mV (n=4). The concentration dependence and voltage dependence of block are shown in Fig 1F and 1G. Submicromolar concentrations were effective at more depolarized potentials (Fig 1G and 1H; IC₅₀=533±254 nmol/L [n=3] at 40 mV). In guinea pig cardiomyocytes, the IC₅₀ value of dofetilide at the higher temperature of 37°C was calculated at 31.0 nmol/L,⁷ but at room temperature 1.0 µmol/L was required to produce significant effects.¹⁵

Dofetilide block of hIRK outward currents (Fig 1C) was also evident in single-channel current recordings (Fig 2). The channels were open at the start of the depolarizing step but closed much more quickly and stayed closed in the presence of dofetilide (Fig 2A). The single-channel current amplitude was unaffected by the drug. The summed single-channel currents mimicked the macropatch currents, with the falling current reflecting the time dependence of the block (Fig 2B).

View larger version (15K): [in this window] [in a new window]   Figure 2. Control single-channel measurements of an inside-out patch (upper three traces) and after bath perfusion with 1 µmol/L dofetilide (lower three traces) (A). Holding potential: -80 mV; test potential: 0 mV (150 milliseconds). B, Averaged currents of 91 (control) and 135 (1.0 µmol/L dofetilide) traces. Bath: EDTA-K+ solution; pipette: low-K+ solution.

The strong voltage dependence of the block and the foreshortening of channel open time (Fig 2A) are consistent with open-channel block. The voltage dependence of block may be rationalized by a single–membrane-binding-site model,¹⁶ in which the blocking site is calculated to be about 85% of the electrical distance across the membrane from the cytoplasmic surface (Fig 1H). The deviation from linearity may be associated with the very small currents recorded at positive potentials or may reflect more than one binding site. For this calculation, we used the measured IC₅₀ values as K_d values.

A prediction of open-channel block is that extracellular K⁺ ions that enter the open channel should relieve the block. This prediction was confirmed by the shift in half-maximal blocking concentrations at higher extracellular K⁺ ion concentrations (Fig 1H).

The strong voltage- and time-dependent block by dofetilide was specific for IRKs as compared with K_v channels. We applied dofetilide at concentrations as high as 10 µmol/L to the cytoplasmic surface of inside-out patches from Xenopus oocytes expressing hK_v1.2, hK_v1.4,¹² hK_v1.5,¹¹ and hK_v2.1.¹³ At submicromolar concentrations no effects were observed, while at 10 µmol/L weak time-independent and voltage-independent block was present (hK_v1.2, 6±5% [n=3]; hK_v1.4, 22±12% [n=4]; hK_v1.5, 9±6% [n=5]; hK_v2.1, 13±8% [n=3]). Experiments with two other Class III methanesulfonanilides, D-sotalol and E-4031, showed that these drugs also blocked hIRK. The K_d values at 40 mV were 717±227 nmol/L (n=4) for D-sotalol and 4.3±3.0 µmol/L for E-4031.

Besides hIRK, dofetilide blocked IRK1.¹⁷ The block was time dependent and voltage dependent, similar to the block of hIRK, and showed the same slow recovery. In contrast ROMK1¹⁸ was blocked by dofetilide only at very positive (80 or 100 mV) potentials, and recovery from block was virtually instantaneous.

Slow Recovery From Block of Dofetilide
A strong feature of dofetilide block is its marked time dependence. Since the heart beats periodically, we wondered whether the drug effects might be cumulative. To test this possibility we measured the rate of recovery from drug block and the offrate at negative potentials, at which the relief of block was readily measured when high extracellular K⁺ concentrations were used to increase inward currents. The offrate and recovery were very slow, even at high extracellular K⁺ (100 mmol/L) (Fig 3A), and became faster at more negative potentials (Fig 3B and 3C). At 5.0 mmol/L [K⁺]_o, the recovery tau was much slower, 1168±553 milliseconds (n=3) at -80 mV recovery potential.

