European Journal of Pharmacology 647 (2010) 13–20

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European Journal of Pharmacology

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Molecular and Cellular Pharmacology

Open channel block of the fast transient outward K+ current by primaquine andchloroquine in rat left ventricular cardiomyocytes

Michael Wagner ⁎, Konstantin Georg Riepe, Esther Eberhardt, Tilmann Volk ⁎Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität, Erlangen-Nürnberg, Erlangen, Germany

⁎ Corresponding authors. Institut für Zelluläre unddrich-Alexander-Universität Erlangen-Nürnberg, WaGermany. T. Volk is to be contacted at Tel.: +49 913122770. M. Wagner, Tel.: +49 9131 85 22303; fax: +49

E-mail addresses: michael.wagner@physiologie2.medtilmann.volk@physiologie2.med.uni-erlangen.de (T. Volk)

0014-2999/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.ejphar.2010.08.007

a b s t r a c t

a r t i c l e i n f o

Article history:Received 12 February 2010Received in revised form 2 July 2010Accepted 12 August 2010Available online 31 August 2010

Keywords:CardiomyocytePrimaquineChloroquineTransient outward K+ currentPatch clampXenopus laevis oocyteKv4.2KChIP2Two-electrode voltage-clamp

Anti-malarial drugs may have severe adverse cardiac effects as a result of their ion channel blockingproperties. Here we investigate the effect of the aminoquinolines primaquine and chloroquine on the fasttransient outward K+ current (Ito) of single epicardial myocytes isolated from the left ventricular free wall offemale Wistar rats. The ruptured-patch whole-cell configuration of the patch-clamp technique was used toinvestigate Ito. At +60 mV, primaquine blocked Ito amplitude (defined as the current inactivating during atest pulse of 600 ms duration) with an IC50 of 118±8 μM. Ito charge was blocked with an IC50 of 33±2 μM(n=42), indicating open channel block. Chloroquine blocked Ito amplitude with an IC50 of 4.6±0.9 mM,while the IC50 for Ito charge was 439±63 μM (n=23). The kinetic analysis of the onset of block revealed Kd

values of 52±8 μM (n=18) and 520±60 μM (n=11) for primaquine and chloroquine, respectively. Bothdrugs significantly accelerated the apparent inactivation time constant of Ito. Steady-state inactivation of Itowas not altered by 30 μM primaquine. In contrast, Ito recovery from inactivation was prolonged with theappearance of an additional long time constant without a change of the short time constant. Exposure to1 mM chloroquine resulted in a right shift of steady-state inactivation, whereas recovery from inactivationwas only mildly affected. Both substances exhibited considerable use dependence. In X. laevis oocytesheterologously expressing hKv4.2+hKChIP2b channels the block by the aminoquinolines was voltagedependent. We conclude that primaquine and chloroquine are open-channel blockers of Ito.

Molekulare Physiologie, Frie-ldstraße 6, 91054 Erlangen,85 24033; fax: +49 9131 859131 85 22770..uni-erlangen.de (M. Wagner),.

l rights reserved.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

The calcium independent transient outward K+ current (Ito) is amajor repolarizing current that contributes to shape and duration ofthe cardiac action potential in rat andman. Furthermore, it adjusts theplateau potential and regulates the action potential-induced Ca2+

influx (Dixon et al., 1996). The transient outward current has beensubdivided into a K+ current component (Ito1 or Ito) and a Ca2+

activated Cl− current (Ito2 or ICl(Ca)) (Zygmunt and Gibbons, 1991).Furthermore, Ito has been shown to consist of a “fast” and a “slow”

component (Ito,f and Ito,s) that can be distinguished based on the speedof their inactivation and recovery from inactivation kinetics (Brah-majothi et al., 1999;Xu et al., 1999). The two components show anoverlapping species distribution as well as an overlapping regionaldistribution within the heart (Nerbonne and Kass, 2005). In themouse ventricle, the highest Ito,f current density has been found inright ventricular and left ventricular subepicardial myocytes, while

Ito,s has been described exclusively in septal myocytes, where it isexpressed alone or in combination with Ito,f (Xu et al., 1999). In ratcardiomyocytes, the contribution of Ito,s to total Ito ranges from 10%in right ventricular myocytes to 20% in septal myocytes (Wickendenet al., 1999). Less information is available regarding the expressionof Ito,s in the left ventricular free wall. Here, a long time constant,which may represent Ito,s, contributes to total recovery from in-activation of Ito between 10% in subepicardial myocytes and 40% insubendocardial myocytes (Volk et al., 2001). For the present studyrat subepicardial left ventricular cardiomyocytes were used sincein these cells ~90% of Ito is represented by Ito,f (Volk et al., 2001). TheIto,f channels are formed by four α-subunits of the Kv4 family (Kv4.2and/or Kv4.3, depending on species) together with the β-subunitKChIP2 and possibly additional subunits, while Kv1.4 channels un-derlie Ito,s (An et al., 2000;Nerbonne and Kass, 2005). To our knowl-edge no comprehensive study on the relative contribution of Ito,fand Ito,s to Ito in rat left ventricular cardiomyocytes has yet beenpublished. Therefore, we decided not to separate these componentsand to simply use the term Ito, which is commonly accepted practicefor rat cardiomyocytes (Nerbonne and Kass, 2005).

Ito is inhibited bymany antiarrhythmic drugs: it is blocked not onlyby a number of class III antiarrhythmics (K+ channel blockers) (Guoet al., 1997;Tamargo et al., 2004), but also by class IV (Ca2+ channelblockers) (Gotoh et al., 1991;Lefevre et al., 1991) and class I drugs

14 M. Wagner et al. / European Journal of Pharmacology 647 (2010) 13–20

(Na+ channel blockers) (Castle and Slawsky, 1993;Slawsky and Castle,1994). The Na+ channel blocker quinidine, a class I drug, is structurallyclosely related to the antimalarial drug quinine, a quinoline derivative.Not surprisingly, quinoline-derived antimalarial drugshavebeenshownto block cardiac ion channels: the 8-aminoquinoline primaquine isa potent Na+ current blocker (Orta-Salazar et al., 2002), while the 4-aminoquinoline chloroquine has been described to block the inwardrectifying K+ current (IK1) (Sanchez-Chapula et al., 2001). Both drugsare widely used antimalarials and while significant cardiotoxicity hasbeen described for chloroquine, less information is available for pri-maquine (White, 2007).Moreover, in vitro, both drugs are used as a toolto study the trafficking of proteins, including ion channels, becausethey block lysosomal degradation (Schwartz et al., 1984;Volk et al.,2004). Therefore a broad knowledge of their pharmacological profile isdesirable. It previously has been reported that Ito was not significantlyblocked by low concentrations of primaquine (Orta-Salazar et al., 2002)and that chloroquine may have an inhibitory effect on Ito (Sanchez-Chapula et al., 2001). However, so far the effects of the aminoquinolinecompounds on Ito have not yet been systematically examined.

