1521-0111/90/2/106–115$25.00 http://dx.doi.org/10.1124/mol.115.102277MOLECULAR PHARMACOLOGY Mol Pharmacol 90:106–115, August 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

K201 (JTV519) is a Ca21-Dependent Blocker of SERCA and aPartial Agonist of Ryanodine Receptors in Striated Muscle

Yuanzhao L. Darcy, Paula L. Diaz-Sylvester, and Julio A. CopelloDepartment of Pharmacology (Y.L.D., P.L.D.-S., J.A.C.) and Center for Clinical Research (P.L.D.-S.), Southern Illinois UniversitySchool of Medicine, Springfield, Illinois

Received October 28, 2015; accepted May 26, 2016

ABSTRACTK201 (JTV-519) may prevent abnormal Ca21 leak from thesarcoplasmic reticulum (SR) in the ischemic heart and skeletalmuscle (SkM) by stabilizing the ryanodine receptors (RyRs; RyR1and RyR2, respectively). We tested direct modulation of the SRCa21-stimulated ATPase (SERCA) and RyRs by K201. In isolatedcardiac and SkM SR microsomes, K201 slowed the rate of SRCa21 loading, suggesting potential SERCA block and/or RyRagonism. K201 displayed Ca21-dependent inhibition of SERCA-dependent ATPase activity, which was measured in microsomesincubated with 200, 2, and 0.25 mM Ca21 and with the half-maximal K201 inhibitory doses (IC50) estimated at 130, 19, and9 mM (cardiac muscle) and 104, 13, and 5 mM (SkM SR). K201($5 mM) increased RyR1-mediated Ca21 release from SkM

microsomes. Maximal K201 doses at 80 mM produced ∼37% ofthe increase in SkM SR Ca21 release observed with the RyRagonist caffeine. K201 ($5mM) increased the open probability (Po)of very active (“high-activity”) RyR1 of SkM reconstituted intobilayers, but it had no effect on “low-activity” channels. Likewise,K201 activated cardiac RyR2 under systolic Ca21 conditions(∼5 mM; channels at Po ∼0.3) but not under diastolic Ca21

conditions (∼100 nM;Po, 0.01). Thus, K201-induced the inhibitionof SR Ca21 leak found in cell-system studies may relate topotentially potent SERCA block under resting Ca21 conditions.SERCA block likely produces mild SR depletion in normal con-ditions but could prevent SR Ca21 overload under pathologic con-ditions, thus precluding abnormal RyR-mediated Ca21 release.

IntroductionK201 (JTV519, a 1-4 benzothiazepine) cardioprotective

properties were first reported by Kaneko and coworkers(Kaneko, 1994; Hachida et al., 1997; Kaneko et al., 2009).Other groups have further confirmed K201’s cardioprotectiveproperties in various heart pathologies, including ischemiaand arrhythmogenesis in various animal models (Kaneko,1994; Ito et al., 2000; Kawabata et al., 2002; Wehrens et al.,2004; Lisy and Burnett, 2006; Loughrey et al., 2007; Otaniet al., 2013), in addition to human cardiac cells (Toischer et al.,2010; Sacherer et al., 2012). The mechanism of action of K201is complex and includes various effects, including block ofannexin V-mediated Ca21 transport (Kaneko et al., 1997) andplasmalemma currents mediated by K1, Na1, or Ca21 chan-nels (Kimura et al., 1999; Loughrey et al., 2007).K201 is also defined as a channel stabilizer of the ryanodine

receptor channel (RyR isoform 2, or RyR2) in the cellularenvironment of heart sarcoplasmic reticulum (SR), preventingabnormal RyR2-mediated leak during diastole (Yano et al.,2003; Wehrens et al., 2005); however, there are conflicting

studies regarding the role of FKBP12.6 and phosphorylationin RyR2-mediated SR Ca21 leak (Yano et al., 2003; Wehrenset al., 2004, 2005; Xiao et al., 2007). Additionally, some studiessuggested that K201 can deplete intracellular Ca21 stores inan ischemic heart model and in smooth muscle cells (Inagakiet al., 2000; Chen et al., 2008), an unexpected property for adrug that prevents RyR2-mediated leak. There are no reportson the direct effect of K201 at the single RyR2 channel level,but [3H] ryanodine binding studies suggest that K201 mayhave a mild effect on RyR2 (Hunt et al., 2007). Furthermore,other targets have been proposed to explain SR depletion orthe lack of SR overload observedwithK201, including SERCA,the Ca21 ATPase of SR (Loughrey et al., 2007).K201 is known to improve the function of the ischemic

skeletal muscle (SkM) (Wehrens et al., 2005). Two studiesassessed the effects of K201 on the RyR isoform 1 (RyR1). Inone study, K201 was found to slightly decrease or increase[3H] ryanodine binding to purified and solubilized RyR1 in theabsence or presence of physiologic levels of Mg21, respectively(Blayney et al., 2010). The other study described a K201-induced increase in SR Ca21 release from microsomes(Almassy et al., 2008). Additionally, in this second study,K201 was found to induce RyR1 channel opening to subcon-ductance states at negative SR holding voltages, and at very

This work was supported by the American Heart Association MidwestAffiliate [AHA-MWA 12180038] and the Eskridge Foundation.

dx.doi.org/10.1124/mol.115.102277.

