State-dependent block of HERG potassium channels by R ...

State-dependent block of HERG potassium channels by R-roscovitine:implications for cancer therapy

Sindura B. Ganapathi,1 Mark Kester,1 and Keith S. Elmslie1,2

Departments of 1Pharmacology and 2Anesthesiology, Penn State College of Medicine, Milton S. Medical Center,Hershey, Pennsylvania

Submitted 11 December 2008; accepted in final form 19 February 2009

Ganapathi SB, Kester M, Elmslie KS. State-dependent block ofHERG potassium channels by R-roscovitine: implications for cancertherapy. Am J Physiol Cell Physiol 296: C701–C710, 2009. Firstpublished February 25, 2009; doi:10.1152/ajpcell.00633.2008.—Hu-man ether-a-go-go-related gene (HERG) potassium channel acts as adelayed rectifier in cardiac myocytes and is an important target forboth pro- and antiarrhythmic drugs. Many drugs have been pulledfrom the market for unintended HERG block causing arrhythmias.Conversely, recent evidence has shown that HERG plays a role in cellproliferation and is overexpressed both in multiple tumor cell linesand in primary tumor cells, which makes HERG an attractive targetfor cancer treatment. Therefore, a drug that can block HERG but thatdoes not induce cardiac arrhythmias would have great therapeuticpotential. Roscovitine is a cyclin-dependent kinase (CDK) inhibitorthat is in phase II clinical trials as an anticancer agent. In the presentstudy we show that R-roscovitine blocks HERG potassium current(human embryonic kidney-293 cells stably expressing HERG) atclinically relevant concentrations. The block (IC50 � 27 �M) wasrapid (� � 20 ms) and reversible (� � 25 ms) and increased withchannel activation, which supports an open channel mechanism.Kinetic study of wild-type and inactivation mutant HERG channelssupported block of activated channels by roscovitine with relativelylittle effect on either closed or inactivated channels. A HERG gatingmodel reproduced all roscovitine effects. Our model of open channelblock by roscovitine may offer an explanation of the lack of arrhyth-mias in clinical trials using roscovitine, which suggests the utility ofa dual CDK/HERG channel block as an adjuvant cancer therapy.

S620T mutant; open channel block; terfenadine; human ether-a-go-go-related gene model

HUMAN ETHER-A-GO-GO-RELATED GENE (HERG) potassium chan-nels (Kv11.1) are a critical regulator of the cardiac actionpotential (cAP) duration. HERG channels display unique ki-netics that allows them to become fully activated during therepolarization phase of cAP (11). These channels activateslowly and inactivate rapidly during the initial phases of cAPto limit the outward potassium flux that would otherwiseprematurely terminate the action potential (11). As repolariza-tion begins, HERG channels rapidly recover from inactivation,and these reactivated channels generate the strong potassiumefflux that completes the repolarization process (34). As aresult, HERG channel block can prolong the cAP, causingacquired long QT syndrome that can lead to fatal torsades depointes (34). HERG is blocked by a diverse group of chemicalcompounds, and many prescription drugs have been removedfrom market because of their HERG-blocking property (34).

These drugs are thought to bind within a large inner cavity(vestibule) formed by the four S6 segments that can trap thedrug when the channel closes (29). Trapping of drugs withinthe inner vestibule increases the block duration, which likelycontributes to the proarrhythmic properties of these drugs.Therefore, a drug that is not trapped by HERG channels mayhave a reduced effect on cardiac rhythm.

In addition to regulating the cAP, HERG channels also playa role in cancer. Multiple hematopoietic cancer cell lines havebeen shown to regulate HERG, and blocking HERG reducesproliferation and tumor invasiveness (1, 24, 28, 41). HERGregulates proliferation in peripheral blood mononuclear cellsand is constitutively expressed in primary human acute my-eloid leukemias (30). In the human colon adenocarcinoma cellline, HT-29, the HERG blocker erythromycin selectively sup-pressed proliferation of cells expressing HERG channels,which resulted in synergistic cytotoxicity effects when eryth-romycin was combined with anticancer agents such as vincris-tine, paclitaxel, and hydroxy-camptothecin (8). Thus, HERGblockers have been proposed as an adjuvant cancer therapy(23, 31). In nonexcitable cells, the steady-state activation andinactivation vs. voltage relationships for HERG currents crossaround �40 mV to reveal a voltage range that generates asteady-state current (window current), which is important inthe maintenance of the resting membrane potential in nonex-citable cells and is thought to play a role in carcinogenesis (2).Together these findings support HERG as an attractive targetfor the antineoplastic therapy (1).

Roscovitine is a cyclin-dependent kinase (CDK) inhibitorthat is currently undergoing phase II clinical trials as ananticancer drug (20, 35). We recently demonstrated that R-roscovitine (Rosc) blocks voltage-dependent potassium chan-nels (Kv1.3, Kv2.1, and Kv4.3) by an open channel mechanism(6). We postulated that Rosc could also block HERG channels.Given that many diverse drugs block HERG channels by anopen channel mechanism, we were not surprised to find thatRosc also blocked open HERG channels. However, unlikeother drugs, Rosc was not trapped by HERG channel closing,which could explain the absence of arrhythmias in recent Roscclinical trials (3, 18). Moreover, our studies support the use ofRosc as an adjuvant therapy for cancer due to the dual block ofCDKs and HERG channels with a reduced proarrhythmogenicpotential.