Here we investigate the effect of primaquine and chloroquine onIto in rat left ventricular subepicardial cardiomyocytes and demon-strate that both drugs exhibit open channel block of Ito. The exper-iments are complemented by an analysis of the effect of primaquineand chloroquine on currents mediated by hKv4.2+hKChIP2bexpressed in Xenopus laevis oocytes.

2. Material and methods

2.1. Isolation of myocytes

Subepicardial myocytes were isolated from the cardiac left ven-tricular free wall of female Wistar rats (~220 g) as described pre-viously (Isenberg and Klöckner, 1982;Wagner et al., 2008). Briefly,after induction of deep anesthesia by i.p. injection of thiopental-sodium (100 mg/kg body mass), the heart was quickly excised andplaced into cold (4 °C) Tyrode's solution where it stopped beatingimmediately. Subsequently, the heart was retrogradely perfused for5 min with modified Tyrode's solution containing 4.5 mM Ca2+ and5 mM EGTA (~1 μM free Ca2+ concentration) supplemented with1 μM insulin (Sigma, Taufkirchen, Germany). The perfusion was con-tinued for 19 min, recirculating 25 ml of the same solution containingcollagenase (CLS type II, 160 U/ml, Biochrom KG, Berlin, Germany)and protease (type XIV, 0.6 U/ml, Sigma). Finally, the heart was per-fused with storage solution (Benitah and Vassort, 1999) containing100 μM Ca2+ for 5 min. Using fine forceps, myocytes were carefullydissected from the subepicardial layer of the left ventricular free walland placed in cell culture dishes containing the same solution. Tissuepieces were minced and gently agitated to obtain single cardiomyo-cytes. After adaption to physiological Ca2+ levels, cells weretransferred to cell culture dishes containing storage solution supple-mented with 100 IU/ml penicillin and 0.1 mg/ml streptomycin, storedat 37 °C in a water saturated atmosphere containing 5% CO2 andused for experiments for up to 36 h. Only quiescent single rod-shaped cells with clear cross striations were used for experiments.The investigation conforms to the EC Directive 86/609/EEC and wasapproved by local authorities.

2.2. Patch-clamp technique

The ruptured-patch whole-cell configuration was used as de-scribed previously (Hamill et al., 1981;Wagner et al., 2008). Currentswere recorded using an EPC-10 amplifier (HEKA Elektronik, Lam-brecht, Germany), controlled by a Pentium-IV based computer andthe PULSE-Software (HEKA Elektronik). Currents were low-passfiltered at 1 kHz and sampled at 5 kHz. Membrane capacitance (Cm)and series resistance (Rs) were calculated using the automated

capacitance compensation procedure of the EPC-10 amplifier. Rs

averaged 5.1±0.1 MΩ (n=227) and was compensated by 85%. Cm

averaged 129.1±2.1 pF (n=227). Pipette potentials were correctedfor the liquid junction potential of 13 mV. All experiments were per-formed at room temperature (22–24 °C). For each set of experiments,myocytes from two to three rats were used. Unless otherwise stated,reported potentials represent pipette potentials.

Since cardiac voltage-gated Na+, Ca2+, and K+ channels activateat overlapping potentials, they need to be carefully experimentallyseparated. It is well established to use divalent cations, e.g. 0.3 mMCd2+, to inhibit the L-type Ca2+ current and to inhibit the voltage-gatedNa+ current by a prepulse (Wagner et al., 2008). The use of Cd2+

or Co2+was impossible in the experiments performed, since they forminsoluble complexes with the phosphate anions contained in thepreparation of primaquine and chloroquine used. Other Ca2+ channelblockers, like dihydropyridines, phenylalkylamines or benzothiaze-pines, are also potent blockers of Ito (Gotoh et al., 1991;Lefevre et al.,1991;Rolf et al., 2000), thereby interfering with the effect of theexamined drugs. Therefore, it was decided not to use inhibitors of Na+

and Ca2+ currents. Instead, tominimize contributions of Na+ and Ca2+

currents to the measured signal, Ito was measured at a test potentialof +60 mV, which is close to the effective reversal potential of thesecurrents.

2.3. Isolation, injection and maintenance of X. laevis oocytes

Female X. laevis were anesthetized by immersion in 0.2% MS-222for 10 min. Ovarian lobes were surgically removed and oocytes wereisolated by enzymatic digestion using collagenase (CLS type II, 260 U/ml, Biochrom KG, Berlin, Germany) in Ca2+ free OR2 solution at 10 °Cfor 3–4 h. We used full-length cDNA transcripts encoding humanKv4.2 (hKv4.2) inserted in pGEM and human KChIP2b (hKChIP2b)included in pGEM-HJ. Linearised plasmids were used as templatesfor cRNA synthesis using the mMessage mMachine Transcription KitT7 (Ambion, Austin, USA). Defolliculated stage V–VI oocytes wereinjected with 0.1 ng hKv4.2+0.5 ng hKChIP2b cRNA. cRNAs weredissolved in RNAse-free water and the total volume injectedwas 50 nlper oocyte. After injection, oocytes were maintained in ND96 solutionand were studied two days after injection.

2.4. Two-electrode voltage-clamp experiments

Oocytes were transferred to a perfusion chamber, continuouslysuperfused with NaCl-95 solution and impaled with electrodes (0.1–1.5 MΩ) filled with 3 M KCl. Whole-cell currents were measured atroom temperature (19–22 °C) with the two-electrode voltage-clamptechnique using an OC-725C amplifier (Warner Instruments Corp.,Hamden, USA) controlled by the Pulse-software (HEKA Elektronik)running on a Pentium IV computer via a LIH-1600 interface (HEKAElektronik). An Ag–AgCl pellet placed directly in the bath solutionserved as reference electrode for the current injection circuit while anadditional Ag–AgCl pellet located close to the oocyte was used tosense the bath potential in order to minimize series resistance errors.Pulsed current data were filtered at 1 kHz and sampled at 5 kHz.