ABBREVIATIONS: [Ca21]cyt, cytosolic Ca21 concentration; CGP, CGP-37157; CPZ, cyclopiazonic acid; DMSO, dimethyl sulfoxide; EGCG,epigallocatechin-3-gallate; FKBP, FK506 binding protein; HA, high activity; Km, dose that includes half-maximal rate; K201, JTV 519, 4-[3-(4-benzylpiperidin-1-yl)propionyl]-7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine; LA, low activity; RR, ruthenium red; ryanodine, 1H-pyrrole-2-carboxylicacid(3S,4R,4aR,6S,6aS,7-S,8R,8aS,8bR,9S,9aS)-dodecahydro-4,6,7,8a,8b,9a-hexahydroxy-3,6a,9-trimethyl-7-(1-methylethyl)-6,9-methanobenzo[1,2]pentaleno[1,6-bc]furan-8-yl ester; RyR, ryanodine receptor; RyR1, skeletal muscle RyR isoform 1; RyR2, cardiac muscle RyR isoform 2; SERCA, sarco/endoplasmicreticulum Ca21 ATPase; SkM, skeletal muscle; SR, sarcoplasmic reticulum; Vm, membrane voltage; Vmax, maximal activity.

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high doses, the drug inhibited the Ca21 ATPase of SR(SERCA1a).We compared, for the first time, the effect of K201 on

SERCA of cardiac muscle and SkM SR microsomes in thepresence of several Ca21 concentrations. Additionally, weassessed the direct effects of K201 on RyR1 and RyR2channels reconstituted into planar lipid bilayers. Experimentsperformed on reconstituted RyR1 channels were comple-mented with Ca21 leak assays using SR microsomes.Our results suggest that K201 stabilization of SR Ca21 leak

in cells cannot be attributed to direct inhibition of RyRs. Weconclude that the effects of K201 on heart and SkM cells can bedue to the drug’s potent inhibitory action on SERCA at restingCa21 concentrations.

Materials and MethodsIsolation of Cardiac and SkM SR Microsomes. All procedures

involving animals were designed conformed to the guidelines of theNational Institutes of Health to minimize pain and suffering andusing protocols (196-05-021 and 196-11-010) reviewed and approvedby the Laboratory Animal Care and Use Committee of SouthernIllinois University School of Medicine, whose animal research proce-dures are certified by the Association for Assessment and Accredita-tion of Laboratory Animal Care (AAALAC 000551) and by the PublicHealth Service (PHS A3209-1).

SkM SR microsomal fraction R2, enriched in longitudinal tubulecontaining high density of SERCA1a (LT fractions) and R4 enriched interminal cisternae, where the RyR1 localize (TC fractions), wereisolated from adult New Zealand male white rabbits, as previouslydescribed by others (Saito et al., 1984; Chu et al., 1988). Cardiac SRmicrosomes containing RyR2 and SERCA2a were prepared fromventricular tissue from male Yorkshire-Landrace crossed breed pigs(age 3 months; weight from 30 to 40 kg) following standard protocols(Chamberlain et al., 1983). Preparations were stored in liquidnitrogen. For experiments, SR microsomes were split into 15 ml or100–600 ml (for bilayer experiments and loading/release/ATPaseassays, respectively) at a concentration of 5–15 mg protein/ml in5 mM imidazol-Cl 290 mM sucrose, pH ∼7, snapped frozen with liquidN2, stored at 280°C and used within 60 days. For experiments,aliquots were quickly thawed in water, incubated in ice, and usedwithin 5 hours.

