The molecular basis of chloroquine block of theinward rectifier Kir2.1 channelAldo A. Rodrıguez-Menchaca*, Ricardo A. Navarro-Polanco*, Tania Ferrer-Villada†, Jason Rupp†, Frank B. Sachse†‡,Martin Tristani-Firouzi†§¶, and Jose A. Sanchez-Chapula*¶

*Unidad de Investigacion ‘‘Carlos Mendez’’ del Centro Universitario de Investigaciones Biomedicas de la Universidad de Colima, Colima, Mexico 28045;and †Nora Eccles Harrison Cardiovascular Research and Training Institute, ‡Bioengineering Department, and §Division of Pediatric Cardiology,University of Utah, Salt Lake City, Utah 84112

Edited by Lily Y. Jan, University of California School of Medicine, San Francisco, CA, and approved December 3, 2007 (received for review August 28, 2007)

Although chloroquine remains an important therapeutic agent fortreatment of malaria in many parts of the world, its safety marginis very narrow. Chloroquine inhibits the cardiac inward rectifier K�

current IK1 and can induce lethal ventricular arrhythmias. In thisstudy, we characterized the biophysical and molecular basis ofchloroquine block of Kir2.1 channels that underlie cardiac IK1. Thevoltage- and K�-dependence of chloroquine block implied that thebinding site was located within the ion-conduction pathway.Site-directed mutagenesis revealed the location of the chloro-quine-binding site within the cytoplasmic pore domain rather thanwithin the transmembrane pore. Molecular modeling suggestedthat chloroquine blocks Kir2.1 channels by plugging the cytoplas-mic conduction pathway, stabilized by negatively charged andaromatic amino acids within a central pocket. Unlike most ion-channel blockers, chloroquine does not bind within the transmem-brane pore and thus can reach its binding site, even while poly-amines remain deeper within the channel vestibule. These findingsexplain how a relatively low-affinity blocker like chloroquine caneffectively block IK1 even in the presence of high-affinity endog-enous blockers. Moreover, our findings provide the structuralframework for the design of safer, alternative compounds that aredevoid of Kir2.1-blocking properties.

IK1 � ion channel � KCNJ2 � malaria � polyamines

Choloroquine remains an important therapeutic agent for theprevention and treatment of malaria in many parts of the

world in addition to adjunct therapy for systemic inflammatorydisorders. Renewed interest in chloroquine has emerged afterrecent reports of its effectiveness in reducing viral loads ofHIV-1 (1, 2). Despite its widespread use, chloroquine has a verynarrow margin of safety. At therapeutic concentrations, chloro-quine causes prolongation of the QT and QRS intervals on thesurface electrocardiogram (3, 4) and at higher concentrationscauses ventricular ectopy and lethal ventricular arrhythmias (5,6). At the cellular level, chloroquine prolongs cardiac actionpotential duration, enhances automaticity, and reduces themaximum diastolic potential (7–9). These cellular and electro-cardiographic side effects occur primarily as the result of chlo-roquine block of the inward rectifier K� current, IK1 (8, 9) andto a lesser degree, block of the rapidly activating delayedrectifier, IKr (9, 10).

In the heart, IK1 is the primary determinant of resting mem-brane potential and contributes to the most terminal phase ofaction-potential repolarization (11). The Kir2.x subfamily ofpotassium channels (Kir2.1, 2.2, and 2.3) underlies cardiac IK1,assembling either as homo- and/or heterotetrameric channels(12–14). However, several lines of evidence suggest that Kir2.1is the primary determinant of IK1 (15–17). For example, muta-tions in the gene encoding Kir2.1 cause Andersen–Tawil syn-drome, a dominantly inherited disorder manifested as ventric-ular arrhythmias, periodic skeletal muscle paralysis, anddsymorphic features (16).

