Kv1.5 Open Channel Block by the Antiarrhythmic Drug Disopyramide: Molecular Determinants of Block

Iván A. Aréchiga1, Gabriel F. Barrio-Echavarria1, Aldo A. Rodríguez-Menchaca1, Eloy G. Moreno-Galindo1, Niels Decher2, Martin Tristani-Firouzi3, José A. Sánchez-Chapula1, and Ricardo A. Navarro-Polanco1,* 1Unidad de Investigación “Carlos Méndez” del Centro de Investigaciones Biomédicas de la Universidad de Colima, Colima, México 2Institute of Physiology, Philipps-University, Marburg, Germany 3Department of Pediatric of Cardiology, University of Utah, Salt Lake City, UT 84132, USA

Received March 26, 2008; Accepted July 9, 2008

Abstract. Kv1.5 is considered to be a potential molecular target for treatment of atrial fibrillation or flutter. Disopyramide is widely used in the treatment of atrial flutter and/or atrial fibrillation. The present study was undertaken to characterize the effects of disopyramide on currents mediated by Kv1.5 channels and to determine the putative binding site involved in the inhibitory effects of disopyramide. Experiments were carried out on wild-type and site directed mutated hKv1.5 channels expressed on HEK 293 cells using the patch-clamp technique. Disopyramide acting from the cytoplasmic side of the membrane produced blocking effects on Kv1.5 that exhibited several features typical of an open channel blocker. Ala-scanning mutagenesis of the Kv1.5 pore domain combined with macroscopic current analysis suggested that disopyramide interacted only with the Val512 residue that faces to the central cavity of the channel. Mutation

of this key residue to Ala caused marked change in the IC50 of disopyramide (22-fold). The single interaction between disopyramide and Val512 in the PVP region is able to change the mechanism of channel closure, reproducing the “foot-in-the-door” phenomenon.

Keywords: potassium channel, ultra rapid delayed rectifier, Kv1.5, antiarrhythmic, disopyramide

Introduction selective block of IKur produced a stronger prolongation of action potential in patients with AF compared to The most common sustained cardiac arrhythmias in patients with sinus rhythm (SR) (7). Class 1A (sodium clinical practice are atrial flutter and/or atrial fibrillation channel blocker) and Class III (potassium channel (AF). The goals of antiarrhythmic therapy are to restore blocker) antiarrhythmic agents reduce the rate of sinus rhythm, to facilitate electrical cardioversion, to recurrence of AF, but are of limited use because of a prevent AF recurrence, and to control ventricular rate variety of potentially adverse effects including ventri- (1). Kv1.5 channels are highly expressed in human atria cular proarrhythmia (1, 8). Disopyramide is a widely (but not ventricle) and conduct the ultrarapid delayed used Class Ia antiarrhythmic agent that is employed in rectifier current (IKur) that contributes to action potential the treatment of AF (9). Disopyramide is a nonspecific repolarization of human atrial myocytes (2 – 4). Thus, channel blocker (9), which blocks cardiac Na+ channels Kv1.5 is a potential target for treatment of atrial to slow conduction (10 – 12). In addition, disopyramide fibrillation or atrial flutter. In different animal models, it blocks repolarizing cardiac K+ channels to prolong was shown that AF could be terminated effectively by action potential duration (13 – 16). selective blockers of IKur (5, 6). Also in human atria, Ongoing studies of potassium channel inhibition by synthetic compounds and toxins provide important *Corresponding author. [email protected] contributions to our understanding of these membrane Published online in J-STAGE proteins. Classical ion channel blockers, such as local doi: 10.1254/jphs.08084FP anesthetics and antiarrhythmics are used both clinically