Measurements of Ca21 Loading/Leak by SRMicrosomes. Ca21

uptake by cardiac or SkM SR microsomes was measured followingpublished protocols (Chamberlain et al., 1984; Chu et al., 1988;Neumannand Copello, 2011; Neumann et al., 2011). Ca21 uptake was initiated byadding 40 nm CaCl2 to a cuvette containing 40–100 mg SR membranesresuspended in 1 ml of buffer [in mM: 100 KH2PO4, 5 MgCl2 (free Mg21

∼0.3 mM), 5 ATP and 0.2 antipyrylazo III; pH 7.0]. Ca21 uptake wasmonitored in SkM (R4 TC fractions) and cardiac SR microsomes witha spectrophotometer (Cory 50, Varian, Walnut Creek, CA) followingchanges in antipyrylazo III absorbance (710–790 nm). Uptake rates(in nMole Ca21 • (mg protein)-1 • min21) were measured in the absence(control) or presence of K201 and/or 5 mM ruthenium red (RR), aspreviously described (Neumann et al., 2011). Ca21 leak from SkM R4fractions preloaded with Ca21 (three pulses of 30 mM Ca21) wasmeasured after stopping SERCA activity with 20 mM cyclopiazonic acid(CPZ) and observing the effects of 1ml dimethyl sulfoxide (DMSO; control)versus K201, both in the absence or presence of 5 mM RR.

Measurements of ATPase Activity in SR Microsomes. Stud-ies of ATPase activity in SR microsomes used a previously describedassay (Chu et al., 1988; Neumann and Copello, 2011; Neumann et al.,2011). SkM SR fractions (10–40 mg) enriched in longitudinal tubule(R2 fractions) or cardiac SR microsomes (300 mg) were incubated withbuffer containing (in mM): 140 KCl, 5 MgCl2, 5 HEPES, 2 phospho-enolpyruvate, 0.3 CaCl2, and BAPTA/BromoBAPTA to obtain free

Ca21 levels of 250, 3, and 0.2 mM, for representation of completesaturation for SERCA binding sites, ∼95% maximal activity (Vmax)and near half-maximal rate (Km), respectively. The buffer alsocontained 3.5 mM A23187, a Ca21 ionophore, to prevent increase inSR lumen Ca21 levels with time owing to SERCA activity. The pHwasadjusted to 7.0 with KOH. The assay also used pyruvate kinase(8.4 U/ml) and lactic dehydrogenase (12 U/ml). The addition of 1 mMATP activates ATPase hydrolysis to ADP. This process is coupled toreactions that regenerateATP fromADP, consuming one equivalent ofNADH (oxidation to NAD1) for every ATP hydrolyzed. Thus, moni-toringNADHdepletion at 340 nm allowed determination of the rate ofATP hydrolysis (Chu et al., 1988; Neumann and Copello, 2011;Neumann et al., 2011). In SkM R2 fractions, ∼99% of the ATPaseactivity is inhibited by addition of the SERCA blocker CPZ (20 mM). Incardiac microsomes, there is a significant CPZ-independent (non–SERCA mediated) component of ATPase activity, which wasinhibited by addition of sodium azide (1 mM) and ouabain (100 mM).In the presence of 250 mMCa21, 75%–85% of the total ATPase activitywas CPZ sensitive; at 0.25 mM (approximately Km for SERCA) Ca21,25%–50% of the ATPase activity is independent of CPZ. At cell restinglevels, SERCA activity is very small (3%–7% of Vmax). Consequently,the CPZ-independent component dominates and prevented quantita-tive analysis of K201 effects on SERCA.

RyR Channel Measurements in Planar Lipid Bilayers. Pla-nar lipid bilayers made of 50% phosphatidylethanolamine, 40%phosphatidylserine, and 10% phosphatidylcholine (Avanti Polar Lip-ids; total 50 mg/ml), were formed on ∼100-mm-diameter circular holesin Teflon septa, separating two 1.2-ml hemichambers (Copello et al.,1997). The trans hemichamber contained 250mMHEPES and 50 mMCa(OH)2 (pH 7.4) and was clamped at 0 mV (Axopatch 200B; MolecularDevices, Sunnivale, CA). The cis compartment (ground) is initiallyfilled with 250mMHEPES and 120mMTris (pH 7.4). While stirring,500–1000 mM CsCl, 1 mM CaCl2, and SR microsomes (5–15 mg) areadded to the cis solution. After a few minutes, RyRs from SR arereconstituted into bilayers with their cytosolic surface facing the cischamber (Copello et al., 1997). Subsequently, the cis chamber wassuperfused (5 minutes at 4 ml/min) with HEPES-Tris solution, andstudies of RyR functionwere carried out in the presence of 1mMBAPTAand dibromo-BAPTA to buffer the free [Ca21] on the cytosolic surface ofthe channel ([Ca21]cyt). Channels used in our experiments displayedconductance (∼90–110 pS), gating (channels are activated by Ca21, ATP,and caffeine, and/or inhibited by Mg21 or tetracaine), and sensitivity toconductance modifiers (peptide blockers, ryanoids, or imperatoxin) thatare characteristics of RyRs (Copello et al., 1997; Sitsapesan andWilliams, 1998; Fill and Copello, 2002).