2.5. Solutions and drugs

Modified Tyrode's solution was used for cell isolation and as bathsolution and contained (in mM): NaCl 138, KCl 4, MgCl2 1, NaH2PO4

0.33, CaCl2 2, glucose 10, HEPES 10 (pH 7.30 with NaOH). Myocytestorage solution contained (in mM): NaCl 130, NaH2PO4 0.4, NaHCO3

5.8, MgCl2 0.5, CaCl2 1, KCl 5.4, glucose 22, HEPES 25 (pH 7.40 withNaOH in the presence of 5% CO2) and supplementedwith 1 μM insulin,1 mg/ml BSA, 100 IU/ml penicillin and 0.1 mg/ml streptomycin. Thepipette solution contained (in mM): Glutamic acid 120, KCl 10, MgCl24, EGTA 10, HEPES 10, Na2ATP 2 (pH 7.20 with KOH). OR2 solution

APrimaquine Chloroquine

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Fig. 1. Inhibition of Ito byprimaquine and chloroquine.A: Representative outward currentsofmyocytes obtained in the absence or presence of primaquine (left panel) or chloroquine(right panel, 0.3 μM, 3 μM, 30 μM, 100 μM, and 1 mM). Starting from a holding potential of-90 mV myocytes were clamped for 600 ms at a test potential of +60mV. Capacitiveartifacts were removed for means of clarity. B: Average concentration-response curves ofIto amplitude (open circles) and Ito charge (filled circles) obtained from experimentssimilar to those shown in A. The left panel displays experiments in the presence ofprimaquine while the right panel depicts experiments in the presence of chloroquine. Itoamplitude was calculated by subtracting the current at the end of the test pulse from thepeak current, while Ito charge was calculated by integrating the inactivating currentcomponent over time. Bothwere normalized to theirmagnitude under control conditions.Error bars represent S.E.M. 23≤n≤42. C: Average concentration-response curves of Ilateamplitude (filled squares) and Ito charge (filled circles) from experiments similar to thoseshown in A. The left panel displays experiments in the presence of primaquine while theright panel depicts experiments in the presence of chloroquine. Ilate amplitude and Itochargewere normalized to theirmagnitude under control conditions. Error bars representS.E.M. 25≤n≤36.

15M. Wagner et al. / European Journal of Pharmacology 647 (2010) 13–20

contained (in mM): NaCl 82.5, KCl 2, MgCl2 1, HEPES 1 (pH 7.40 withNaOH). ND96 solution contained (in mM): NaCl 96, KCl 2, MgCl2 1,CaCl2 1.8, HEPES 5 (pH 7.40withNaOH), supplementedwith 100 U/mlpenicillin and 0.1 mg/ml streptomycin (Sigma). NaCl-95 solutioncontained (inmM):NaCl 95, KCl 4,MgCl2 1, CaCl2 1, HEPES10, (pH7.40with Tris). Primaquine bisphosphate and chloroquine bisphosphatewere obtained from Sigma and were freshly dissolved in the bathsolution on each day of experiments. pH was readjusted with NaOH.

2.6. Data analysis and statistics

Data were analyzed using the PULSE-FIT software (HEKA Elek-tronik), IGOR Pro (WaveMetrics, Lake Oswego, USA) and MicrosoftExcel (Microsoft Corporation, Redmond, USA) as described previously(Volk et al., 2001).

Voltage dependence of block was assessed by fitting the Woodhullequation

QB = QC = 1− B½ �= B½ � + Kd⋅expð−z⋅δ⋅F⋅V = ðR⋅TÞÞð Þ

(QB, charge in the presence of primaquine, QC, charge undercontrol conditions, [B], primaquine concentration, Kd, equilibriumdissociation constant, z, charge of primaquine (+1 at an assumedintracellular pH of 7.2) or chloroquine (+1.66 at pH 7.2), δ, relativeelectrical distance, F, Faraday's constant, V, membrane potential, R,ideal gas constant, T, absolute temperature) to the relative charge notblocked by primaquine or chloroquine (Woodhull, 1973). The chargeof the aminoquinolines at a given pH was calculated usingMarvinSketch (ChemAxon, Budapest, Hungary).

Data are given as mean±S.E.M. Calculations were performedusing Prism (GraphPad, San Diego, USA) and statistical significancewas evaluated by the appropriate version of Student's t-test or one-way ANOVA followed by a Newman–Keuls post-hoc test when morethan two groups were compared. The Dunnett's Multiple Comparisontest was used when values were compared to control only. Pb0.05was considered statistically significant.

3. Results

3.1. Inhibition of Ito by primaquine and chloroquine

Fig. 1A shows representative outward currents from myocytesexposed to increasing concentrations of primaquine (left panel) orchloroquine (right panel). Outward K+ currents were elicited by a600 ms long voltage step to +60 mV from a holding potential of−90 mV. Under control conditions, a rapidly activating outwardcurrent is visible which subsequently inactivates with a remainingslowly or non-inactivating current component. In this study, Itowas assessed as the difference between the peak current and thecurrent at the end of the voltage step. The current at the end ofthe voltage step was designated Ilate and consists of slowly and non-inactivating current components. In the cardiomyocytes isolatedfrom the subepicardial layer of the left ventricular free wall used forthe concentration-response curves, Ito current density at +60 mVaveraged 47.4±2.0 pApF−1 (n=69) while Ilate was 9.2±0.4 pApF−1

(n=69). Primaquine as well as chloroquine inhibited both, Ito andIlate, in a concentration dependent manner. Average concentration–response curves for Ito and Ilate are depicted in Fig. 1B and C. By fittingthe Hill equation (Hill, 1910) to the concentration–response curve ofeach individual cell, IC50 values were obtained for Ito currentamplitude inhibition by primaquine and chloroquine which averaged117.6±8.1 μM (n=42) and 4.6±0.9 mM (n=23), respectively(Table 1). To assess steady-state block, the integral under theinactivating current component between the peak current and thecurrent level at the end of the test pulse was calculated (Ito charge).Like Ito amplitude, Ito charge was inhibited in a concentration

dependent manner (Fig. 1B). However, the IC50 values for primaquine(33.3±1.8 μM, n=42, Pb0.001) and for chloroquine (438.8±6.3 μM,n=23, Pb0.001) were approximately one order of magnitude lowerfor Ito charge than the corresponding IC50 values for Ito amplitude(Table 1). This suggests that both aminoquinolines preferentiallyblock open channels. The IC50 for Ilate inhibition was in a similar rangeas that for Ito charge with 31.6±2.3 μM (n=36) for primaquine and389.1±42.6 μM (n=25) for chloroquine (Table 1). The chloroquine-induced block was far from complete even at the highest concentra-tion used (1 mM). Therefore, the IC50 values for chloroquine had to becalculated under the assumption that a complete block can beachieved at very high concentrations. To further investigate theeffects of the aminoquinolines on Ito, concentrations of 30 μMprimaquine and of 1 mM chloroquine were selected, since theseconcentrations exhibited a substantial inhibition of Ito (~30% ofcurrent amplitude) with enough residual current to allow a robustevaluation of the data.

3.2. Influence on the kinetic properties of Ito

Ito inactivated rapidly with a time constant of ~30 ms (Table 2). Ascan be seen in Fig. 2A, primaquine (left panel) as well as chloroquine

Table 1Concentration-dependent inhibition of Ito by primaquine and chloroquine.