View larger version (36K): [in this window] [in a new window]   Figure 3. Recovery from block by dofetilide in hIRK. A, Left: control measurement of an inside-out macropatch. Right: slow release from block by dofetilide at negative return potentials. Holding potential: 0 mV; prepulse: -80 mV, 100 milliseconds; test pulse: 40 mV, 400 milliseconds; return pulse: 0 to -100 mV in 20-mV steps. B, Slow recovery from block at -40 mV. Holding potential: -60 mV; test pulse: 100 mV, 400 milliseconds; delays to return pulse of 100 mV, 50 milliseconds were 50 to 950 milliseconds in 100-millisecond increments. C, Multiple measurements of recovery at different recovery potentials in a single patch (10 µmol/L dofetilide). Inset: Recovery time constants were fitted by (Irec-I0)/(Imax-I0)=1-exp(-t/tau), where Irec is the peak current after the time of recovery, I0 is the unblocked current at the end of the test pulse, Imax is the peak current of the test pulse, and tau is the time constant of recovery. Bath: EDTA-K+ solution; pipette: 100 mmol/L K+ solution.

Dofetilide Block in the Presence of Physiological Cytoplasmic Blockers
hIRK is 70% identical to IRK1⁷ and has similar inward rectifier properties. IRK1 is blocked by cytoplasmic Mg²⁺ and polyamines. For IRK1, the IC₅₀ for block by Mg²⁺ at 40 mV is 17 µmol/L.⁹ The IC₅₀s for the block by the polyamines spermine and spermidine are 8 and 18 nmol/L, respectively. We calculated the IC₅₀ for SPD block of hIRK to be 23±6.6 nmol/L (n=3) at +40 mV membrane potential. In three experiments Mg²⁺ at 0.187 mmol/L produced 86±6.6% block at +20 mV.

Because cytoplasmic blockers are physiologically significant it was important to evaluate dofetilide blockade of hIRK in the presence of Mg²⁺ or SPD. We used repetitive stimulations at 1.1 Hz to simulate the beating heart,¹⁹ and as expected from the slow recovery, the block was progressive (Fig 4). After 20 pulses we found the block to be 42.0±5.5% (n=3) in the presence of Mg²⁺ at 0.187 mmol/L (free Mg²⁺, about 10 times its IC₅₀; Fig 4A) and 26.5±1.0% (n=2) for SPD at 0.1 µmol/L (about 5 times its IC₅₀; Fig 4B).

View larger version (17K): [in this window] [in a new window]   Figure 4. Dofetilide block of hIRK currents from an excised inside-out macropatch in the presence of 1.0 mmol/L Mg-ATP (0.19 mmol/L free Mg2+) (A) or 0.1 µmol/L SPD (B). Repetitive pulses were used to simulate heart rate and were delivered at 1.1 Hz. Holding potential: -80 mV; conditioning pulse (CP): 20 mV, 400 milliseconds, followed by a return pulse to -80 mV for 500 milliseconds; number of CPs, n; test pulse 1: 20 mV, 400 milliseconds; intervening return pulse: -120 mV, 1.0 second; test pulse 2: 20 mV, 400 milliseconds. C, Block of outward current and inward current in the presence of 10 µmol/L SPD. Holding potential: -75 mV; ramp: -75 to +45 mV, 400 milliseconds; return potential: -95 mV, 1000 milliseconds. Bath: EDTA-K+ solution; pipette: low K+ solution.