49 50 IA Aréchiga et al and as molecular probes for studying ion channel external solution contained 130 mM NaCl, 4 mM KCl, function. Drug-binding from the internal side takes place 1 mM MgCl2, 10 mM Hepes, 1.8 mM CaCl2, and 10 mM inside a large water-filled inner cavity and requires a glucose (pH was adjusted to 7.35 with NaOH). Patch change in protein conformation to allow entrance of pipettes were filled with 5 mM K4BAPTA, 100 mM the blocker. Disopyramide blocks various potassium KCl, 10 mM Hepes, 5 mM MgCl2, 5 mM ATP-K2 (pH currents like the transient outward current Ito (15) and adjusted to 7.2 with KOH). The holding potential was the delayed rectifying outward current IKr (13, 14, 16). −80 mV. The interpulse interval for all the protocols Disopyramide is suggested to bind only to open channels was 15 s to allow channels to fully recover from and either has extremely low affinity or is unable to bind inactivation between pulses. To obtain current–voltage to closed channels (15 – 17). To our knowledge, the relationships and activation curves, 200-ms voltage effect of disopyramide on IKur or Kv1.5 has not been steps were applied in 10-mV increments to potentials studied. that varied from −60 to +60 mV, followed by repolariza- Identification of the drug binding site and understand- tion to −40 mV. The ratio of current in the presence of ing of the channel blocker binding mode may help to drug divided by current before drug (Idrug/Icontrol) was promote the development of in silico prediction methods determined to calculate the fraction of unblocked current for identifying potential Kv1.5 channel blockers. as a function of time. The time constant of current block

Quinidine has been reported to block Kv1.5 by interact- ( b) was determined by fitting Idrug/Icontrol to a mono- ing primarily with residues in the S6 domain (18). exponential function. The voltage dependence of Kv1.5 Recently, Decher et al. (19, 20) used an alanine- channel activation (with or without disopyramide) was scanning mutagenesis approach to define residues in the determined from tail current analyses at −40 mV. The pore of Kv1.5 that interact with S0100176 and resulting relationship between test voltage (Vt) and AVE0118, two novel antiarrhythmic agents. Mutation of normalized tail current (In) was fit to a Boltzmann residues located at the base of the pore helix and the S6 equation to obtain the halfpoint (V1/2) and slope factor domain had the greatest effect on reducing the potency (k): In = 1 / {1 + exp[(V1/2 − Vt) /k]}. Other voltage pulse of these Kv1.5 channel blockers. In the present study, protocols are described under Results and Figure we investigated the effects of disopyramide on currents legends. mediated by Kv1.5 channels expressed in a mammalian cell line, with the following aims: i) to characterize Data analyses the nature of the inhibitory action on Kv1.5 by dis- pCLAMP 8 (Molecular Devices, Sunnyvale, CA, opyramide; and ii) to define the molecular basis of USA) and Origin 7 (OriginLab Corp, Northampton, disopyramide block of Kv1.5 channels. MA, USA) software were used for data acquisition and analysis on a personal computer. All the fitting Materials and Methods procedures were based on the simplex algorithm. The

concentration required for 50% block of current (IC50) Molecular biology was determined from Hill plots using 3 – 5 concentra- Wild-type (WT) and mutant Kv1.5 (KCNA5) cDNA tions of drug for each mutant (5 – 12 cells/point). were generously donated by Dr. Michael Sanguinetti Results are each reported as the mean ± S.E.M. from the University of Utah and Dr. Niels Decher (n = number of cells). Statistical differences between (Aventis Pharma GmbH, Frankfurt, Germany). WT and mutant channels were evaluated by ANOVA and Bonferroni’s test. Significance was assumed for Transfection of HEK 293 cells P<0.05. HEK 293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with Drugs

10% horse serum at 37°C in an air 5% CO2 incubator. Disopyramide (Sigma-Aldrich, Mexico City, Mexico) Cells were transiently transfected using Lipofectamine was prepared daily, dissolved at the desired concentra- 2000 (Invitrogen, Mexico City, Mexico) according to tions in the external or internal solution. the supplier’s directions. Soluble GFP was coexpressed with the channel subunits to identify cells for voltage Results clamp experiments. Voltage-dependent block of Kv1.5 channels by dis- Voltage clamp protocols opyramide Currents were recorded at room temperature (23°C The effect of disopyramide on Kv1.5 was character- –25°C) with standard patch clamp techniques. The ized by recording currents in a voltage range from −60 to Disopyramide Blocking of hKv1.5 51