IC50 (μM) Hill coefficient Onset of block kinetics

Current Charge Current Charge n Kd (μM) K+1 (μM−1 s−1) K−1 (s−1) n

PrimaquineIto 117.6±8.1 33.3±1.8a 1.0±0.0 1.0±0.0 42 51.8±8.2 3.5±0.3 160.8±16.3 18Ilate 31.6±2.3 1.0±0.1 36

ChloroquineIto 4566±897 438.9±62.9a 0.9±0.2 0.6±0.1 23 520.4±59.5 0.2±0.0 78.1±8.9 11Ilate 389.1±42.6 0.6±0.3 25

Half maximal inhibitory concentration (IC50) and Hill coefficient were calculated from concentration-response curves in eachmyocyte. Currents were assessed as peak current minuscurrent at the end of the test pulse. Charge was calculated as the integral of the inactivating current components between peak current and the end of the voltage pulse. Kd,equilibrium dissociation constant calculated from the onset of block kinetics in each myocyte. K+1, K−1, association and dissociation rate constants of the onset of block.

a Pb0.001 charge vs. current; n, number of cells examined.

16 M. Wagner et al. / European Journal of Pharmacology 647 (2010) 13–20

(right panel) accelerated the apparent inactivation of Ito: primaquinesignificantly accelerated Ito inactivation starting at a concentration of3 μM, whereas chloroquine did not affect Ito inactivation at concen-trations lower than 100 μM. The time constants for all examinedconcentrations are given in Table 2.

Fig. 2B shows the steady-state inactivation of Ito. Details aregiven in Table 3. In control cells, half maximal inactivation wasobserved at ~−58 mV. Since the bath solution did not contain Cd2+,half maximal inactivation of Ito occurred at markedly more negativevoltages compared to other studies (Agus et al., 1991;Volk et al.,2001). 30 μM primaquine (left panel) did not affect Ito steady-stateinactivation. 1000 μM chloroquine (right panel) however resulted in asignificant rightward shift of Ito inactivation from −57.2±0.7 mV(n=11) to−52.6±0.8 mV (n=14, Pb0.001). The slope factors werenot affected by the drugs used.

Recovery from inactivation (Fig. 2C) of Ito followed a bi-ex-ponential time course: more than 85% of Ito recovered quickly with atime constant of ~60 ms, while the remaining current recovered witha longer time constant in the range of seconds (Table 3). In cellsexposed to 30 μM primaquine (left panel), these time constants werenot significantly altered. Instead, a third time constant of ~360 msappeared and accounted for ~20% of recovery, probably reflecting theunbinding of primaquine from the resting state of the channel. Thissuggests that primaquine not only blocks open but also inactivated Itochannels. Overall, this resulted in amarkedprolongationof Ito recoveryfrom inactivation (Fig. 2C). Chloroquine (right panel) only mildlyaffected Ito recovery; the appearance of a third time constant was notevident. However, the long time constant of recoverywas significantlyaccelerated and accounted for a marginally (n.s.) larger part ofrecovery. This may indicate that also in the case of chloroquine, anintermediate time constant is present, but cannot be separated fromthe long time constant due to its small contribution to total recovery.

3.3. Use dependence of the aminoquinoline-induced block of Ito

A prolonged recovery from inactivation suggests use dependenceof block. Fig. 3A demonstrates representative Ito traces elicited by

Table 2Inactivation time constants of Ito in cardiomyocytes exposed to the aminoquinolines.

0 0.3 3

[Primaquine] (μM)τ60mV (ms) 26.7±0.7 25.7±0.6 23.9±0.7a

[Chloroquine] (μM)τ60mV (ms) 38.9±1.1 36.9±1.3 37.8±1.5

τ60mV, inactivation time constants of Ito calculated by fitting a bi-exponential function to thholding potential of−90 mV in each myocyte. The second time constant served to eliminatefurther examined.

a Pb0.01 vs. 0 μM primaquine; n, number of myocytes examined.

pulse trains to +60 mV at 2 Hz measured in a control myocyte and amyocyte exposed to 30 μM primaquine. Ito block by primaquine wascharacterized by a marked use dependence at 5 Hz (Pb0.01 at thesecond pulse, Pb0.001 from the third pulse onward, Fig. 3D leftpanel), 2 Hz (Pb0.001 from the second pulse onward, Fig. 3C leftpanel) and at 0.5 Hz (Pb0.05 at the fifth pulse, Pb0.01 from the sixthpulse onward, Fig. 3B left panel). As expected from the recovery frominactivation kinetics, chloroquine exhibited weaker use dependence:no use dependence was observed at 0.5 Hz (Fig 3B, right panel).However, at 2 Hz (Pb0.001 from the secondpulse onward, Fig. 3C rightpanel) and at 5 Hz (Pb0.01 from the secondpulse onward, Fig. 3D rightpanel) the chloroquine block of Ito was also use dependent.

3.4. Analysis of the onset-kinetics of block

In the presence of open channel block it is possible to directlymonitor the onset of block and, by analyzing its kinetics, to derivethe equilibrium binding constant (Kd) assuming a pseudo-first-orderkinetics (Caballero et al., 2004). A concise description of theunderlying theory can be found in O'Shannessy, et al. (1993). Theonset-kinetics of the primaquine- and chloroquine-induced blockwere analyzed by calculating the ratio between the aminoquinoline-sensitive current and the current under control conditions (fractionalblock, FB=(IC− IAQ)/IC). Subsequently, a monoexponential functionwas fitted to the time course of the fractional block (Fig. 4A+B, leftpanels), thus obtaining the time constant of the onset of block (τblock).For each cell, the rate constant of block (1/τblock) was plotted againstthe drug concentration (primaquine: 20–100 μM, chloroquine 200–1000 μM). Linear regression (average values: Fig. 4A+B, right panels)yielded the apparent rate constants of association (K+1, slope) anddissociation (K−1, y-intercept). The equilibrium dissociation constantwas calculated as Kd=K−1 / K+1 and averaged 51.8±8.2 μM(n=18)for primaquine and 520.4±59.5 μM (n=11) for chloroquine. Thesevalues are in good agreement with the IC50 calculated from Ito chargefor both drugs (Tables 1 and 2). The lower potency of chloroquinecompared to primaquine can be explained by the approximately 20-fold lower association rate constant. This together with the lower

30 100 1000 n

15.4±0.5a 8.4±0.4a 3.9±0.3a 41

34.4±1.8 23.6±1.5a 9.9±0.6a 24

e decay of the outward current elicited by a 600 ms voltage step to +60 mV from thepotential contaminations by more slowly inactivating current components and was not