Channel recordings (4–8 minutes in duration in each condition) werefiltered at 1 kHz, digitized at 20 kHz with a Digidata 1360 (MolecularDevices), stored, and analyzed using pClamp10 software (Axon Instru-ments) as described before (Copello et al., 1997; Diaz-Sylvester et al.,2011). The presence and abundance of substates and/or flicker blockwere estimated as previously described (Porta et al., 2008). Briefly, all-points current-amplitude histograms (bandwidth 5 0.01 pA) wereobtained from digitally filtered (500 Hz) single-channel recordings(2 minutes each, obtained at voltage ranging from 140 to 260 mV). Inall control conditions, we detected only peaks corresponding to thebaseline and full openings. Each component was fitted with a Gaussianfunction using the Levenberg-Marquardt method, and an estimation ofprobability was obtained. Only with K201 and 260 mV, intermediatestateswere observed and their probabilitywas estimated by subtractingfrom the area of the curve the fits to the baseline and full openings fromcontrol experiments.

Drugs and Chemicals. CaCl2 standard for calibration was fromWorld Precision Instruments, Inc. (Sarasota, FL). Phospholipids wereobtained from Avanti (Alabaster, AL). Cyclopiazonic acid was fromTocris Bioscience (Avonmouth, Bristol, UK). One set of experimentswas carried out with K201 (Aetas PharmaCo. Ltd., Tokyo, Japan), butmost of the studies were carried out with K201 from Tocris Bioscience(Bristol,UK) orSigma-Aldrich (St. Louis,MO).No significantdifferences

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were observed in data collection from the K201 drug batches. All otheragents were obtained from Sigma-Aldrich-Fluka.

Statistical Analysis. Data are presented as means 6 S.E.M. ofn measurements. Statistical comparisons between groups wereperformed with a paired t test or with analysis of variance test forcomparison of multiple groups versus a control. Differences wereconsidered statistically significant at P, 0.05. The theoretical curvesof Ca21 loading and ATPase activity as a function of K201 dose wereobtained from the fit, using SigmaPlot10 (Systat Software Inc., SanJose, CA), of eq.1:

V ðATPase  or  Loading  rateÞ5 Vcont×½K201�fIC50 1 ½K201�g (1)

Previously, the Hill equation was used to fit the data, but theestimates of Hill coefficient (n) for K201 inhibition of SR Ca21 loadingor ATPase activity only varied from 0.76 to 1.2. The n ∼ 1 coefficientvalue in all curve fittings suggests minor, if any, cooperativity in K201action. Consequently, a Hill coefficient of n 5 1 was assumed for allcurves.

ResultsK201 Inhibits Ca21 Loading and SERCA-Mediated

ATPase Activity in Cardiac and SkM SR Microsomes.Net SR Ca21 uptake by SR microsomes represents thenumerical difference between the active, SERCA-mediated,Ca21 influx and the passive Ca21 efflux, dependent mainly onRyRs activity (Barg et al., 1997; Chamberlain et al., 1984).Figure 1A represents a sample UV recording with SkM SRmicrosomes. Under control conditions (DMSO), a Ca21 pulseproduced a rapid spike in the absorbance value, followed by aslow, gradual decrease in Ca2 as it is uptaken by the SRmicrosomes. Figure 1, A (top panel) and B, shows that K201(80 mM) decreased SR Ca21 loading rates similarly to the RyRagonist caffeine (10 mM). Figure 1D shows changes in the rateof SR Ca21 loading into SkM microsomes in response toincreasing doses of K201, from where the IC50 was estimatedat 27.5 mM. Figure 1, C and E, shows similar findings incardiac SR microsomes; K201 inhibited SR Ca21 loading withan IC50 of 40.5 mM. To discern whether K201 decreases SRCa21 loading rates by stimulating Ca21 efflux through RyR1,inhibiting Ca21 influx through SERCA or a combination ofbothmechanisms,we conducted our experiments in the absenceand presence of the well known RyR blocker RR (5 mM).Figure 1A (lower panel, RR) as well as Fig. 1, B and C (RR),show that RR did not prevent the inhibitory action of 80 mMK201 on the rate of loading while, as expected, it abolished theeffect of 10 mM caffeine. These results may indicate that K201either renders the RyRs insensitive to block by RR or that K201might directly inhibit SERCA.Effects of K201 on SERCA-Mediated ATPase Activity

of SkM and Cardiac SR Microsomes. The effects of K201on SERCA were studied by measuring ATPase activity usingan assay that follows the decrease of NADHabsorbance, whichis coupledmole tomole to the consumption of ATP by ATPases(see Materials and Methods). Figure 2 show examples ofrecordings of [NADH] versus time (A) and summary data (B)of the rate of ATPase activity. These assays were performed byincubating cardiac SR microsomes with bathing solutionscontaining either 200 mM, 2 mM, or 0.25 mM Ca21. Figure 2Aa1shows recordings with 200 mMCa21 under control conditions, inthe presence of K201 (at 4, 16, or 80 mM), as well as in the