Fig. 1. Disopyramide effect on WT Kv1.5 channel. Whole cell currents recorded from HEK-293 cells before (A) and after (B) extracellular application of disopyramide (100 µM). Cur- rents were elicited by 200-ms depolarizations from a holding potential of −80 mV to test potentials between −60 to +60 mV in 10-mV steps. Test potential was followed by a repolarizing step to −40 mV to record deactivation currents. C: I-V relationships for currents measured at the end of the 200-ms test pulse before and after application of 100 and 300 µM disopyramide (n = 5). D: Fractional block of WT Kv1.5 currents, measured at the end of 200-ms pulses and plotted as a function of test potential. E: Acti- vation curves generated with tail currents in the absence or presence of drug. Tail currents were normalized to the peak value under control conditions for each cell. The smooth curves were obtained by fitting average data to a Boltzmann function. F: Tail currents recorded in control conditions and in the presence of disopyramide.

+60 mV, applied in 10-mV increments from a holding disopyramide, Fig. 1E). Figure 1F shows the super- potential of −80 mV. In the absence of drug, Kv1.5 position of the tail currents obtained at −40 mV after a slowly inactivates during the test pulse (Fig. 1A). In the 200-ms depolarization to +60 mV under control condi- presence of drug, the current appeared to inactivate tions and in the presence of 100 μM disopyramide. In much faster and more thoroughly, but this effect more control, the tail declined with a time constant of likely represents open channel block (Fig. 1B). Dis- 27.1 ± 5.1 ms. After exposure to disopyramide, the opyramide caused a voltage-dependent decrease in decline of the tail current was slower than in the control Kv1.5 currents elicited by depolarizations between −10 with a time constant of 60.1 ± 8.7 ms, resulting in the and +10 mV. For depolarizations greater than +10 mV, “crossover” phenomenon. current reduction was no longer voltage-dependent (Fig. 1: C and D). The lack of an increase in block at Disopyramide acts from the cytoplasmic membrane higher potentials suggests that this compound does not surface preferentially block Kv1.5 channels in the inactivated To determine whether disopyramide acts from the state. Disopyramide caused a slight hyperpolarizing extracellular or intracellular membrane surface, we shift in the voltage dependence of activation (V1/2 compared the concentration–response curves following −8.0 ± 0.5 mV before and −13.9 ± 0.8 mV after 100 μM extracellular (whole-cell patch clamp technique) and

Fig. 2. Disopyramide blocks the hKv1.5 channel from the intracellular face as an open channel blocker. A: Concentration- dependence of disopyramide-induced block of hKv1.5 channels in whole cell and inside-out configurations. The conti- nuous line represents the fit of the experimental data to a Hill equation. B: Onset of hKv1.5 channel block by 100 µM disopyramide assessed during depolarizing voltage step to +60 mV. 52 IA Aréchiga et al cytoplasmic application (excised inside-out patches)

(Fig. 2A). The IC50 obtained from cytoplasmic application (IC50 = 25 ± 4.8 μM) was four times lower than that for external application (IC50 = 92.5 ± 9.9 μM). This result further suggests that disopyramide acts from the inside of the membrane.

Disopyramide is an open channel blocker of Kv1.5 To determine whether disopyramide is a Kv1.5 open channel blocker, we recorded Kv1.5 currents before and after a pulse-free period of incubation with 100 μM of disopyramide. Figure 2B (inset) presents superimposed Kv1.5 currents elicited at a test potential of +60 mV before and after exposure to 100 μM disopyramide for 10 min without pulsing at a holding potential of −80 mV.