MATERIALS AND METHODS

Cell culture. Human embryonic kidney (HEK)-293 cells stablyexpressing HERG (a gift from Dr. Eckhard Ficker, MetroHealthMedical Center, Cleveland, OH) were maintained in DMEM �glutamax media (Invitrogen) supplemented with 10% FBS, 100�antibiotic-antimycotic, and the selection agent G418. Chinese hamster

Address for reprint requests and other correspondence: K. S. Elmslie,Dept. of Anesthesiology, Penn State College of Medicine, Milton S.Hershey Medical Center, 500 University Dr., Hershey, PA 17033 (e-mail:[email protected]).

Am J Physiol Cell Physiol 296: C701–C710, 2009.First published February 25, 2009; doi:10.1152/ajpcell.00633.2008.

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ovary (CHO) cells were used for transient transfection of S620Tmutant (see below) and were maintained in F12 � glutamax media(Invitrogen) supplemented with 7.5% FBS and 100� antibiotic-antimycotic (without G418). For electrophysiology, cells were har-vested using 0.25% trypsin/EDTA (Invitrogen) and plated into 35-mmculture dishes that served as the recording chamber (6).

Transient transfection. The HERG mutant S620T cDNA cloneused for transient transfection was very generously provided by Dr.Shetuan Zhang (Queen’s University, Kingston, Ontario, Canada).Transient transfection of CHO cells utilized FuGENE 6 (RocheApplied Science) following the manufacturer’s instructions. MutantcDNA (0.9 �g) with 0.1 �g of green fluorescent protein (GFP) wascombined with FuGENE 6 at a 6:1 reagent to cDNA ratio. GFP-expressing cells were selected for study.

Electrophysiological recording. Membrane currents were recordedin whole cell patch-clamp mode at room temperature (�23°C).Electrodes were pulled from Schott 8250 glass (inside diameter 0.90mm, outside diameter 1.50 mm; Garner Glass, Claremont, CA) usinga Flaming/Brown P-97 pipette puller (Sutter Instrument, San Rafael,CA). Electrodes typically had a resistance of 1–3 M�. Series resis-tance was compensated at �80%. Currents were amplified and filteredusing an Axopatch 200A amplifier (Molecular Devices, Sunnyvale,CA) and digitized with either a 12-bit A/D converter (GW Instru-ments, Somerville, MA) or an ITC-18 (Instrutech, Bellmore, NY)after analog filtering with the amplifier’s four-pole low-pass Besselfilter.

HERG current comprised 90% of the potassium current in thestably transfected HEK-293 cells since 0.1 �M terfenadine (Terf)blocked 91 4% of the tail current at �40 mV (n � 22). Theendogenous potassium current expressed by some HEK-293 cells wasnot affected by 0.1 �M Terf (�4 11% effect on tail current at �40mV, n � 9).

Solutions. The internal solution contained (in mM) 120 KCl, 6MgCl2, 10 N-methyl-D-glucamine (NMG) �HEPES, 5 NMG2 �EGTA,5 Tris2 �ATP, and 0.3 Tris2 �GTP. The pH was adjusted to 7.4 usingNMG base and the osmolarity was 304 mosM. The externalsolution contained (in mM) 140 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2,10 NMG �HEPES, and 10 glucose. The pH was adjusted to 7.4using NMG base, and the osmolarity was 313 mosM.

Data acquisition and analysis. Data were taken using either S3 orS5 (software developed by Dr. Stephen Ikeda, National Institutes ofHealth, National Institute on Alcohol Abuse and Alcoholism, Be-thesda, MD) on a Macintosh computer (Apple Computer, Cupertino,CA). IgorPro software (WaveMetrics, Lake Oswego, OR) was used tomeasure current amplitudes and to fit (Marquardt-Levenberg algo-rithm) equations to currents and data plots.

The step time course and trapping protocols were leak subtractedusing scaled hyperpolarizing pulses of one-fourth amplitude (P/4). Forall other protocols, leak current was estimated by linear regression ofthe nearest current-voltage (I-V) relationship over voltages (�80 to�40 mV) that did not generate HERG current. Data were not used ifthe holding current differed by 10% between the I-V used togenerate leak and the protocol for which that leak was to be used forsubtraction. Solutions were applied using a gravity-fed flow systemwith six inputs and a single output. The exchange time for this systemis 1–2 s. For examining the time course of drug effects, cells werepulsed to �20 mV (1-s duration) once every 20 s, and currents weremeasured from peak tail currents at �40 mV following the �20-mVstep (the step time course protocol). Running this protocol undercontrol conditions revealed a slow time-dependent loss of HERGcurrent in most cells (rundown), which appeared to result in the partialrecovery of current amplitude on washout of roscovitine. If uncom-pensated, rundown would contribute to an overestimate of Rosc-induced inhibition. Since rundown cannot be independently measuredduring drug-induced inhibition, we estimated the effect of rundown byaveraging current measurements (MControl) taken just before (MBefore)and on recovery from (MRecov) Rosc-induced inhibition (MDrug) [i.e.,

MControl � (MBefore � MRecov)/2]. We have previously found thatusing an average of measurements before and on recovery from drugapplication for MControl was best for compensating for the effect ofrundown on fractional inhibition (14). Fractional inhibition was cal-culated from (MControl � MDrug)/MControl. MBefore, MDrug, and MRecov

were each the average of three tail current measurements taken before,during, and on recovery from drug application, respectively.

Ratio currents provide a method to observe the change of inhibitionwith time during voltage steps (15). The ratio current was calculatedfrom (IControl � IDrug)/(IControl), where IControl is the average of IBefore

and IRecov. IBefore, IDrug, and IRecov are each the average of threecurrent traces before, during, and on recovery from drug application,respectively. Data are reported as means SD.