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Fig. 2. Effect of primaquine and chloroquine on the kinetic properties of Ito. A: Itoinactivation under control conditions and in the presence of 30 μM primaquine (leftpanel) or 1 mM chloroquine (right panel). Each current trace was normalized to itspeak to compare the inactivation kinetics. Capacitive artifacts were removed for meansof clarity. B: Voltage dependence of steady-state inactivation of Ito in myocytes exposedto 30 μM primaquine (left panel) and 1 mM chloroquine (right panel). Steady-stateinactivation was assessed using a double pulse protocol. A prepulse of 600 ms durationto conditioning potentials between −80 mV and +10 mV was followed by a 600 mstest pulse to+60 mV. Ito was calculated by subtracting the current at the end of the testpulse from the peak current. Each current was normalized to the current measuredafter a conditioning potential of -90 mV. Average relative current is plotted versus theconditioning potential. Cycle length was 3 s. Filled circles depict control conditionswhile open circles depict the respective drug. Error bars represent S.E.M. 11≤n≤17.C: Recovery from inactivation was examined using a double pulse protocol. Ito wasinactivated by a prepulse of 600 ms duration to +60 mV which was followed by anexponentially rising duration at -90 mV during which Ito could recover. The extent ofrecovery was evaluated by a second pulse to +60 mV. Relative current was calculatedby dividing this Ito amplitude by the Ito amplitude elicited by the prepulse. The averagerelative current is depicted versus the duration at −90 mV. Please note that theduration is plotted on a logarithmic scale. Cycle length was 10 s. Filled circles depictcontrol conditions while open circles depict the respective drug. Error bars representS.E.M. 7≤n≤13.

17M. Wagner et al. / European Journal of Pharmacology 647 (2010) 13–20

dissociation rate constant of chlorquine, which is only about halfthat of primaquine, explains the nearly 10-fold higher Kd ofchloroquine.

3.5. Voltage dependence of the aminoquinoline-induced block ofhKv4.2+hKChIP2b channels

In rat, the main contributors to native Ito channels are Kv4.2 andKChIP2. Since voltage-dependent properties of block could not beexamined in cardiomyocytes under our experimental conditions, weassessed the voltage dependence of the aminoquinoline-inducedblock in oocytes heterologously coexpressing hKv4.2 and hKChIP2b.Fig. 5A displays representative currents from an oocyte expressinghKv4.2+hKChIP2b recorded under control conditions (left panel)and in the presence of 100 μM primaquine (right panel). Similar to theeffect on Ito in cardiomyocytes, primaquine and chloroquine accelerated

the inactivation of hKv4.2+hKChIP2b currents (data not shown). At+60 mV the outward current (peak current minus current at the endof the test pulse) averaged 24.4±2.1 μA (n=15). The IC50 for steady-state block was 61.8±8.6 μM (n=7) for primaquine and 1.8±0.6 mM(n=8) for chloroquine. Both values are higher than the IC50 observed incardiomyocytes for the inhibition of Ito by primaquine and chloroquine.

Fig. 5B summarizes voltage dependence of activation and steady-state inactivation, together with the voltage-dependent properties ofblock. To characterize steady-state block, charge was used to assessthe voltage dependence of block. Activation and steady-stateinactivation were calculated from the time-integral of the inactivatingcurrent. Half maximal activation occurred at 16.0±1.7 mV (n=15).The slope factor of the Boltzmann equation was 28.5±1.1 mV(n=15). Primaquine and chloroquine are positively charged at phys-iological pH. This could give rise to a voltage dependence of block,since the blocker may be driven into the channel (or away from it) bythe membrane potential. From −20 mV to +60 mV, the relativecharge not blocked by primaquine or chloroquine decreased withvoltage, indicating a strong voltage dependence of block (Fig. 5B andC, filled and open triangles). For 30 μM and for 100 μM primaquine,the Woodhull equation (Woodhull, 1973) was fitted to the voltagedependence of the relative charge not blocked by primaquine in eachexperiment (Fig. 5B), yielding a relative electrical distance of thebinding site of 0.29±0.04 and 0.38±0.05, respectively (n=7). Thepredicted Kd at 0 mV was 111.9±13.4 μM for 30 μM and 70.1±8.5 μM for 100 μM primaquine (n=7). These values were slightlylower than the IC50 calculated at a test potential of 0 mV from theindividual concentration–response curves (162.0±16.5 μM, n=7).This indicates that the interaction of primaquine with the hKv4.2+hKChIP2b channels is in general agreement with the Woodhullformalism but probably somewhat more complex than accounted forin this model. Similar fits were performed for 1 mM and 5 mMchloroquine (Fig 5C). Here the relative electrical distance amounted to0.32±0.05 (1 mM, n=8) and 0.38±0.04 (5 mM, n=7) with Kd

values of 10.3±3.0 mM (1 mM, n=8) and 10.5±5.2 mM (5 mM,n=7). This again is in good agreement with the IC50 obtained fromindividual concentration–response performed at 0 mV (8.8±1.2 mM,n=4).

In the oocyte system we also examined whether hKv4.2+KChIP2bchannels are blocked at negative voltages, where inactivation occurs inthe absence of channel activation (Fig. 5B, diamonds). Steady-stateinactivation started at −60 mV and half maximal inactivation wasmeasured at −46.9±0.8 mV (n=15). The slope factor was −3.9±0.1 mV (n=15). The relative block increased with inactivation: theremaining charge at conditioning potentials of −50 mV (Pb0.01 for30 μM primaquine) and −40 mV (Pb0.01 for both 30 μM and 100 μM,n=7; Pb0.01 for 1 mM chloroquine (n=8); Pb0.05 for 5 mM chlo-roquine) was significantly different from the charge measured at aconditioning potential of−90 mV. A similar behavior was found, whencurrent instead of charge was assessed (data not shown). This findingsupports the idea thathKv4.2+hKChIP2bchannels are not only blockedin the open but also in the inactivated state by both aminoquinolines.

4. Discussion

In this study we analyzed the effects of two anti-malarial drugs,the aminoquinolines primaquine and chloroquine, on the transientoutward K+ current in rat left ventricular cardiomyocytes. In additionwe investigated the voltage-dependence of the aminoquinoline-induced block of hKv4.2+hKChIP2b channels heterologouslyexpressed in X. laevis oocytes. Our results demonstrate that bothdrugs block Ito channels and suggest open channel block as theunderlying mechanism. These results complement the description ofprimaquine as a Na+ channel blocker (Orta-Salazar et al., 2002) andare in line with an earlier report that chloroquine weakly blocks Ito(Sanchez-Chapula et al., 2001).

Table 3Effect of the aminoquinolines on steady-state inactivation and recovery from inactivation of Ito.