presence of cyclopiazonic acid (CPZ), a SERCA inhibitor. Asshown, the decreases in NADH absorbance with K201 aremore similar to those under control conditions than to CPZ.Figure 2B (squares) shows that at 200 mM Ca21, whereSERCA Ca21 binding sites are saturated, K201 (at doses upto 80 mM) hadminor effects on ATPase activity of cardiac SR.Figure 2C (squares) shows analogous effects of K201 onATPase activity of SkM SR microsomes in the presence of200 mM Ca21. We estimated similar IC50 values for cardiacand SkM SERCA-mediated ATPase activities (130 and104 mM, respectively; Fig. 2, B and C). At 2 mM Ca21, whereSERCA still retains ∼90% of maximal activity (Vmax), K201was much more efficacious, as suggested in the recordings(Fig. 2Aa2) and summary data (Fig. 2, B and C, triangles). TheIC50 values estimated from these data were 17.8 mM and13.3 mM for cardiac and SkM microsomes, respectively. At0.25mMCa21, slightly above cell resting levels, K201 efficacyto inhibit SERCA increased further, and the ATPase activitywas significantly reduced at all K201 concentrations tested(Fig. 2, Aa3, B, and C, circles). IC50 values were 8.8 and 4.9 mMfor cardiac and SkM microsomes respectively. Overall, theseresults indicated that K201 is a Ca21-dependent blocker ofSERCA, having a similar potency in both SkM and cardiac SRmicrosomes. Inferring from the data, K201 inhibition ofSERCA could be further magnified under low, resting cell,Ca21 conditions (40–80 nM).K201 is a Partial Agonist of SkM RyR1 and Cardiac

RyR2. We tested the effects of K201 on RyR1-mediated SRleak from SkM TC microsomes (R4 fractions). SR microsomeswere loaded with Ca21 (∼1.5 mM Ca21/mg SR protein). Wemeasured the effects of K201 on the release (or leak) of Ca21

after the addition of 20 mM of the SERCA inhibitor CPZ.Figure 3A showed a sample assay of K201 effects on Ca21 leakat various conditions (summarized in Fig. 3B). K201 was apartially effective RyR agonist since the maximal concentra-tions of K201 tested (80 mM) did not reach the Ca21 releasevalues observed with 10 mM caffeine (Fig. 3B). K201 partialagonism on RyR1-mediated Ca21 leak was not significantlyaffected by the addition of calmodulin (1 mM) (n5 4; results notshown). K201 did not affect the action of caffeine (Fig. 3B),whereas agents that induce subconductance states (“substates”),such as ryanodine or imperatoxin-A, decreased the rate of Ca21

release since these substates decrease the current amplitude ofcaffeine-activated RyR1. Our results also demonstrated thatK201 is unable to counteract RyR1 block byRR.Moreover, K201was also unable to counteract RyR1 block by 500 mMTetracaine(n 5 3; results not shown).The effects of K201 on channel activity of RyR1 from rabbit

SkM and RyR2 from pig heart were studied after reconstitu-tion of the channels from SR membranes into planar lipidbilayers. In all experiments, we used 50 mM Ca21 (in the transchamber) at a pH of 7.4. Inmost cases, the bilayermembranewasclamped at 0 mV. For SkM RyR1, we carried out experiments inthe presence of Mg21/ATP and ∼2 mM cytosolic [Ca21]. Underthese conditions, RyR1 had been classified into two groups: highactivity (HA) channels,with openprobability, Po, ranging from0.1to0.6, and lowactivity (LA) channelswithPo,0.01 (Copello et al.,1997, 2002). Fig. 3Cpresented an example ofHARyR1 recordingsin the absence or presence of K201. Figure 3D shows summarydata indicating thatwith the addition of K201, the Po ofHA-RyR1channels increased. In contrast, LA-RyR1 remained inactive.Figure 3, E andF, shows that the increase inPo induced byK201