Computer simulations. Simulated currents were generated usingAxovacs 3 (written by Stephen W. Jones, Case Western ReserveUniversity, Cleveland, OH) running on a Dell Inspiron 640m com-puter (Dell Computer, Round Rock, TX). Voltage (V)-dependent rateconstants (kx) in the model were calculated from

kx � Ax exp�VzxF/RT� (1)

where Ax is the rate constant at 0 mV, zx is the charge moved, and R,T, and F are the gas constant, absolute temperature, and Faraday’sconstant, respectively. The ionic concentrations for the current simu-lations were [K�]o � 5 and [K�]i � 140 mM.

Drugs. R-roscovitine was purchased from LC Labs (Woburn, MA).Indirubin was obtained from EMD Biosciences. Terfenadine, NMG,KCl, MgCl2, HEPES, EGTA, Tris2 �ATP, Tris2 �GTP, NaCl, CaCl2,and glucose were purchased from Sigma (St. Louis, MO). Terf, Rosc,and indirubin were dissolved in DMSO to make a stock solution of 50mM. For experiments using these drugs, all external solutions con-tained identical concentrations of DMSO.

RESULTS

Rosc inhibits HERG current. HERG current block by manydifferent drug classes is characterized by slow development(2–5 min) and a slow recovery (10 min) (17, 40). This typeof block is exemplified by the antihistamine Terf (32), whichachieved steady-state block in 1–2 min and showed incompleterecovery even after a 9-min washout period (see Fig. 1). Thisis contrasted by Rosc, which blocked HERG current in �20 sand completely recovered �20 s following termination of drugapplication (Fig. 1). The speed of Rosc block suggested aunique mechanism relative to other blockers (e.g., Terf) andthat kinase inhibition is not involved (5, 6). This hypothesiswas further tested by examining the effect of the structurallyunrelated CDK inhibitor indirubin-3 -monoxime (Indir), whichhas a similar affinity for CDK1 (0.2 vs. 0.5 �M) and CDK5(0.1 vs. 0.2 �M) relative to Rosc (22). Indir (30 �M) showeda slower block and recovery compared with Rosc. Thus, Roscappears to directly interact with HERG channels to blockpotassium flux.

The Rosc dose-response relationship was measured from thepeak tail current (�60 mV) following a 1-s step to �20 mV(20-s interval) to activate HERG channels (Fig. 2). Roscinhibited HERG current in a dose-dependent manner with littleor no effect at 1 �M and almost complete block at 100 �M.The IC50 was calculated using the Hill equation to be 27 �M(n � 6–10) (Fig. 2B). This apparent affinity was similar to thatfor neuronal delayed rectifier potassium current (IC50 � 23�M), but lower than that for expressed Kv2.1 (IC50 � 10 �M)(6). The Hill coefficient was 1.4, which indicates a smallpositive cooperativity in the Rosc block. The dose-responserelationship measured from step current (at the end of the 1-s

C702 HERG BLOCK BY R-ROSCOVITINE

AJP-Cell Physiol • VOL 296 • APRIL 2009 • www.ajpcell.org

step) showed an IC50 � 35 �M and a Hill coefficient of 1.7.These higher values at a voltage where more HERG channelsare inactivated could indicate that Rosc differentially affectsinactivated channels (see below).

Rosc block depends on HERG channel activation. The speedand dose-response relationship of block provide importantclues regarding possible mechanisms. To continue investigat-ing the blocking mechanism, we recorded the HERG I-Vrelationship by stepping the voltage from �60 to �40 mV(20-mV increments) and measuring current from both step (at4 s) and tail currents (peak) (Fig. 3). The step current showedslow activation at negative voltages and a bell-shaped I-Vrelationship that is typical of HERG channels. This shaperesults from channels inactivating more completely at depolar-ized voltages to reduce current measured during the step (33,36). Repolarization to �40 mV induced the characteristicallylarge tail currents that result from the rapid recovery frominactivation. A plot of peak tail current vs. voltage showed thatthe threshold for current activation was �20 mV and thatmaximum activation was achieved at �20 mV (Fig. 3C). Rosc(30 �M) reversibly blocked HERG current, but the effectdepended on the degree of depolarization (Fig. 3D). The blockwas weaker at �20 mV (mean � 30 11%, n � 7) and

reached a maximum at �20 mV (mean � 49 5%, n � 7),which corresponded with maximal activation (Fig. 3). Theblock was associated with a small but significant left-shift inthe voltage dependence of activation (shift in V1/2 � �2 2mV; P � 0.01, t-test paired samples, n � 10). These effectscould be explained if Rosc block either was correlated withHERG channel activation or exhibited weak intrinsic voltagedependence.

Interestingly, the voltage dependence of block was reversedwhen examining the effect of Rosc on tail currents measured atdifferent voltages (Fig. 4A). HERG current was activated by avoltage step to �60 mV and tail currents measured at voltagesranging from �20 to �100 mV. The inhibition increased from26% at �20 mV to 43% at �40 mV and 47% at �60 mV (P �0.05 for �20 vs. �40 mV, and �20 vs. �60 mV, n � 5,ANOVA) (Fig. 4B). This combined with the result from the I-Vprotocol (discussed above) demonstrates that Rosc block is notvoltage-dependent per se, but primarily depends on the avail-ability of activated HERG channels.