The initial value of the Idrug/Icontrol relationship (Fig. 2B) was close to 1, suggesting a tonic block of <5% and Fig. 3. External and internal comparative block of WT Kv1.5 by ± disopyramide. Successive 6-s recordings at +60 mV in whole cell (A) the remainder of the steady-state block of 55 7% and inside-out (B) configurations. At the time indicated by the solid developed during the pulse. line, the substances were applied during 2 s at the indicated concen- Next, we compared the effects of internal and external trations. Arrows indicate the drug application on and off. application of disopyramide during a depolarizing pulse. Disopyramide (100 μM) was applied for 2 s during a 10-s depolarizing pulse to +60 mV, to the external side Alanine-scanning mutagenesis of Kv1.5 pore residues of the membrane in the whole cell configuration Sixteen residues located in the S6 domain or between (Fig. 3A) or to the intracellular side of an excised inside- the pore helix and the selectivity filter were chosen for out patch (Fig. 3B). When applied to the external side of the alanine-scan based on their proximity to the central the membrane, disopyramide caused a 12% reduction in cavity, as previously described (19, 20). Disopyramide current and did not reach a steady-state. When applied to was applied at a concentration 1 mM and Kv1.5 current the internal side of the membrane, disopyramide caused inhibition was assessed at the end of 200 ms test pulses a 63% reduction in current that was complete in to +60 mV. Because the P513A mutation caused a large 215 ± 28 ms (n = 4). depolarizing shift in the voltage dependence of activation (20), block was assessed at a test potential of

Fig. 4. Alanine-scanning mutagenesis of the pore helix and S6 domain of Kv1.5 to identify amino acid residues that modulate channel block by disopyramide. A: Super- imposed current traces recorded during 200-ms test depolarizations to +60 mV from a holding potential of −80 mV in the absence (c) or presence (d) of disopyramide for WT (left), I502A (middle), and V512A (right) Kv1.5 channels. B: Inhibi- tion of Kv1.5 by disopyramide was calcu- lated as the percent of reduction of the current at the end of a 200-ms pulse applied at +60 mV (n = 5 – 7). C: Changes in IC50 caused by mutations at the putative Kv1.5 binding sites of disopyramide. NT, not tested residues; NE, non-expressing mutant channel; *P<0.05, compared with WT hKv1.5. Disopyramide Blocking of hKv1.5 53

+80 mV for this mutant. Disopyramide (1 mM) caused segment and/or regions near the selectivity filter (28 – an 85% reduction in WT Kv1.5 current (Fig. 4A). The 33). For the Kv1.5 channel blockers quinidine, benzo- potency of disopyramide block was significantly caine, and bupivacaine, a residue at the base of the pore reduced (P<0.05) by three mutant Kv1.5 channels helix (Thr479) and residues in the S6 domain (Thr507, (Ile502, Leu506, and Val512) (Fig. 4: A and B). The Leu510, and Val514) were identified as potential drug-