A hallmark of Kir2.1 channels is strong inward rectification; thatis, the preferential conduction of inward compared with outwardcurrent (18–20). Inward rectification is not an inherent property ofthe channel protein itself but reflects strong voltage dependence ofchannel block by intracellular cations such as Mg2� and polyamines(putrescine, spermidine, and spermine). The apparent voltagedependence of block is derived from displacement of K� ions acrossthe electrical field by the blocking agent (21). Negatively chargedamino acid residues in the pore-lining M2 helix (D172) and thecytoplasmic pore (E224, D255, D259, and E299) are critical forvoltage-dependent block of Kir2.1 channels (21–25). Polyamineblock likely involves sequential binding to a shallow site in thecytoplasmic pore, followed by progression to a deeper site in thepore-lining M2 helix, displacing K� ions to produce the observedvoltage-dependent block (26, 27).

In this study, we characterized the biophysical and molecularbasis of chloroquine block of Kir2.1 channels. Chloroquineblocked Kir2.1 channels from the cytoplasmic surface in avoltage- and K�-dependent manner. Chloroquine reached itsbinding site, even when Kir2.1 channels were preblocked bypolyamines. Based on site-directed mutagenesis and molecularmodeling, we propose that chloroquine blocks Kir2.1 channels byplugging the cytoplasmic conduction pathway, stabilized bynegatively charged and aromatic amino acids. Our findingsexplain how a relatively low-affinity blocker like chloroquine caneffectively block IK1 even in the presence of high-affinity en-dogenous blockers and provide a structural framework for thedesign of safer, alternative compounds that lack Kir2.1-blockingproperties.

ResultsChloroquine Preferentially Blocks Outward Current Through Kir2.1Channels. The effects of chloroquine on Kir2.1 currents were firststudied in the whole-cell configuration in transfected HEK293cells. In control conditions, hyper- and depolarizing pulses of 4-sduration between �140 and 0 mV elicited large inward currents,but small outward currents, typical of the strong inward rectifierKir2.1 channel (Fig. 1A). Chloroquine significantly decreasedcurrent amplitude in a voltage-dependent manner with prefer-ential inhibition of outward, compared with inward current (Fig.1 A and B). A slow recovery from block was observed duringhyperpolarizing pulses to negative membrane potentials (Fig.

Author contributions: A.A.R.-M., M.T.-F., and J.A.S.-C. designed research; A.A.R.-M., R.A.N.-P., T.F.-V., and F.B.S. performed research; J.R. contributed new reagents/analytic tools;A.A.R.-M., R.A.N.-P., T.F.-V., M.T.-F., and J.A.S.-C. analyzed data; and A.A.R.-M., R.A.N.-P.,M.T.-F., and J.A.S.-C. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

¶To whom correspondence may be addressed. E-mail: mfirouzi@cvrti.utah.edu andsancheza@cgic.ucol.mx.

This article contains supporting information online at www.pnas.org/cgi/content/full/0708153105/DC1.

© 2008 by The National Academy of Sciences of the USA

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1A). The effect of chloroquine on Kir2.1 channels at concen-trations �10 �M was completely reversible after 20 min ofwashout, whereas at higher concentrations it was only partiallyreversible (data not shown).

To determine the effect of chloroquine on Kir2.1 current in aphysiologically relevant setting, we used an action potentialvoltage clamp protocol. In control conditions, Kir2.1 current wasnoted to be small or absent during the plateau phase of the actionpotential, rapidly increased during the terminal phase of repo-larization, and declined in early diastole. Chloroquine inhibitedthe peak outward current in a concentration-dependent manner(Fig. 1C). The IC50 of Kir2.1 current elicited by the actionpotential voltage clamp was 8.7 � 0.9 �M with a Hill coefficientof 1.0 (n � 5 cells, Fig. 1D).