Inactivated HERG channels are relatively insensitive toRosc. The increase in block with step voltage could result fromRosc block of inactivated HERG channels, which should berevealed by examining inactivation kinetics. Using the tail I-Vprotocol (described above), the recovery from inactivation timeconstant (�) was determined by fitting the initial rising phase oftail current with a single exponential equation (33, 36, 47).Rosc increased the recovery from inactivation � at voltagesranging from �20 mV to �40 mV, but not at �80 and �100mV (Fig. 4C). The acceleration of recovery was observed onlyat voltages where � was 6–8 ms, which could indicate thatRosc affects a process other than inactivation. The develop-

Fig. 2. Concentration-dependent block of HERG by Rosc. A: a representativecell shows the time course of inhibition for 1, 3, 10, 30, and 100 �M Rosc. Stepcurrents (E) were measured at the end of the 1-s step to �20 mV. The voltageprotocol and peak tail current measurements (F) are the same as shown in Fig.1B. B: dose-response relationship showing the fractional block of tail currentsby Rosc. The smooth solid line is a fit using the Hill equation to generateIC50 � 27 �M and Hill coefficient � 1.4, while the dashed line shows the fitwith Hill coefficient fixed to 1 (n � 6–10).

Fig. 1. Comparison of the effects of roscovitine, indirubin, and terfenadine onhuman ether-a-go-go-related gene (HERG). A: peak tail currents were mea-sured from a representative cell (same as in B) showing the time course ofinhibition and recovery from inhibition for 30 �M R-roscovitine (Rosc), 30�M indirubin-3 -monoxime (Indir), and 0.1 �M terfenadine (Terf). On aver-age, inhibitions were 49 9% (SD) for Rosc (n � 10), 61 11% for Indir(n � 4), and 89 7% (n � 6) for Terf. Step currents were generated by 1-ssteps to �20 mV, and tail currents were generated by a 1-s repolarization to�60 mV. The interval between sweeps was 20 s. Note the differences in thespeed of inhibition induced in each of these drugs. B: representative currenttraces from A with the voltage protocol shown below. The arrowhead indicatesthe point at which peak tail currents were measured. Cntl, control; Recov,recovery.

C703HERG BLOCK BY R-ROSCOVITINE


ment of inactivation was measured using an inactivation pro-tocol where voltage was stepped to �60 mV for 500 ms toinactivate channels, hyperpolarized to �100 mV for 5 ms torecover channels from inactivation, and returned to voltagesranging from �20 to �60 mV, which was where the inacti-vation � was measured (45). Rosc (30 �M) significantlyincreased the speed of inactivation at voltages � �20 mV, butnot at �20 to �60 mV where inactivation is extremely fast(Fig. 4C). Again, the differential effect of Rosc on inactivationkinetics may indicate that a separate process (e.g., open stateblock) is being affected.

Rapid Rosc application supports open state block. To furtherinvestigate the possibility of open channel block, we used ourrapid exchange flow system (6) to apply and remove Roscduring a single 10-s tail current at �40 mV (Fig. 5A). Rosc (30�M) reversibly blocked the tail current by 38 8% (n � 4).Thus, open HERG channels are blocked by Rosc. To comparethe effect of Rosc on inactivated vs. open channels in the samecell, we used another protocol where HERG currents wereelicited by an 8-s step to 60 mV to inactivate the channelsfollowed by repolarization to �40 mV where majority ofHERG channels recover from inactivation and enter the openstate (Fig. 5B). Rosc (30 �M) was applied toward the end ofthe step to �60 mV and continued through the repolarizationto �40 mV. Rosc only induced a small 14 7% (n � 7)inhibition at �60 mV, which increased to 29 7% (P � 0.05)on hyperpolarization to �40 mV. Thus, Rosc preferentiallyblocks open HERG channels with a significantly lower affinityfor inactivated HERG channels.

If the HERG inactivated state is Rosc insensitive, thenequivalent block of step current and tail current should beobserved from inactivation-impaired mutants. This hypothesiswas tested by measuring the effect of Rosc on the inactivation-

impaired HERG mutant S620T (Fig. 5C), which showed largestep currents and relatively smaller tail currents (due to lack ofinactivation). Rosc was applied in similar manner as shown inFig. 5B. In contrast to the wild-type HERG, the step currentblock was not significantly different from that for peak tailcurrent (20 6% and 23 6% for step current and peak tailcurrent, respectively, P � 0.45, n � 5), which supports ourhypothesis of similar block for both step and the tail current.Thus, Rosc appears to preferentially block the HERG channelopen state with relatively little effect on inactivated HERGchannels.

Rapid Rosc-blocking kinetics. The absence of an effect ofRosc on inactivation kinetics at extreme voltages combinedwith the smaller block induced by Rosc when applied at �60mV supports our conclusion that inactivated HERG channelsare weakly affected. If this were true, we should be able tomodulate the magnitude of block by altering the fraction ofinactivated channels. We tested this idea by examining theeffect of Rosc on HERG current elicited by a protocol consist-ing of a 3-s step to �20 mV, followed by a 500-ms repolar-ization to �40 mV and a subsequent depolarization back to�20 mV for 3 s (Fig. 6A). Under control conditions (in theabsence of Rosc), the current increased on repolarization to�40 mV (� � 13 2 ms, n � 5) and rapidly spiked when thevoltage was returned to �20 mV. The amplitude of this spikerepresents increased driving force (at �20 mV) on K� movingthrough the channels that recovered from inactivation at �40mV, while the rapid decline in current results from inactivationat �20 mV (� � 8.5 1.2 ms, n � 4). The Rosc effect wascalculated from ratio currents generated by (Cntl � Rosc)/Cntl,where Cntl is the average of currents recorded immediatelybefore and on recovery from 30 �M Rosc application (Fig. 6,B–D). As predicted, the percent inhibition increased from 26