Control 30 μM Primaquine Control 1 mM Chloroquine

Steady-state inactivationV1/2 (mV) −58.6±0.6 −59.1±0.6 −57.2±0.7 −52.6±0.8b

Slope factor (mV) −5.2±0.2 −4.6±0.2 −4.4±0.2 −4.6±0.2n 17 11 11 14

Recovery from inactivationτ1 (ms) 67.8±4.1 64.9±4.2 55.4±2.6 54.4±2.5τ2 (ms) 358.6±21.6τ3 (s) 2.18±0.5 2.2±1.1 3.1±0.8 1.2±0.3a

% τ1 86.3±5.0 56.8±2.9 90.0±2.9 87.3±1.5% τ2 20.1±7.5% τ3 13.7±5.0 23.1±6.4 10.0±2.9 12.7±1.5n 8 7 11 13

Half maximal inactivation (V1/2) and the slope factor were calculated by fitting the Boltzmann equation to the steady-state inactivation curve of Ito in each myocyte. Recovery frominactivation was assessed by fitting a bi-exponential or, when possible, a triple-exponential function to the recovery of Ito in each myocyte. τ1–τ3 designate the obtained timeconstants, % τ1–% τ3 represent their contribution to total recovery. n, number of myocytes examined.

a Pb0.05, b Pb0.001 chloroquine vs. control; n, number of cells examined.

18 M. Wagner et al. / European Journal of Pharmacology 647 (2010) 13–20

4.1. Open channel block of Ito by the aminoquinolines

Several findings indicate open channel block of Ito by the examinedcompounds. Both blockers inhibit Ito charge with a tenfold higherpotency than Ito amplitude and concentration-dependently acceleratethe apparent inactivation time constant. By analyzing the timedependence of block, it was directly possible to monitor the onset ofblock (Fig. 4). Calculating the equilibrium dissociation constant fromthe kinetics of the onset of block resulted in similar values as those forthe IC50. Taken together these findings strongly suggest open channelblock. Primaquine also markedly prolonged recovery from steady-state inactivation (Fig. 2) by adding an additional time constant to therecovery kinetics. The appearance of an additional time constant hasbeen suggested to represent the unbinding of the blocker form theresting state of the channel (Slawsky and Castle, 1994). A slowing ofthe recovery kinetics should result in use dependence of block, whichwas demonstrated for primaquine (Fig. 3). Chloroquine also displayeduse dependence of block at higher frequencies, but recovery frominactivation was only marginally affected (Fig. 2) and we could notdetect the appearance of a third time constant of recovery. However,since the degree of use dependence of chloroquine was small, therelative contribution of a potential third component of recoverymighthave been below the threshold of detection by triple-exponentialfitting. A minor contribution of a time constant representing thedissociation of chloroquine from the channel to total recovery mayexplain the smaller degree of use dependence of the chloroquine-induced block compared to the block by primaquine.

So far we have argued that primaquine and chloroquine are openchannel blockers, binding to the open, but not or substantially lessto the closed Ito channel. The slowing of recovery observed forprimaquine and the use-dependent block of both compounds indicatethat a substantial block of inactivated channels also occurs. However,these results do not rule out a very slow dissociation of the drugs frominactivated channels. When the inactivated channel is blocked, a shiftof the steady-state inactivation curve tomore negative potentials maybe expected, caused by a block of the inactivated channels in additionto the voltage-dependent inactivation (Radicke et al., 2008). Theminimal, insignificant, leftward shift of the inactivation curve forprimaquine neither supports nor contradicts this conclusion. How-ever, the hypothesis that inactivated channels are blocked is stronglysupported by our oocyte expression studies. Here we found thatsteady-state inactivation of hKv4.2+hKChIP2b channels went alongwith an increase in steady-state block. This indicates that channelswere partly blocked when they became inactivated during theconditioning step, thereby reducing peak current as well as chargeduring the test pulse. It is quite likely that native Ito channels exhibit a

similar behavior in cardiomyocytes, too. The rightward shift in steady-state inactivation induced by chloroquine in cardiomyocytes is un-expected and seems to contradict the block of inactivated channels bychloroquine. However, for this experiment, a relatively high concen-tration of chloroquine (1 mM) was used (in the corresponding ex-periments with primaquine only 30 μM was employed), which mayhave unspecific effects. For example chloroquine may affect thesurface potential of the cell membrane in a similar way as describedfor divalent cations like Cd2+ or Ca2+ (Agus et al., 1991), therebyshifting the inactivation curve to more positive potentials.

4.2. Voltage dependence of block

To characterize the voltage dependence of block, the oocytesystem was used. Whereas the IC50 for primaquine was similar incardiomyocytes and in cloned hKv4.2+hKChIP2b channels expressedin X. laevis oocytes, the IC50 of chloroquine was more than four timeshigher in oocytes than in cardiomyocytes. It is not clear whether this iscaused by a different composition of the heterologously expressedchannels (e.g. a lack of a subunit that contributes to the native Ito incardiomyocytes) or by other factors. Nevertheless, also for chloro-quine the data obtained from the oocytes expressing hKv4.2+hKChIP2b gives insight into the voltage-dependent properties ofblock thatmost likely are also true for the native Ito in cardiomyocytes.The increasing block of hKv4.2+hKChIP2b channels at positivemembrane potentials is consistent with a positive blocking ionmoving from the inside into the channel pore. Moreover, this suggeststhat the aminoquinolines do not act from the extracellular side of themembrane (where they are applied), but need to cross the membraneto block the pore from the inner side. Although the IC50 and calculatedKd differed one order of magnitude between primaquine and chlo-roquine, the relative electrical distances derived by the Woodhullequationwere similar. This may imply a common binding site for bothaminoquinolines.

4.3. Limitations

In this study some experimental compromises had to be made.Despite the complex nature of the total outward current present inrat ventricular cardiomyocytes (Himmel et al., 1999;Wickenden et al.,1999), we used a relatively simple method to identify Ito: the dif-ference of the peak current minus the current at the end of a 600 msvoltage pulse. This reliable and widely used method is appropriate toassess Ito in rat subepicardial myocytes under the chosen conditions,because in these cells Ito represents by far the largest outward currentcomponent (Volk et al., 2001;Nerbonne and Kass, 2005). Since this

Fig. 4. Onset of block kinetics of primaquine and chloroquine. A: The left panel depicts thedevelopment of the Ito block by different concentrations of primaquine (20 μM, 40 μM,60 μM, 80 μM, and 100 μM) in a representative cardiomyocyte. Relative blockwas assessedby dividing the primaquine-sensitive current by the current recorded under controlconditions. Amonoexponential functionwasfitted to thedevelopment of block to calculateits time constant. Please note that the time axis is limited to the initial 100 msof the600 mslong test pulse to +60mV. In the right panel, the average reciprocal of the time constant(rate of block) is plotted versus the respective primaquine concentration. The straight linerepresents the linear regression of the rate of block vs. the primaquine concentration.Dashed curves indicate the 95 % confidence intervals. Error bars represent S.E.M. n=18.B:Developmentof the Ito blockbydifferent concentrationsof chloroquine (200 μM,400 μM,600 μM, 800 μM, and 1000 μM) in a representative cardiomyocyte (left panel) andregression of the average rate of block vs. the chloroquine concentration (right panel).Dashed curves indicate the 95 % confidence intervals. Error bars represent S.E.M. n=11.