Chloroquine Blocks from the Cytoplasmic Surface. To determinewhether chloroquine preferentially blocks from the cytoplasmicor extracellular surface, we compared the rate of chloroquineblock in excised inside-out patches with that observed in thewhole-cell configuration. When applied to the cytoplasmic faceof excised inside-out patches, chloroquine produced steady-stateblock in �15 s, compared with �8 min. when applied to theexternal solution in the whole-cell configuration [supportinginformation (SI) Fig. 6). Thus, chloroquine accesses its bindingsite by entering the channel pore from the cytoplasmic surface.

Voltage Dependence of Chloroquine Block. To determine the volt-age dependence of chloroquine block, we measured the onset ofblock in excised patches where the competing effects of endog-enous blockers are absent. Like whole-cell experiments, appli-cation of chloroquine in excised inside-out patches causedpronounced dose-dependent block of outward currents (SI Fig.7). The onset of chloroquine block (�block) was determined byfitting current traces to a monoexponential function and plottingthe values vs. membrane potential (SI Fig. 7B). The voltagedependence of chloroquine block suggested that chloroquine

may block Kir2.1 channels within the channel pore similar toother cationic blockers, such as Mg2� and polyamines.

To estimate the distance that chloroquine penetrates thechannel pore, we plotted the fraction of unblocked current in thepresence of various chloroquine concentrations against mem-brane voltage (SI Fig. 7D). The data were fit to a Woodhullequation (28) to yield an apparent Kd (at 0 mV) of 27 � 12 �Mwith an apparent valence (Z) of 2.1 � 0.6 (n � 6). The apparentvalence is a measure of the ability of a blocker to displace K� ionsacross the membrane electrical field and an indirect measure ofthe distance penetrated by the blocker. The low valence suggeststhat chloroquine does not extensively traverse into the channelpore and therefore displaces less K� ions than polyamines, suchas spermine [valence �5 (21, 26)].

Unblock of Chloroquine at Voltages Negative to EK is [K�]o Dependent.A common feature of many charged particles that block ionchannels by a pore-plugging mechanism is that unblock isaccelerated at elevated [K�]o, the so-called ‘‘knock-off’’ phe-nomenon (26). Chloroquine unblock depended on both voltageand [K�]o (Fig. 2). In whole-cell experiments, increasing [K�]o

resulted in increased rate of chloroquine unblock (�unblock),consistent with a knock-off phenomenon (26). For example,�unblock in the presence of [K�]o � 4 mM was 16-fold slower than[K�]o � 75 mM as measured at �100 mV (Fig. 2B). Thisphenomenon explains, in part, the more complete chloroquineunblock observed in excised patches where [K�]o � 150 mM(SI Fig. 6C).

In summary, chloroquine block shares many features of classicintracellular ionic pore blockers, including voltage-dependentblock and unblock as well as a knock-off phenomenon in thepresence of elevated [K�]o. The apparent valence of chloroquineblock suggests that it penetrates the ion-conduction pathway todisplace K� ions but perhaps not as deeply as polyamines. Takentogether, these observations suggest that the chloroquine-binding site is located within the ion-conduction pathway.

Fig. 1. Chloroquine preferentially blocks outward current through Kir2.1 channels. (A) Effect of chloroquine on Kir2.1 channels expressed in HEK293 cells. Kir2.1 current elicited by 4-sec pulses from a holding potential of �80 mV to test potentials from �140 to 0 mV, applied in 10-mV increments, in absence (control)and presence of chloroquine 3 and 30 �M. (B) Normalized current-voltage relationship for currents measured at the end of 4-sec pulses. (C) Currents elicited byaction potential command signals as voltage protocol, before and after application of 1, 3, 10, and 30 �M chloroquine. (D) Concentration–response relationshipfor Kir2.1 current inhibition. Steady-state peak-current amplitudes (shown in C) for each concentration of chloroquine normalized to control. Mean values wereplotted against chloroquine concentration and fitted with the Hill equation. Mean IC50 was 8.7 � 0.9 �M, and the Hill coefficient nH � 1.0 (n � 5). CQ, chloroquine.