Fig. 3. Rosc block increases with step depolarization. A: HERGcurrents from a representative cell are shown for voltage stepsto �20, 0, and �20 mV. The tail currents were generated onhyperpolarization to �40 mV. The Rosc concentration was 30�M. Control (Cntl; before Rosc application), Rosc (duringRosc application), and recovery (Recov; on recovery fromRosc application) currents are represented. The arrowheadindicates the point at which peak tail currents were measured.B: the step current vs. step voltage relationship (I-V) is shownfor the representative cell from A. Step currents were measuredat the end of the 4-s step ranging from �60 to �40 mV(20-mV increments). Values are shown for control (‚), 30 �MRosc (F), and on recovery (ƒ). C: peak tail currents are plottedvs. step voltage (symbols same as in B) for the same represen-tative cell (A and B). The smooth lines represent single Bolt-zmann fits to generate V0.5 � �7, �11, and �7 mV, slope �7, 7, and 7, and maximum current � 1.7, 0.8, and 1.5 nA forcontrol, 30 �M Rosc, and recovery, respectively. D: compar-ison of the percent inhibition of peak tail currents induced by30 �M Rosc shows significantly lower inhibition at �20 vs. 0(b), �20 (c), and �40 mV (d) (P � 0.01 for each comparison,ANOVA; n � 6).



5% to 37 4% and back to 24 5% for �20, �40 and�20-mV steps (n � 4), respectively (Fig. 6, B and D). We nextdetermined the blocking and unblocking � from the inhibitiontime course. The kinetics were not limited by our applicationsystem since these measurements were obtained in the constantpresence of 30 �M Rosc (applied at least 20 s before initiation

Fig. 5. Rosc blocks open, but not inactivated, HERG channels. A: HERGchannels were activated by a 1-s step to �60 mV, and tail current wasmeasured during a 10-s step to �40 mV. Three pulses were given at 1-minintervals between the pulses. During the tail current of the second pulse (*), 30�M Rosc was rapidly applied and removed for the duration shown by the blackbar. The remaining two traces show the current without Rosc application thatwere recorded 1 min before and 1 min following the Rosc-exposed trace. Onaverage, Rosc blocked the HERG tail current by 38 8% (n � 4). B: currentwas elicited by an 8-s step to �60 mV, and tail currents were measured at �40mV. Rosc (30 �M) was rapidly applied during the step and continued into thetail as indicated by the black bar. The superimposed control and recoverycurrents were recorded 1 min before and after the Rosc-exposed trace,respectively. On average, Rosc block of HERG current at the end of thevoltage step to �60 mV was 14 7%, which increased to 29 7% (P � 0.05)on hyperpolarization to �40 mV (n � 7). The arrowheads on the voltageprotocol indicate the approximate points at which the step and tail currentswere measured. C: the same experiment presented in B was repeated with theinactivation-impaired HERG mutant S620T. The superimposed control currentwas recorded 1 min before the Rosc-exposed current (*). Unlike the wild-typeHERG, S620T currents were equally blocked at �40 mV (step) and �40 mV(peak tail) by application of Rosc (20 6% and 23 6% for step and peaktail currents, respectively, P � 0.45, n � 5). The arrowheads on the voltageprotocol indicate the approximate points at which the step and peak tailcurrents were measured.

Fig. 4. Increased Rosc block of tail current with hyperpolarization. A: the peaktail current vs. tail voltage relationship is shown for a representative cell. Peaktail currents were measured at voltages ranging from 20 to �100 mV (20-mVincrements) following a 1-s step to 60 mV to activate HERG channels (inset)and are plotted vs. tail voltage for control (‚), 30 �M Rosc (F), and recovery(ƒ). The arrowhead on the voltage protocol indicates the point at which the tailcurrent was measured. B: percent inhibition of IHERG by 30 �M Rosc from thevoltage protocol shown in A. Percent inhibition at different tail voltages isplotted vs. tail voltage. Inhibition at 20 mV was significantly different (*P �0.05, n � 5) from that at �40 and �60 mV (ANOVA). C: Rosc decreasesinactivation kinetics only at intermediate voltages. The recovery from inacti-vation � (circles, n � 6) was measured (single exponential equation) from theraising phase of the tail current upon hyperpolarization from �60 mV to theindicated voltage (x-axis). The development of inactivation � (squares, n � 4)was measured from a triple-pulse protocol where voltage was stepped to �60(500 ms), �100 (10 ms), and the indicated voltage (x-axis) for 250 ms. Asingle exponential equation was used to fit inactivation during the third voltagestep. Data are shown for control (average of values before and after Rosc) andduring the application of 30 �M Rosc (open and closed symbols, respectively)(**P � 0.01).



of the 3-step voltage protocol). The speed of block (� � 29 10 ms; n � 4) on stepping from �20 to �40 mV was slowerthan that of the recovery from inactivation at �40 mV (� �13 2 ms, n � 5), which suggests that the channels need torecover from inactivation before Rosc can block. Unblock wasalso rapid (� � 22 11, n � 4) when the voltage was steppedback to �20 mV. Given the rapid transition of channelsbetween blocked and unblocked states, there appear to beminimal barriers to Rosc binding and unbinding as HERGchannels move between the open and inactivated states.