Fig. 3. Use dependence of the Ito block by primaquine and chloroquine. A: Representativeoutward currents elicited by pulse trains of 2 Hz in a myocyte under control conditions(upper panel) and a myocyte exposed to 30 μM primaquine (lower panel). From theholding potential of−90 mV, cellswere clamped to the test potential+60mV for 100 ms,followed by 400 ms at the holding potential. The interpulse interval is not displayed formeans of clarity. The average normalized Ito elicited at 0.5 Hz (B), 2 Hz (C) or 5 Hz (D) isplotted versus the pulse number. Cells were exposed to control solution, to 30 μMprimaquine (left panels) or to 1 mM chloroquine (right panels). Ito was quantified as thecurrent inactivatingwithin 100 msandnormalized to themagnitudemeasured at the startof the pulse train. Error bars represent S.E.M. 7≤n≤25.

19M. Wagner et al. / European Journal of Pharmacology 647 (2010) 13–20

study focuses on Ito, a complete characterization of the various othercomponents of outward current is beyond its scope. However, whenexamining the recovery from inactivation, it was noticed that morethan 85% of Ito recovers with a time constant of 50–70 ms (Table 3)which is characteristic for Ito,f (Nerbonne and Kass, 2005). Theremaining part is likely to consist of Ito,s and the portion of the delayedrectifier current that inactivates within 600 ms. The current desig-nated Ilate is likely to consist of the remaining part of the delayedrectifier current and the non-inactivating current component. The

Ca2+ activated chloride current (Ito2 or IClCa) is unlikely to havecontributed to the outward current, since the intracellular calciumwas buffered by the presence of 10 mM EGTA in the pipette solution(Zygmunt and Gibbons, 1991).

The analysis of the onset of block kinetics is also dominated by Ito,fas the most prominent current component. However, here the con-tamination by other current components is larger, since the analysiswas performed by monoexponential approximation of the block ofthe total outward current. In addition to blocking the pore, theaminoquinolines may interfere with the gating kinetics of thechannels, e.g. by inducing changes in the inactivation process. More-over, changes in the inactivation process of the channels cannot beseparated from block of the pore andmay therefore distort the results.The best approach to overcome these problems would have beento analyze the onset of block in a homogeneous population ofheterologously expressed channels engineered to lack inactivation.However, this was beyond the scope of the present study.

5. Conclusion

Taken together our results demonstrate that, compared to thestructurally related quinidine, which blocks Ito at concentrations ofapproximately 3 μM (Slawsky and Castle, 1994), the 8-aminoquino-line primaquine exhibits a 10-fold lower potency and the 4-aminoquinoline chloroquine an approximately 100-fold lower poten-cy. The mechanism of block is consistent with the block of open aswell as of inactivated channels from the inside of the membrane bythe positively charged blocker.

Conflict of interest

None.

A

B

0

100 µM Primaquine

0100 ms

5 µA

Control

C

-80 -60 -40 -20 0 20 40 60 80 -80 -60 -40 -20 0 20 40 60 800

0.5

1.0

Vm (mV) Vm (mV)

Rel

ativ

e C

harg

e

0

0.5

1.0R

elat

ive

Cha

rge

****

**

**

*

Fig. 5. Voltage-dependent block of hKv4.2+hKChIP2b currents by primaquine. A:Representative outward currents recorded from X. laevis oocytes expressing hKv4.2+hKChIP2b obtained in the absence (left panel) or presence (right panel) of 100 μMprimaquine. From the holding potential of -90 mV, oocytes were clamped for 1000 ms totest potentials between−80mV and+60mV in steps of 20mV. Cycle lengthwas 3 s. Thegraphs were restricted to the first 600 ms of each trace and capacitive artifacts wereremoved for means of clarity. B: Average voltage dependence of activation (circles) andsteady-state inactivation (squares) of hKv4.2+hKChIP2b currents expressed in X. laevisoocytes under control conditions. Activation was calculated from recordings similar asshown in A (left panel). For each voltage the charge of the inactivating current wasmeasured, divided by the driving force (thus eliciting the time-integral of theconductance) and normalized to the time-integral of the conductance calculated at+60 mV. Triangles depict the average relative charge after the application of 30 μM(filledtriangles) and 100 μM (open triangles) primaquine as a function of the test potential.Steady-state inactivation was assessed using a double pulse protocol. A prepulse of1000 msduration to conditioningpotentials between−90 mVand+10 mVwas followedby a 1000 ms test pulse to+60mV. For each voltage the charge of the inactivating currentwas calculated and was normalized to the chargemeasured after a conditioning potentialof −90 mV. Average relative charge is plotted versus the conditioning potential. Cyclelength was 3 s. Diamonds depict the average relative charge after the application of 30 μM(filled diamonds) and 100 μM (open diamonds) primaquine as a function of the con-ditioning potential. Error bars represent S.E.M. ** Pb0.01 vs. −90 mV, n=7. C: Averagevoltage dependence of activation (circles) and steady state inactivation (squares) ofhKv4.2+hKChIP2b currents expressed in X. laevis oocytes under control conditions.Activation and steady-state inactivation were assessed as described in B. Triangles depictthe average relative charge after the application of 1 mM(filled triangles) and 5 mM(opentriangles) chloroquine as a function of the test potential. Diamonds depict the averagerelative charge after the application of 1 mM (filled diamonds) and 5 mM (opendiamonds) chloroquine as a function of the conditioning potential. Error bars representS.E.M. * Pb0.05, ** Pb0.01 vs. −90 mV, 6≤n≤8.

20 M. Wagner et al. / European Journal of Pharmacology 647 (2010) 13–20

Acknowledgements

We gratefully acknowledge the expert technical assistance ofCéline Grüninger, Jessica Rinke and Ralf Rinke. We are indebted toDr. Christoph Korbmacher for carefully reading the manuscript andmany helpful comments. This work was supported by the Johannesund Frieda Marohn-Stiftung.

References

Agus, Z.S., Dukes, I.D., Morad, M., 1991. Divalent cations modulate the transientoutward current in rat ventricular myocytes. Am. J. Physiol. 261, C310–C318.

An, W.F., Bowlby, M.R., Betty, M., Cao, J., Ling, H.P., Mendoza, G., Hinson, J.W., Mattsson,K.I., Strassle, B.W., Trimmer, J.S., Rhodes, K.J., 2000. Modulation of A-type potassiumchannels by a family of calcium sensors. Nature 403, 553–556.