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Spermine Does Not Protect Kir2.1 Channels from Chloroquine Block. Ifthe chloroquine-binding site is located within the ion-conductionpathway, we wondered whether the initial binding of polyaminesmight protect Kir2.1 channels from chloroquine block. Toaddress this question, spermine and chloroquine were sequen-tially applied to the cytoplasmic face of excised inside-outpatches (Fig. 3). The application of 10 �M spermine duringdepolarization resulted in rapid block, with rapid unblock duringmembrane hyperpolarization (Fig. 3, trace A). Application ofchloroquine also rapidly blocked outward current but resulted inslow unblock during hyperpolarization (Fig. 3, trace B). Whenspermine application was followed by chloroquine, unblockduring hyperpolarization was slow (Fig. 3, trace C), indicatingthat chloroquine block predominated. Likewise, endogenousKir2.1 channel blockers (e.g., polyamines and Mg2�) did notprotect channels from chloroquine block (SI Fig. 8). Taken

together, these experiments imply that chloroquine binds to asite distinct from that of spermine and other endogenous Kir2.1channel blockers and that chloroquine can bind to its site evenwith spermine present within the channel vestibule.

Chloroquine-Binding Site Is Within the Kir2.1 Cytoplasmic Pore. Todetermine the binding site for chloroquine within the Kir2.1pore, we sequentially mutated residues in the pore-lining M2helix (G164–M180) to Ala and assessed the degree of chloro-quine block. All Ala-substitutions (G164A–M180A) expressedfunctional Kir2.1 channels that were effectively blocked by 30�M chloroquine (Fig. 4A). This observation was consistent withthe relatively low apparent valence (�2) of chloroquine block,suggesting that chloroquine does not penetrate deeply into the

Fig. 2. Unblock of chloroquine at voltages negative to EK is voltage- and[K�]o-dependent. (A) Normalized current transients elicited by voltage stepfrom 0 to �140 mV in the presence of 10 �M chloroquine and 4, 20, and 75 mMextracellular K�. The curves superimposed on the current transients are mono-exponential fits. The time constants of chloroquine unblock were 830 � 103,218 � 15, and 14 � 3 ms (mean � SEM; n � 4–5) for 4, 20, and 75 mMextracellular K�, respectively. (B) Voltage-dependence of chloroquine un-block at various extracellular K�. I/O, inside-out patch.

Fig. 3. Spermine does not protect Kir2.1 channels from chloroquine block.In excised, inside-out patches, the membrane was depolarized to �50 mV,followed by cytoplasmic surface application of spermine (10 �M), chloroquine(10 �M), and combination of spermine, followed by chloroquine. Membranehyperpolarization to �80 mV elicited nearly instantaneous inward currents inthe presence of spermine alone (rapid spermine unblock) but slowly increas-ing inward currents in the presence of chloroquine (slow chloroquine unblock)despite preapplication of spermine.

Fig. 4. Ala-scanning mutagenesis of Kir2.1 transmembrane and cytoplasmicpore regions. (A) Ala-scanning mutagenesis of transmembrane pore (M2segment) of Kir2.1 channel. The fraction of current inhibited by 30 �Mchloroquine at �50 mV (excised inside-out patches) is plotted as a bar graphfor WT and each of the Ala mutants. (B) Ala-scanning mutagenesis of theKir2.1 cytoplasmic domain. Data generated as described in A. *, P � 0.0001, byusing one-way ANOVA and Holm–Sidak multiple comparison procedure (Sig-maStat v3.5; Systat). (C) Concentration–effect relationship for current inhibi-tion of Kir2.1 cytoplasmic domain mutants by chloroquine. The IC50 as deter-mined by fits to a Hill equation was 1.1 � 0.2 �M for Kir2.1 WT (n � 8), 2.8 �0.2 �M for D255A (n � 6), 15.1 � 1.9 �M for F254A (n � 5), 100 � 16 �M forE299A (n � 5), 307 � 30 for D259A (n � 5), and 698 � 49 for E224A (n � 6).