Rosc does not block closed HERG channels. In calculatingthe ratio currents, we noticed that the time course of block atthe onset of the first step to �20 mV was surprisingly complex,with the inhibition initially increasing then decreasing (Fig.6B). Early in the step, block increased from near 0% to a peakof 56 21% with � � 18 7 ms (n � 9). The block thendecreased more slowly to a minimum of 28 8% (� � 117 44 ms, n � 7) (Fig. 6B). We speculate that the initial increasein inhibition on stepping the voltage to �20 mV results fromthe rapid block of HERG channels and subsequent decrease ininhibition results from relief of block on inactivation, which isstrongly supported by our kinetic model (Fig. 9D). As a directtest of this hypothesis, we ran the same experiment using theinactivation-impaired S620T HERG mutant (Fig. 6, C and D).The ratio currents from the S620T mutant showed the initialrise in the inhibition as observed with wild-type HERG chan-nels, but unlike in the wild type, the inhibition remainedrelatively stable during the voltage step. Furthermore, therewas no change in fractional inhibition on return to �40 mV forcurrents generated by the S620T mutant (Fig. 6D). The slowdecline in the ratio current observed with S620T currents likelyresults from potassium depletion from the cells during theselong voltage steps. This depletion would be stronger for controland recovery currents relative to the inhibited currents in Rosc,which explains the slow decline in apparent inhibition. Large

potassium currents are known to deplete small HEK-293 cellsof intracellular potassium (19), and we have observed obviouscurrent reductions during 5-s steps to voltages that maximallyactivate S620T channels. The current reductions were associ-ated with a depolarization of the reversal potential as expectedfor intracellular potassium depletion. Together, these resultsdemonstrate that Rosc preferentially blocks activated HERGchannels with little or no affinity for either closed or inacti-vated channels.

Rosc is not trapped in the inner vestibule of HERG. ManyHERG blockers like Terf bind in the inner vestibule of theHERG and become trapped by channel closing, which ac-counts for their slow recovery from block (Fig. 1). To deter-mine whether Rosc can be trapped in closed channels, wehyperpolarized to �120 mV where the deactivation was suf-ficiently fast, � � 15 2 ms (80% of channels; n � 4), tocapture Rosc if it was binding within the inner vestibule(Fig. 7). (The remaining 20% of channels deactivated with � �68 7; n � 4.) However, ratio currents showed the sameincrease in block with each step in the presence of Rosc, andrecovery on Rosc washout was just as rapid as when thedeactivation voltage was �40 mV. Thus, Rosc does not appearto be trapped by HERG channel closing.

HERG channel model reproduces Rosc block. Our resultsstrongly support preferential block of activated HERG chan-nels by Rosc. A gating model (Fig. 8) based on Wang et al.(42) was developed to further test this mechanism. The rateconstant (A, s�1) and charge moved (z) for each transition aregiven in Table 1. The binding rate constants (k5, k6, and k7)have units of �M�1 s�1. In the diagram shown in Fig. 8, C1–3,O, and I represent closed, open, and inactivated states, respec-tively; CR, OR, and IR are Rosc-bound closed, open, andinactivated states, respectively. All horizontal transitions arevoltage dependent except C27C3 (42), and the vertical tran-sitions involve Rosc binding and unbinding.

Fig. 6. Inactivated channels are insensitive to Rosc. A: HERG currents were activated by a 3-s step to 20-mV, followed by a 1-s hyperpolarization to �40 mVand a return to �20 mV. Representative currents from a single cell are shown. B: the ratio current generated from the currents in A is displayed as percentinhibition by 30 �M Rosc and shows rapid block at the onset of the voltage step from �80 to �20 mV (1); slower unblock during the sustained �20-mV step(2); rapid reblock on hyperpolarization to �40 mV (3); and rapid unblock on returning to �20 mV (4). The ratio current was generated by the formula (Cntl �Rosc)/Cntl, where Cntl is the average of currents before and on recovery from Rosc application. The smooth gray lines are single exponential fits to obtain the� for initial block, slower unblock during the step to �20 mV, increased block at �40 mV, and decreased block at �20 mV. C: 30 �M Rosc inhibits both stepand tail currents (from a representative cell) obtained from inactivation-impaired mutant S620T. D: ratio currents obtained for currents shown in C. Notice thatthe inhibition increases with current activation and remains relatively stable during the �20-mV step. The arrow marks the repolarization to �40 mV to highlightthe absence of the increase of inhibition observed in wild-type HERG.



We have modified the rate constants from the original model(42) to fit our data and added three Rosc-bound states (CR, OR,and IR; Fig. 8). The major modifications were to increase thecharge moved between the open and closed state (C37O) to fitthe voltage dependence of deactivation �, and to increase therate of I3O to fit the recovery from inactivation � (Table 1;

supplemental Fig. 1; the online version of this article containssupplemental data).