Benitah, J.P., Vassort, G., 1999. Aldosterone upregulates Ca2+ current in adult ratcardiomyocytes. Circ. Res. 85, 1139–1145.

Brahmajothi, M.V., Campbell, D.L., Rasmusson, R.L., Morales, M.J., Trimmer, J.S.,Nerbonne, J.M., Strauss, H.C., 1999. Distinct transient outward potassium current(Ito) phenotypes and distribution of fast-inactivating potassium channel alphasubunits in ferret left ventricular myocytes. J. Gen. Physiol. 113, 581–600.

Caballero, R., Gomez, R., Nunez, L., Moreno, I., Tamargo, J., Delpon, E., 2004. Diltiazeminhibits hKv1.5 and Kv4.3 currents at therapeutic concentrations. Cardiovasc. Res.64, 457–466.

Castle, N.A., Slawsky, M.T., 1993. Characterization of 4-aminopyridine block of thetransient outward K+current in adult rat ventricular myocytes. J. Pharmacol. Exp.Ther. 265, 1450–1459.

Dixon, J.E., Shi,W.,Wang, H.S., McDonald, C., Yu, H.,Wymore, R.S., Cohen, I.S., McKinnon,D., 1996. Role of the Kv4.3 K+channel in ventricular muscle. A molecular correlatefor the transient outward current. Circ. Res. 79, 659–668.

Gotoh, Y., Imaizumi, Y., Watanabe, M., Shibata, E.F., Clark, R.B., Giles, W.R., 1991.Inhibition of transient outward K+current by DHP Ca2+ antagonists and agonistsin rabbit cardiac myocytes. Am. J. Physiol. 260, H1737–H1742.

Guo, W., Kamiya, K., Toyama, J., 1997. Evidences of antagonism between amiodaroneand triiodothyronine on the K+channel activities of cultured rat cardiomyocytes.J. Mol. Cell. Cardiol. 29, 617–627.

Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J., 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-freemembrane patches. Pflugers Arch 391, 85–100.

Hill, A.V., 1910. The possible effects of the aggregation of the molecules of haemoglobinon its dissociation curves. J. Physiol. 40, iv–vii.

Himmel, H.M.,Wettwer, E., Li, Q., Ravens, U., 1999. Four different components contributeto outward current in rat ventricular myocytes. Am. J. Physiol. 277, H107–H118.

Isenberg, G., Klöckner, U., 1982. Calcium tolerant ventricular myocytes prepared bypreincubation in a “KB medium”. Pflugers Arch 395, 6–18.

Lefevre, I.A., Coulombe, A., Coraboeuf, E., 1991. The calcium antagonist D600 inhibitscalcium-independent transient outward current in isolated rat ventricularmyocytes. J. Physiol. 432, 65–80.

Nerbonne, J.M., Kass, R.S., 2005. Molecular physiology of cardiac repolarization. Physiol.Rev. 85, 1205–1253.

Orta-Salazar, G., Bouchard, R.A., Morales-Salgado, F., Salinas-Stefanon, E.M., 2002.Inhibition of cardiac Na+current by primaquine. Br. J. Pharmacol. 135, 751–763.

O'Shannessy, D.J., Brigham-Burke, M., Soneson, K.K., Hensley, P., Brooks, I., 1993.Determination of rate and equilibrium binding constants for macromolecularinteractions using surface plasmon resonance: use of nonlinear least squares analysismethods. Anal. Biochem. 212, 457–468.

Radicke, S., Vaquero, M., Caballero, R., Gomez, R., Nunez, L., Tamargo, J., Ravens, U.,Wettwer, E., Delpon, E., 2008. Effects of MiRP1 and DPP6 b-subunits on the blockadeinduced by flecainide of Kv4.3/KChIP2 channels. Br. J. Pharmacol. 154, 774–786.

Rolf, S., Haverkamp, W., Borggrefe, M., Musshoff, U., Eckardt, L., Mergenthaler, J.,Snyders, D.J., Pongs, O., Speckmann, E.J., Breithardt, G., Madeja, M., 2000. Effects ofantiarrhythmic drugs on cloned cardiac voltage-gated potassium channelsexpressed in Xenopus oocytes. Naunyn Schmiedebergs Arch Pharmacol 362, 22–31.

Sanchez-Chapula, J.A., Salinas-Stefanon, E., Torres-Jacome, J., avides-Haro, D.E.,Navarro-Polanco, R.A., 2001. Blockade of currents by the antimalarial drugchloroquine in feline ventricular myocytes. J. Pharmacol. Exp. Ther. 297, 437–445.

Schwartz, A.L., Bolognesi, A., Fridovich, S.E., 1984. Recycling of the asialoglycoproteinreceptor and the effect of lysosomotropic amines in hepatoma cells. J. Cell Biol. 98,732–738.

Slawsky,M.T., Castle, N.A., 1994. K+channel blocking actions of flecainide comparedwiththose of propafenone and quinidine in adult rat ventricular myocytes. J. Pharmacol.Exp. Ther. 269, 66–74.

Tamargo, J., Caballero, R., Gomez, R., Valenzuela, C., Delpon, E., 2004. Pharmacology ofcardiac potassium channels. Cardiovasc. Res. 62, 9–33.

Volk, T., Nguyen, T.H.D., Schultz, J.H., Faulhaber, J., Ehmke, H., 2001. Regional alterationsof repolarizing K+currents among the left ventricular wall of rats with ascendingaortic stenosis. J. Physiol. 530, 443–455.

Volk, T., Konstas, A.A., Bassalay, P., Ehmke, H., Korbmacher, C., 2004. Extracellular Na+removal attenuates rundown of the epithelial Na+-channel (ENaC) by reducingthe rate of channel retrieval. Pflugers Arch. 447, 884–894.

Wagner, M., Rudakova, E., Volk, T., 2008. Aldosterone-induced changes in the cardiacL-type Ca2+ current can be prevented by antioxidants in vitro and are absent inrats on low salt diet. Pflugers Arch. 457, 339–349.

White, N.J., 2007. Cardiotoxicity of antimalarial drugs. Lancet Infect. Dis. 7, 549–558.Wickenden, A.D., Jegla, T.J., Kaprielian, R., Backx, P.H., 1999. Regional contributions of

Kv1.4, Kv4.2, and Kv4.3 to transient outward K+current in rat ventricle. Am. J.Physiol. 276, H1599–H1607.

Woodhull, A.M., 1973. Ionic blockage of sodium channels in nerve. J. Gen. Physiol. 61,687–708.

Xu, H., Guo, W., Nerbonne, J.M., 1999. Four kinetically distinct depolarization-activatedK+currents in adult mouse ventricular myocytes. J. Gen. Physiol. 113, 661–678.

Zygmunt, A.C., Gibbons, W.R., 1991. Calcium-activated chloride current in rabbitventricular myocytes. Circ. Res. 68, 424–437.