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channel vestibule. Next, we selectively mutated amino acids thatare predicted to face the cytoplasmic pore based on the crystalstructure of the Kir2.1 cytoplasmic domain (29). E224A, D259A,and E299A Kir2.1 channels were markedly resistant to chloro-quine block (Fig. 4B and SI Fig. 9). The potency of chloroquineblock was reduced 635-fold for E224A Kir2.1 channels, 100-foldfor E299A, 307-fold for D259A, 14-fold for F254A, and 3-fold forD255A channels. The rank order of importance of Kir2.1channel residues critical for chloroquine block, E224 � D259 �E299 � F254 � D255, generally follows the contribution of eachresidue to polyamine block (25, 30). The exception is D172A,which is critical for high-affinity polyamine binding but not forbinding of chloroquine.

Molecular Modeling of Chloroquine Within the Kir2.1 CytoplasmicDomain. Molecular modeling was used to define the lowestenergy-minimized docking for chloroquine within the conduc-tion pathway of the Kir2.1 cytoplasmic domain (Fig. 5 B and C).

In the lowest energy pose, the positively charged alkylaminonitrogen of chloroquine (Fig. 5A) is coordinated by an electro-negative ring formed by the eight glutamic acid side chains ofresidues 224 and 299 (Fig. 5C). An additional charge–pairinteraction is predicted between the positively charged quinolinering nitrogen and the aspartic acid side chain at position 259.Given the asymmetric structure of chloroquine, this interactionis limited to D259 from a single monomer. The aromatic sidechain of Phe at position 254 is directed perpendicular to thequinoline ring (Fig. 5B), suggesting that T-stacking may stabilizechloroquine within the binding pocket.

DiscussionThe inward rectifier potassium current IK1 plays a critical role inthe stabilization of the resting membrane potential and in thelate repolarization phase of the cardiac action potential (11).Gain- and loss-of-function mutations in the gene encodingKir2.1 cause short QT and Andersen–Tawil syndrome, respec-tively, predisposing affected individuals to ventricular arrhyth-mias (16, 31, 32). Recently, it has been suggested that IK1 playsan important role in high-frequency rotor stabilization andventricular fibrillation dynamics (33). Thus, modulation of IK1has important implications for electrical stability in cardiactissue. In this context, chloroquine block of Kir2.1 channels hassignificant therapeutic ramifications.

Therapeutic doses of chloroquine typically result in plasmaconcentrations of 2–3 �M, but can achieve peak levels up to 5�M (34). In a large prospective study of acute chloroquineoverdose, blood levels ranged from 40 to 80 �M (5). Thus, thechloroquine concentrations used in the present study (0.3–30�M) and the IC50 for block of Kir2.1 current (�9 �M) are withina clinically relevant range.

Chloroquine is a 4-aminoquinoline derivative with two sites ofionization (Fig. 5A). The pKa values of the alkylamino and aminoquinoline ring groups (10.4 and 8.4, respectively) predict that thedrug would be 99.9% charged at normal intracellular pH. Similarto other cationic pore blockers, such as quaternary ammoniumcompounds and piperazine (35, 36), the voltage and K� depen-dence suggested that chloroquine blocks Kir2.1 channels fromwithin the conduction pathway. Although the vast majority ofion-channel blockers exert their effects by binding within thetransmembrane pore, this is not the case for chloroquine. Thelow apparent valence of chloroquine block suggested that chlo-roquine does not penetrate deep into the transmembrane pore.Moreover, spermine was unable to protect Kir2.1 channels fromchloroquine blockade. Finally, mutation of residues within thecytoplasmic, but not the transmembrane, pore markedly reducedchloroquine block.