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in HA-RyR1 is associated with subtle changes in channelkinetics. As previously done, we fit the distribution of RyR2openings with two open and two closed dwell time components,which is an approximation (Diaz-Sylvester et al., 2011). In thepresence of K201, the two dwell open times (to1 and to2) aresimilar in value to those under control conditions. Still, theprobability of the longer openings increases (Fig. 3). K201 alsoinduces a significant decrease in the closed dwell times, tc1 (from13.9 to 3.11milliseconds) and tc2 (from 71.6 to 22.8milliseconds)(Fig. 3).Figure 4 summarizes our results with RyR2 channels. A

set of experiments was carried out in the presence of cytosolic∼5 mM [Ca21], where channels displayed moderate Po. Here,wewere able to determine that K201 at doses$5mMincreasesthe activity of RyR2 (Fig. 4, A and C, triangles). At holding

voltages of 0 mV, K201 did not affect channel currentamplitudes and did not induce any type of channel openingsto subconductance states (Fig. 4, A, D, and E). Therefore, weconclude that, under these conditions, K201 only affected RyR2channel gating characteristics. Still, the highest dose of K201we tested (80 mM; close to the maximal solubility of K201 inaqueous solutions) resulted in a Po ∼ 0.8, a value significantlylower than the Po ∼0.99 obtained with caffeine under the sameconditions (Fig. 4, A and C). We also tested the effect of K201 onchannels incubated with cytosolic [Ca21] ∼100 nM in thepresence of Mg21/ATP 5 mM. As previously reported (Copelloet al., 1997, 2002; Porta et al., 2011), RyR2 display sporadicopenings and are quiescent most of the time under theseconditions (Po , 0.01). Aliquot additions of K201 (from 1.5 to80 mM) did not alter RyR2 gating kinetics or channel activity

Fig. 1. K201 inhibits SR Ca2+ uptake. SRmicrosomes (from rabbit SkM and pig heart) were incubated in phosphate buffer containing ATP/Mg with 2 mlof K201 in DMSO (final K201 levels range from 1.5 to 80 mM) or with 2 ml of DMSO (control) and in the absence or presence of 5 mMRR or 10mM caffeine.SR Ca2+ loading was started by increasing Ca2+ in the cuvette to 30 mM. (A) An example of Ca2+ uptake by rabbit SkM microsomes measured in theabsence (top panel) or presence (lower panel) of 5 mMRR under control conditions, 80 mMK201, or 10 mM caffeine. (B and C) Effect of 80 mMK201 and of10mM caffeine on the rate of SR Ca2+ loading by rabbit SkM (B) and cardiac (C) SRmicrosomes in the absence or presence of RR; data are mean6 S.E.Mof n = 4 experiments. (D and E) Loading rates of SkM (D) and cardiac (E) SR microsomes as a function of K201 concentration. Experimental data(n = 4 experiments in each condition) as in (A) were fitted by a single exponential function from which the initial rate of Ca2+ uptake was derived (seeMaterials andMethods). From the data in (D), an IC50 of 27.56 2.4mMwas estimated for K201 in SkMmicrosomes. From the data in (E), we estimated anIC50 of 40.5 6 4.3 mM for K201 in cardiac microsomes.

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(values ofPo, 0.01were recorded even in the presence of 80mMK201; Fig. 4, B and C, filled circles). Caffeine (10 mM) was stillcapable of activating the channels to Po ∼0.9 (Fig. 4, B and C).These results indicate that K201 has no effect on RyR2-dependent Ca21 release under resting cell conditions.Previous reports indicated that K201 strengthens FKBP12.6

binding to RyR2 and FKBP12.6 is a requirement for K201-induced channel gating stabilization of RyR2 inmyocytes (Yanoet al., 2003; Wehrens et al., 2005). In vitro studies, however,suggest that K201 increasing FKBP12.6 rate of dissociationfrom RyRs (Blayney et al., 2010). Here, we tested if the effect ofK201 on RyRs could be related to FKBP12.6 dissociation. Asseen in Fig. 4D, with the addition of 20 mM K201, RyR2 Po

increased; however, after the addition of FKBP12.6 (20 mM)channel activity did not change, suggesting that the action ofK201 is independent of FKBP12.6.K201 induced subconductance states (substates) during open-

ings of purified RyR1, but only when channels are held at

negative SR voltages and not with positive voltages; Vm ∼0 mV(the putative SR voltage in cells) could not be tested due to thesymmetrical ionic conditions (Almassy et al., 2008). Most of ourexperiments were conducted at 0mV, but we occasionally testedvoltages ranging from140 to215 using our usual cations (Tris1

in cis and Ca21 in trans solution). We have already reportedthat, in our conditions, substates were rare (substate P# 0.002)both in the presence or absence of FKBP12.6 (Barg et al., 1997;Copello et al., 1997). A very low frequency of substates was alsoobserved after K201 was added. We occasionally tested voltagesranging from 140 to 215 using our usual cations (Tris1 in cisand Ca21 in trans solution) and again K201 did not induce asignificantly higher probability of substates under theseconditions.We have previously described a subpopulation of RyR2 that

gates mostly to long openings (i.e., they do not display shortflickering events) (Diaz-Sylvester et al., 2011).We performed aset of experiments using this population to increase the