In another set of experiments we used very high concentrations of SPD (10 µmol/L), which for inside-out excised macropatches mimicked (Fig 4C) the current-voltage relationship for hIRK observed during recordings from whole cells (Fig 1B) or cell-attached patches.²⁰ Block of outward hIRK currents by dofetilide (10 µmol/L) at -55 mV was 19±6.5% (n=7), and block of inward currents at -95 mV was 10±2.8% (n=7) (measured at the peak current at -95 mV). At the hyperpolarizing potential the slow offrate is apparent (Fig 4C). We also did experiments with SPD (10 µmol/L) plus Mg²⁺ (0.187 mmol/L) and found similar current reductions (18.0±3.1% [n=5] at -55 mV and 15.2±3.0% [n=5] at -95 mV). The combined experiments were fewer in number because of the rundown associated with Mg²⁺.²¹

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our experiments show that the human inward rectifier K⁺ channel hIRK is blocked by dofetilide and other methanesulfonanilides such as E-4031 and D-sotalol. Besides hERG², hIRK is the only molecular target to be identified for this group of Class III antiarrhythmics.

Dofetilide may block open hIRK channels because open times are shortened. In addition, block is enhanced by depolarization. For the latter to be consistent with open-channel block, we assume that the protonated form of the drug is responsible and that access occurs via a cytoplasmic pathway. This assumption is not unreasonable, because at pH 7.4 the amine function of dofetilide is 25% protonated.²³ If the neutral form of the drug permeates the cell membrane, then its positively charged form would produce the appropriate voltage-dependent block. There are precedents for our interpretation. Open-channel block has previously been proposed for dofetilide's actions,¹⁹ and cytoplasmic access was assumed for guinea pig cardiomyocytes because several minutes were required for extracellularly applied dofetilide to block I_Kr.¹⁵

Recovery from block of dofetilide in hIRK is very slow even at hyperpolarized potentials (Fig 3B) and even in the presence of high concentrations of cytoplasmic blockers (Fig 4C). Slow recovery from dofetilide block has also been reported for I_Kr in rabbit ventricular myocytes.²⁴ As a result, at rates comparable to the heart rate, the effect of dofetilide on hIRK is use dependent. Recently it was demonstrated that the prolongation of the guinea pig CAP by Class III methanesulfonanilides is also use dependent.¹⁹

We demonstrated that dofetilide reduced significantly the outward current of hIRK in the presence of concentrations of Mg²⁺ and SPD that mimicked the currents recorded in the whole-cell configuration (Fig 4C). However, in the presence of these cytoplasmic blockers the drug's affinity for hIRK was reduced (cf Figs 1G and 4C)_. Nevertheless, the effects on hIRK are significant and may be even more pronounced under pathophysiological conditions in which the concentrations of these blockers might be reduced. Furthermore, the slow removal of the drug from the channel has important consequences in the rhythmically beating heart. The time dependence of dofetilide block is about 100 times slower than the so-called "intrinsic gating" of IRKs produced by polyamines.^{9 25} Consequently, after exposure to dofetilide the channel remains blocked during the repolarizing phase of the CAP. Since outward current through IRKs produces terminal repolarization and regulates resting membrane potential, this result may have important consequences. On the one hand, dofetilide block may have the desired effect of prolonging the CAP. On the other, there may be an additional effect on diastolic resting potential and cardiac excitability.

	Selected Abbreviations and Acronyms

 

CAP = cardiac action potential hERG = human ether-related gene hIRK = human IRK channel hKv = human cardiac Kv channel IK = repolarizing outward cardiac K+ current IKr = rapidly activating component of IK IRK = inward rectifier K+ channel IRK1 = IRK cloned from a mouse macrophage cell line Kv = voltage-dependent K+ channel ROMK1 = IRK cloned from rat kidney outer medulla SPD = spermidine

	Acknowledgments

 
Dr Kiehn was supported by a grant of the Deutsche Forschungsgemeinschaft, Dr Wible by National Institutes of Health grant HL-37044, Dr Ficker by a grant from the Alexander von Humboldt Foundation, Dr Taglialatela by a grant from the Texas Heart Association (94G218) and a grant from Telethon (Italy) N. 748, and Dr Brown by National Institutes of Health grants HL-37044 and HL-36930.

Received April 3, 1995; accepted August 28, 1995.

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