Molecular modeling defined a low-energy docking conforma-tion that suggested charge–pair interactions between the posi-tively charged nitrogens of chloroquine and specific acidic aminoacids lining the cytoplasmic conduction pathway. In this pose,the alkylamino nitrogen occupies the center of the conductionpathway, thereby plugging the conduction pore. Of the interac-tions predicted by the docking model, E224 and D259, likelyform the primary chloroquine-binding sites through electrostaticinteractions with the alkylamino and quinoline ring nitrogens,respectively.

Although molecular modeling identified electrostatic interac-tions that are consistent with the site-directed mutagenesisanalysis, several important limitations exist. First, we selectedthe lowest energy-minimized docking for presentation, althoughother solutions are possible. Second, the Kir2.1 cytoplasmicdomain was crystallized without the native transmembranechannel structure and in the absence of the membrane-delimitedsecond messenger phosphatidylinositol 4,5-bisphosphate (PIP2).Thus, the precise location of the critical amino acids in thefull-length channel may differ from their position in the isolated

Fig. 5. Docking of chloroquine within the Kir2.1 cytoplasmic domain. (ALeft) Chemical structure of chloroquine. Quinoline ring and alkylamino nitro-gen ionization sites are depicted by � sign. Atoms are colored as follows:nitrogen, blue; chlorine, red; carbon, green; hydrogen, gray. (Right) Trans-membrane (KirBac1.1) and cytoplasmic domain (Kir2.1) highlighting positionsin Kir2.1 critical for polyamine block; D172 (blue), E224 (red), E299 (yellow),and D259 (orange). (B) Enlarged view of dotted box from A illustrating a singleKir2.1 cytoplasmic domain subunit (25) and docking of chloroquine relative topositions E224, E299, and D259. The aromatic side chain of F254 is shown inball and stick format, colored in orange. (C) Stereo view (‘‘Top-down’’) of thetetrameric Kir2.1 cytoplasmic domain showing chloroquine plugging theconduction pathway. F254 side chain depicted as in B.

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cytoplasmic domain crystal structure. Finally, it is not knownwhether the conformational changes induced by PIP2 binding(25, 37) alter the orientation of residues lining the cytoplasmicpore. The atomic forces that stabilize chloroquine within thecytoplasmic binding site will be defined ultimately by cocrystal-lization of chloroquine with the full-length Kir2.1 channel andPIP2.

An intriguing question is how a relatively low-affinity cationicdrug such as chloroquine may effectively block Kir2.1 channelseven in the presence of high-affinity endogenous blockers. Wepropose that chloroquine and polyamines compete for access tothe key cytoplasmic pore domain acidic residues. At depolarizedpotentials, polyamines preferentially occupy their high-affinitybinding site within the channel vestibule, thus chloroquine canaccess the cytoplasmic pore even while polyamine is bound deepwithin the channel. Because chloroquine unblock is slow relativeto polyamine unblock, chloroquine block predominates at phys-iological membrane potentials.

Although chloroquine use is declining in many parts of theworld because of chloroquine-resistant malaria, new therapeuticindications are arising, most notably for the treatment of HIV.The narrow safety margin of chloroquine could limit its thera-peutic potential. Identification of the chloroquine-binding sitewithin the cytoplasmic pore domain will allow for the design ofsafer chloroquine analogs devoid of Kir2.1 channel blockade.

Materials and MethodsMolecular Biology and Cell Transfection. Kir2.1 cDNA (kind gift of C. Vanden-berg, University of California, Santa Barbara) was subcloned into pcDNA3.1(�)plasmid (Invitrogen). Mutations were made by using the QuikChange Site-Directed Mutagenesis kit (Stratagene). All mutations were confirmed by directDNA sequencing. Kir2.1 constructs were expressed in HEK293 cells as described(38). Cells were transfected with Lipofectamine 2000 reagent (Invitrogen)according to manufacturer instructions.