IC50 for disopyramide was determined for these mutant binding sites (18, 25, 34 – 37). Interestingly, the channels (Fig. 4C). The V512A mutant caused a 22-fold resolved Kv1.2 structure predicts that these S6 residues increase in the IC50 for disopyramide (IC50 = 2007 ± point away from the central cavity (38). Several residues 321 μM), while I502A and L506A increased the IC50 have been identified within the pore helix and S6 domain sixfold (IC50 = 545 ± 137 μM) and fivefold (IC50 = 499 ± of Kv1.5 as putative common drug-binding sites for a 119 μM), respectively (Fig. 4C). number of novel antiarrhythmic agents (19, 20, 39, 40). Most of those residues face toward the central cavity of Discussion the channel. Unlike all the Kv1.5 channel blockers studied to date, The class Ia antiarrhythmic drug disopyramide is used residues at the base of the pore helix were not important for the maintenance of sinus rhythm in patients with molecular determinants of disopyramide block. Muta- AF and for the prevention of ventricular tachycardia or tions of T479 and/or T480 significantly reduced sensi- fibrillation (9). In cardiac myocytes, disopyramide is tivity to AVE0118, S0100176, ICAGEN-4, S9947, and known to antagonize actions of acetylcholine (ACh) vernakalant (19, 20, 39, 40), but did not appreciably inhibiting the G protein-gated potassium current (IKACh) influence disopyramide block. This observation suggests (21, 22), in addition to depress directly the inward Na+ that disopyramide does not bind as deep within the currents (23, 24). In the present work, we found that central cavity to reach residues at the base of the pore disopyramide blocked Kv1.5 channels in a similar helix, as reported for other Kv1.5 channel blockers. manner to that described for quinidine (25). Block by Our mutagenesis scan identified Val512 as the primary disopyramide exhibited several features typical of an molecular determinant for disopyramide block with open channel blocker. First, block mainly occurred after lesser contributions from Ile502 and Leu506. Val512 is channel opening and increased sharply in the voltage predicted to face the central cavity of the channel pore range of channel activation. Second, slowed deactivation and forms the conserved Pro-Val-Pro motif. Why does and tail current crossover in the presence of disopyra- disopyramide induce a “foot-in-the-door” phenomenon, mide was observed, suggesting that drug binding while S0100176 does not, despite sharing Val512 as impeded closure of the activation gate. This is similar an important drug-binding site? Based on molecular to the so-called “foot-in-the-door” mechanism first modeling, Decher et al. proposed that the S0100176 hypothesized by Armstrong (26) to describe the kinetics compound occupied a space between the base of the of unblock of squid axon K+ channels by tetraethylam- pore helix and the Pro-Val-Pro motif, allowing the monium derivatives. Third, cytoplasmic application of activation gate to fully close, trapping the molecule disopyramide produced rapid block of Kv1.5 currents, within the pore cavity. Because disopyramide does not while extracellular application caused slower onset and bind to the base of the pore helix, it might straddle the less pronounced block. Finally, the IC50 for channel Pro-Val-Pro motif to impede channel closure. block was fourfold lower when disopyramide was The side chains of the two other residues identified as applied to the cytoplasmic face of inside-out patches as putative binding sites, Ile502 and Leu506, are positioned compared to external application in the whole-cell away from the central cavity (19, 20). Mutation of these configuration. Therapeutic doses of disopyramide typi- residues caused smaller shifts in IC50 for disopyramide, cally result in plasma concentrations of 9 – 22 μM (27); compared to Val512. Interestingly, drug-affinities of at these concentrations, the Kv1.5 current is significantly AVE0118, vernakalan, and flecainide were also de- blocked (IC50 = 25 ± 4.8 μM), suggesting a clinically creased by the I502A mutation (19, 40). The interacting relevant effect. mechanism of disopyramide with these residues is not The disopyramide molecule has an amino group with clear. However, it has been suggested that substitution of a pKa of 10.4. At physiologic pH, more than 99.9% of large hydrophobic residues by Ala produce a local the molecules are charged, which hinders the passage of alteration affecting the side chains orientation of the drug across the cellular membrane to account for the nearby residues and consequently disturbing the inter- slower rate of onset for externally applied disopyramide. actions with drugs (19). Numerous studies have shown that blockers of Na+, The observation that disopyramide interacts primarily Ca2+, and K+ channels bind to residues within the S6 with a single residue (Val512) may explain its low 54 IA Aréchiga et al potency relative to ICAGEN-4, S9947, S0100176, and U, Allessie MA. ‘‘Early’’ class III drugs for the treatment of AVE0118 that interact with several residues within the atrial fibrillation: efficacy and atrial selectivity of AVE0118 in central cavity. High-potency hERG blockers simulta- remodeled atria of the goat. Circulation. 2004;110:1717–1724. neously interact with a number of residues located in 7 Wettwer E, Hala O, Christ T, Heubach JF, Dobrev D, Knaut M, et al. Role of IKur in controlling action potential shape and the S6 domain and near the pore helix (33, 41). But most contractility in the human atrium: influence of chronic atrial of the low-potency hERG blockers like propafenone fibrillation. Circulation. 2004;110:2299–2306. 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