Fig. 2. K201 inhibits SERCA in a Ca2+-dependent manner. (A) Decrease in NADH absorption at 340 nm as a function of time (indicative of equimolar ATPconsumption by ATPase activity) by cardiac SR microsomes and at three Ca2+ concentrations: 200 mM (a1), 2 mM (a2), and 0.25 mM (a3). As shown, K201inhibited the decrease inNADH levels (ΔNADH)more strongly at lowCa2+.On the contrary, 20mMCPZ is a strong SERCA inhibitor at all Ca2+ concentrations,which leaves only a small residual fraction of SERCA-independent ATPase activity (similar in magnitude at the three [Ca2+] concentrations tested). (B and C)ATPase activity of cardiacSRmicrosomes (B) andSkMR2microsomal fraction enriched in longitudinal tubule (C) as a function ofK201 concentrations (rangingfrom 1.5 to 80 mM). Dose-response curves to K201 were built at 200 mM (squares) 2 mM (triangles), and 0.25 mM Ca2+ (circles). Data are shown asmeans 6 S.E.M of n = 4 experiments. From the data, we estimated K201 IC50 for SERCA at high Ca2+ concentrations (130 6 11.8 mM and 104 610 mM, respectively, for cardiac and SkM), mid- Ca2+ levels (17.8 6 1.5 mM and 13.3 6 2.5 mM), and low Ca2+ (8.8 6 1.0 mM and 4.9 6 0.7 mM).

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Fig. 3. K201 activates skeletal muscle RyR1. (A) Ca2+ leak from SkMmicrosomes (R4 fractions enriched in terminal cisternae) was induced by blockingthe SERCApumpwith cyclopiazonic acid (CPZ, 20mM).Measurements were performed under control conditions and upon the addition of 0.4, 2, 4, 16, and80 mMK201. SR Ca2+ leak was also measured in the presence of the RyR1 agonist caffeine (10 mM) or with 10 mMRR (which inhibits RyR1). (B) SR Ca2+

leak rate in the presence of K201 versus control (n = 4 experiments in each condition). Leak rate significantly increased from control values by 107%6 8%in the presence of 80mMK201. Still, the leak increased by 292%6 25%with caffeine, a significantly stronger agonist thanK201. Note also that K201 doesnot interfere with the effects of caffeine. RR significantly decreased the rate of SR Ca2+ leak, in which the addition of K201 has no effects. (C) K201 (40 mMbut not 2 mM) activated a skeletal HA RyR1 reconstituted into planar lipid bilayers from TC microsomes. Recordings were carried out at 0-mVtransmembrane voltage with 2 mMcytosolic free Ca2+ concentration and 5mM ofMg2+/ATP (for details, seeMaterials andMethods). Openings are shownas discrete upward deflections of the current. (D) Dose-response of K201 action on LA (n = 4) and HA RyR1 (n = 5) in planar lipid bilayers. Values aremeans 6 S.E.M. *P , 0.05 compared with absence of K201 (n = 4–6 paired experiments). For HA, we estimated an EC50 of 29.0 6 3.5 mM. (E and F)Histograms for open and closed dwell-time distribution (left and right charts, respectively) were obtained from 4-minute recordings of channels undercontrol conditions (black outlines) and in the presence of 40 mMK201 (gray outlines). Two exponential components were used to fit openings and closures.Under control conditions, dwell open times were to1 = 3.526 0.08millisecond (71%6 4%) and to2 = 26.86 0.2millisecond (29%6 4%). Closed times were

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visibility of any K201-induced changes in conductance. Evenminor K201 effects would be visible when minimizing noisefrom flicker short openings/closures, which is found undercontrol conditions in most other RyRs (Copello et al., 1997;Diaz-Sylvester et al., 2011). We used Cs1 (instead of Tris1) inour cytosolic (cis) solution to increase RyR2 channel amplitudeat 0 and 215 mV, as well as to generate measurable cis-to-trans Cs1 currents (i.e., 2.3 pA cytosol to lumen) at the SRvoltage (Vm) of 260 mV. Figure 4E shows current-amplitudeplots that do not evidence any K201 induced change inconductance, substates, or increase in flickering in the rangeof 10 mV to 215 mV. No substates were observed with K201at 120 and 140 mV (results not shown). In the amplitudedistribution of channel openings (filtered at 500 Hz), welldefined open and closed conductance states generate twoGaussian distributions. The use of different scales at differentvoltages (Fig. 4E) makes the standard deviation of thedistributions appear to increase at negative voltages; how-ever, this visual effect resulted from a decreased signal(decreasing RyR2 current amplitude from 6.5 to 3.6 and2.3 pA at 0, 215, and 260 mV, respectively) to constant noise(the intrinsic electrical noise of the bilayer system after500-Hz filtering). Still, it is clear that the probability ofintermediate states is negligible (∼0), with a Vm of 0 mVand 215 mV (Fig. 4E). This is in agreement with all otherrecordings at 0 mV using Tris1 in our cytosolic solutions. Incontrast, the high frequency of flicker closures and interme-diate states was observed at 260 mV in the presence of highK-201 levels. This flicker block correlates with a ∼10% probabil-ity of intermediate states of conductance (Fig. 4E), which is aneffect of filtering very short-lived closures, as well as, somesubstates (Colquhoun and Sigworth, 1995). Thus, our observa-tion of K201 having a blocking action atVm5260mV coincides,in part with previous findings (Almassy et al., 2008) andemphasizes the fact thatK201has a complexaction that includesvoltage-dependent components; however, that we found nosignificant increase in flickering events when Vm $ 215 mV(Fig. 4E), which includes 0 mV, the putative SR voltage in cells(Mejía-Alvarez et al., 1999; Fill and Copello, 2002).