Current Recordings in HEK293 Cells. Macroscopic currents were recorded in thewhole-cell and inside-out configurations of the patch-clamp technique (39) byusing an Axopatch-200B amplifier (Molecular Devices). Data acquisition andcommand potentials were controlled by pClamp 8.0 software (MolecularDevices). Patch pipettes with a resistance of 1–2 M� were made from boro-silicate capillary glass (WPI). For whole-cell recordings, the internal solutioncontained 100 mM KCl, 10 mM Hepes, 5 mM K4BAPTA, 5 mM K2ATP, and 1 mMMgCl2 (pH 7.2). The standard bath solution contained 130 mM NaCl, 4 mM KCl,1.8 mM CaCl2, 1 mM MgCl2, 10 mM Hepes and 10 mM glucose (pH 7.4).Inside-out patches were recorded by using Mg2�- and spermine-free solutionon both sides of the patch containing 123 mM KCl, 5 mM K2EDTA, 7.2 mM

K2HPO4, and 8 mM KH2PO4 (pH 7.2). Action potential voltage clamp recordingswere performed in HEK293 cells by using the action potential recorded froman isolated feline subendocardial ventricular myocyte as the command signal.Currents elicited in response to the action potential voltage clamp wererecorded under control conditions in the presence of chloroquine and afterapplication of 2 mM BaCl2. Kir2.1 currents are represented as Ba2�-subtractedcurrents elicited by the action potential voltage clamp.

Drugs. Chloroquine (Sigma) was dissolved directly in the external solution atthe desired concentration. HEK293 cells were exposed to chloroquine solu-tions until steady-state effects were achieved. The effects of chloroquine weremeasured 8–10 min after bath application in whole-cell experiments and 15sec after application in excised inside-out patches. To determine the concen-tration–effect relationships, a single cell was exposed to cumulative concen-trations of chloroquine.

Data Analysis. Data are present as mean � SEM (n � number of cells). pClamp8.0 software (Molecular Devices) was used to perform nonlinear least-squareskinetic analyses of time-dependent currents. The fractional block of current( f ) was plotted as a function of drug concentration ([D]) and the data fit witha Hill equation: f � 1/{1 � (IC50/[D]nh} to determine the half-maximal inhibitoryconcentration (IC50) and the Hill coefficient, nh. The voltage dependence ofblock was measured by fitting fractional unblock as a function of voltage witha Woodhull equation of the form Iblock/Icontrol � 1/(1�([chloroquine]/Kd

(0)e�zFV/RT)), where V � test potential, Kd (0) � dissociation constant at 0 mV,z � valence of block, and F, R, and T have their usual meanings.

Molecular Modeling and Ligand Docking. Molecular modeling was based on thecrystal structure of cytoplasmic Kir2.1 domains (PDB ID Code 1U4F) and astructural model of choloroquine (DrugBank, http://redpoll.pharmacy.ualber-ta.ca/drugbank/) after adjusting its ionization for pKa � 8.04. The docking wasinitiated from random configurations (n � 50) of the ligand in the cytoplasmiccavity of the Kir2.1 tetramer and performed by using simulated annealingtechniques with 1,000 stages, an initial temperature of 500 K, and a finaltemperature of 300 K. The consistent valence force field (CVFF) was used tocalculate conformational energies. Both ligand- and cavity-facing residues ofthe receptor were kept flexible during the minimization procedure. Thelowest energy-minimized conformation was selected for presentation. Mod-eling, docking, and visualization were performed with Insight II MolecularModeling System (version 2005; Accelrys) on a four-processor workstationwith Linux operating system.

ACKNOWLEDGMENTS. This work was supported by a Secretaria de EducacionPublica (SEP)-Consejo Nacional de Ciencia y Tecnologıa (CONACYT) (Mexico)Grant 2004-C01-47577 (to J.A.S.-C), Fondo Ramon Alvarez-Buylla de Aldana(Universidad de Colima), a National Heart, Lung, and Blood Institute/NationalInstitutes of Health Grant HL075536 (to M.T.-F.) and by the Nora EcclesHarrison Cardiovascular Research and Training Institute.

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