We began the model with a single Rosc blocked state thatwas accessible only from the open state (OR). This wasintended to reproduce the apparent open state binding prefer-ence of Rosc. However, the main problem with this simplemodel was that deactivation and inactivation kinetics weredrastically slowed by Rosc (not shown), which was not ob-served experimentally. The slow kinetics resulted from themass-action movement of Rosc-bound channels into the openstate before either inactivating or closing (depending on thevoltage), which suggested that additional exits from the Rosc-bound state were needed. The addition of an inactivatedblocked state (IR) helped to resolve the problems with inacti-vation kinetics, while an additional closed blocked state (CB)was required to resolve the problem of roscovitine-inducedslowed deactivation. Altering the affinity of the CB state forroscovitine had little affect on the simulated currents so the rateconstants were kept the same as O7OR. However, Roscbinding to the IR state needed to be reduced to match our data(Fig. 5B). This was accomplished by increasing the unbindingrate 5� (IR3 I), while the IR3OR rate was increased 5� tomaintain microscopic reversibility. The simulated current re-produced our results very well (Fig. 9 and supplemental dataFig. 1). The IC50 for simulated tail current block was 30 �Mvs. 27 �M for the experimental data. The block in the modelwas modulated by voltage as in our experimental results (cf.data in Figs. 3, 4, 6, 9, and supplemental Fig. 1), which arisesfrom both the requirement of channel activation for bindingand the decreased affinity of inactivated channels for Rosc. Aswe observed in our recordings, the inhibition increased withstep depolarization during the I-V protocol (Fig. 9B) and alsoincreased with tail hyperpolarization during the tail I-V proto-col (supplemental Fig. 1C). The model closely mimicked thekinetics of Rosc block as channels moved between the openand inactivated states (cf. Figs. 6 and 9C). The model ratiocurrents have a blocking � � 16 ms (cf. 29 10 ms for ourexperimental data) for the increased block on a voltage changefrom �20 to �40 mV, and an unblocking � � 16 ms (cf. 22 11 ms) on return to �20 mV (Fig. 9D). The model alsoreproduces the complex block on stepping the voltage from theholding potential to �20 mV (cf. Figs. 6B and 9D). The initialincrease in block during activation had a � � 16 ms (cf. 18 7 ms) and the subsequent decline in block had a � � 160 ms(cf. 117 44 ms). The model strongly supports our conclu-sions that Rosc blocks by preferentially binding to activated,

Fig. 7. Rosc is not trapped by HERG channel closing at �120 mV. A: peak tailcurrents were measured at �120 mV following 700-ms voltage steps to �20mV. The interval between sweeps was 20 s. C (control), R1 (Rosc sweep 1),R2 (Rosc sweep 2), and Re (recovery) indicate sweeps shown in B. B: currenttraces are shown before, two times during, and on recovery from 30 �M Roscalong with the voltage protocol. C: ratio currents are depicted for both R1 andR2 currents (successive current traces in Rosc). Note the rapid onset ofinhibition at the beginning of both R1 and R2, which shows that Rosc unboundduring the 20-s interval between sweeps.

Fig. 8. Gating model [based on Wang et al. (42)] of mechanism of preferentialblock of activated HERG channels by Rosc.

Table 1. Model parameters

Forward A (k1—k9) z (k1—k9) Backward A (k�1—k�9) z (k�1—k�9)

k1 2 0.6 k�1 50 �0.6k2 10 k�2 10k3 14 1.5 k�3 0.1 �1.5k4 90 0.6 k�4 20 �0.6k5 30�R� k�5 500k6 30�R� k�6 500k7 30�R� k�7 2500k8 14 1.5 k�8 0.1 �1.5k9 90 0.6 k�9 100 �0.6

A (s�1) is the rate constant at 0 mV and z is the charge moved. �R� is theroscovitine concentration (in �M). The units of A for k5, k6, and k7 are �M�1

s�1. See model diagram in Fig. 8.



but not inactivated, HERG channels. Thus, Rosc therapy mayblock the open HERG channels (the steady-state windowcurrent) that are crucial to proliferation of certain cancers (1, 24).

DISCUSSION

In this study, we examined the Rosc block of HERG chan-nels using kinetics, mutants, and modeling to determine thatRosc blocks by preferentially binding to activated channelswith little or no effect on inactivated channels. Supportingevidence includes increased inhibition with channel activation,rapid block of open channels, and increased block on repolar-ization where open probability increases following recoveryfrom inactivation (36, 43). The block does not appear to bevoltage dependent per se since the inhibition did not changeover voltages that generated maximal activation. Instead, theapparent voltage dependence results from the preferentialblock of activated channels by Rosc. Unlike many otherHERG-blocking drugs, inactivated channels are relatively in-sensitive to Rosc. The block during an inactivating step wassignificantly less compared with the block during peak tailcurrent (after the channels are recovered from inactivation).However, this difference in block was not observed for cur-rents generated by the inactivation-impaired S620T mutant.The preference of Rosc for activated channels combined withthe unique HERG channel inactivation kinetics permitted us toexamine the block/unblock process for channels moving be-tween the open and inactivated states (Fig. 6, A and B). Thisanalysis demonstrated the rapid association/dissociation ofRosc with HERG channels, which was reproduced by ourmodel and further supports preferential block of activatedHERG channels.

Comparison with other HERG blockers. Several drugs, in-cluding terfenadine, have been removed from the market be-cause they block HERG to induce cardiac arrhythmias (34).These drugs along with class III antiarrhythmic drugs, such asdofetilide, block by binding within the inner vestibule ofHERG near the selectivity filter (21, 27, 34). Binding to thissite generally results in 1) a preference for these drugs to bindto the open state and 2) a slow recovery from block, both of

which results in use-dependent block (17, 37, 40). While Roscdoes require channel opening to block, use dependence was notobserved, which could result from its rapid block/unblockkinetics. There are some drugs that also rapidly block HERGlike spironolactone (� � 350 ms), fluoxetine (estimated � �400 ms), and fluvoxamine (� � 7.5 ms). However, there areclear differences in the block by these drugs with that inducedby Rosc. Fluvoxamine appeared to block closed HERG chan-nels since the majority of channels were blocked at the earliesttime point measured (10 ms), and additional data furthersupported closed channel block (26). Another difference offluvoxamine block from that induced by Rosc was that inacti-vated HERG channels also appeared to be blocked. Fluoxetineappeared to block open channels like Rosc, but it had a long(50 min) washout period (38). Spironolactone and its metabo-lite canrenoic acid blocked rapidly, but like fluvoxamine,appeared to block closed HERG channels (7).

We used the CDK inhibitor Indir to determine whether rapidblocking kinetics were common to all CDK inhibitors. UnlikeRosc, the slower kinetics of the Indir block of HERG currentare consistent with kinase inhibition, but further work isrequired to determine the mechanism. Another kinase inhibitorbisindolylmaleimide I (BIM 1), which inhibits protein kinaseC, has been shown to block HERG channels in a kinase-independent manner (39). However, there were several differ-ences between the block induced by BIM I and Rosc. BIM Iappeared to block both open and inactivated HERG channels,and recovery from BIM I block was incomplete even after 20min of washout. In addition, BIM I block was use dependent(39), which we have not observed with Rosc. Therefore, Roscshows unique preference for open compared with either closedor inactivated HERG channels and rapid blocking kinetics withno use dependence.

Physiological/pathophysiological relevance. HERG chan-nels are blocked by structurally diverse molecules many ofwhich have been shown to induce acquired long QT syndrome.As a result, many studies have investigated the mechanism ofHERG block, and development of molecules that do not blockHERG is of considerable interest for drug development (34).

Fig. 9. A gating model reproduces Rosc block of HERGchannels. A: currents were simulated using a 4-s step to theindicated voltage (x-axis) followed by a 1-s step to �40mV. Peak tail currents were measured during the �40-mVstep and normalized to the current following the �60-mVstep. The simulated data are shown for control (‚) and 30�M Rosc (F). The smooth lines are single Boltzmannequation fits to the experimental data to illustrate the closecorrespondence with the simulation data. B: the percentinhibition of tail current vs. step voltage by 30 �M Rosc isshown for both simulated (■ ) and experimental data(�, reproduced from Fig. 3D). C: the time course of 30 �MRosc block of simulated current is shown for a 3-s step to�20 mV, which is followed by a 50-ms step to �40 mV anda subsequent return to �20 mV for 3 s. The simulatedcurrents are shown for control (gray trace) and Rosc (blacktrace). D: the ratio current is shown as percent inhibition andwas calculated from the above records in C (cf. Fig 6B).



However, HERG is a desirable target for block to facilitatetreatment of certain cancers (9). We propose that it may bepossible to block HERG channels in the cancer cells withoutsignificantly increasing the risk for cardiac arrhythmias.

Rosc is currently undergoing phase II clinical trials as ananticancer drug, and the clinically relevant dosing range hasbeen shown to be 10–50 �M (12, 13, 25). However, cardiacarrhythmias have not been reported as a side effect in thesepatients (3, 18) even though we find the IC50 for HERG blockto be �30 �M. We believe that rapid Rosc-blocking proper-ties, combined with the inability of HERG channels to trapRosc, are the reason that cardiac arrhythmias have not beendescribed in clinical studies. These properties allow Rosc tobind and unbind within a single cardiac cycle so that the blockis similar for each cAP and does not increase with time (no usedependence). In addition, the absence of use-dependent blockmeans that HERG block will not increase with heart rate,which will help to limit the proarrhythmic potential of Rosc.Furthermore, Rosc has been shown to block cardiac L-channels(6, 44), and it has been reported that L-type calcium channel(CaV1.2) block can compensate for the proarrhythmic effectsof HERG block (4, 10, 46). While it is still early in humantesting, the absence of arrhythmias is encouraging and suggeststhat Rosc may block activated HERG channels with minimalcardiovascular side effects.

Potassium channels are of considerable interest as therapeu-tic targets for cancer chemotherapy (16). HERG channels areoverexpressed in many types of neoplastic cell lines andprimary tumors (1). The HERG channels in these cells generatea small but steady-state current (window current) that isthought to play a role in proliferation, tumor invasiveness, andtumor neoangiogenesis (1). Blocking the steady-state currentcould be an effective way to treat these cancers. The combinedblock of both CDKs and HERG channels (as well as other Kvchannels) should enhance Rosc as an anticancer therapy, whilethe inhibition of L-type calcium currents along with the rapidHERG-blocking kinetics may limit cardiovascular side effects.Therefore, the development of state-specific blockers that candifferentially affect HERG current in nonexcitable cancer cellsvs. excitable cells, like cardiac myocytes, can offer a uniquetherapeutic advantage. This could lead to the development ofdrugs that specifically block HERG channels in transformed(cancer) cells to further minimize potential side effects inducedby drug block of HERG channels in nontransformed or excit-able cells.

ACKNOWLEDGMENTS

The authors thank Drs. Eckhard Ficker (MetroHealth Medical Center,Cleveland, OH) and Shetuan Zhang (Queen’s University, Kingston, Ontario,Canada) for the gift of cell lines and clones that were crucial to the completionof this work.

GRANTS

This work was supported in part by an American Heart AssociationPre-doctoral fellowship (0715336U) to S. B. Ganapathi; by National Heart,Lung, and Blood Institute Grants RO1-HL-066371 and RO1-HL-076789 to M.Kester; and by a grant from the Pennsylvania (PA) Department of Health usingTobacco Settlement Funds to K. S. Elmslie. The PA Department of Healthspecifically disclaims responsibility for analyses, interpretations, and conclu-sions presented here.

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