DiscussionWe found that K201 blocks SERCA ATPase activity with

similar characteristics and affinity in cardiac and SkM SRmicrosomes. K201 acts as a Ca21-dependent SERCA blocker,which has increased potency when Ca21 levels decrease tothose observed in resting cells. K201 also acts as a partialRyR1 and RyR2 agonist, which would increase the Po ofpartially active channels but would not activate channels thatare in the closed state (such as LA RyR1 or RyR2 under Ca21

resting conditions). K201 also induces RyR2 flicker block atnegative SR voltages, which has unclear significance for thedrug action in the cellular environment.Ca21-Dependent Blocker of SERCA is a Novel Char-

acteristic of K201. With incubating Ca21 levels of 100, 2,and 0.25 mM, we estimated that the corresponding IC50 valuesof K201 ATPase inhibition were within ∼130–104, 18–13, and

9–5 mM (cardiac vs. SkM respectively). Previous studies withcardiomyocytes have shown that K201 induces SR Ca21

depletion (Inagaki et al., 2000; Loughrey et al., 2007; Chenet al., 2008). The decrease in SR content could be attributed toother K201 targets in plasmalemma, including block of L-typeCa21 channels (Kimura et al., 1999; Inagaki et al., 2000) and/or inhibition of SERCA-mediated Ca21 uptake (Inagaki et al.,2000; Loughrey et al., 2007). Indeed, K201 (3 mM) inhibited by∼30% the Vmax of Ca

21 uptake in aggregates of permeabilizedcardiomyocytes, which was measured in the range of 0.2 and2.5 mM Ca21 (Loughrey et al., 2007). These data correlaterelatively well with the results we obtained when measuringATPase activity carried out in the presence of 2 mM Ca21,indicating that SERCA is also an important cellular target forK201. Our data suggest that K201 potency would be evenhigher to inhibit SERCA ATPase activity in the presence of0.05 mM Ca21 (resting cell levels).Many SERCA blockers have been previously identified,

including the widely used thapsigargin and cyclopiazonic acid;however, most display much milder Ca21 dependency in theireffects on ATPase activity, despite the marked Ca21 depen-dence of normal SERCA function (Seidler et al., 1989;Wuytack et al., 2002; Zafar et al., 2008; Michelangeli andEast, 2011; Soler et al., 2012). It is possible that themechanism of action of K201’s is similar to that ofepigallocatechin-3-gallate (EGCG), a drug that also showsmarked Ca21 dependence in its SERCA block, attributed toEGCG interfering with Ca21 binding being counteracted byaccumulation of Ca21-bound SERCA conformations with in-creasing Ca21 levels (Soler et al., 2012). Various benzothiaze-pines, including K201, are known to target cellular paths thatmodulate intracellular Ca21 homeostasis, as previously dis-cussed (Neumann et al., 2011). Still, there is only one reportfrom our group where CGP37157 (which is a 4,1 benzothiaze-pine) was found to be a SERCAblocker (Neumann et al., 2011).More recent ATPase testing has revealed that CGP andvarious CGP analogs are also Ca21 dependent SERCA block-ers (Darcy YL and Copello JA, unpublished results).K201 inhibits SERCA1a (main isoform in fast twitch SkM)

and SERCA2a (main isoform in heart SR microsomes) withsimilar potency, suggesting that the action of K201 is notmediated by phospholamban, which is not expressed in thefast-twitch SkM SR used here (Jorgensen and Jones, 1986);however, a previous study conducted with SkM SR micro-somes found an IC50∼110mM for K201 inhibition of SERCA1aATPase activity in 2 mM Ca21 (Almassy et al., 2008). Thesevalues are almost 10 times higher compared with the resultsreported here and the estimates of IC50 based on extrapolationfrom the effect of K201 on SERCA mediated Ca21 uptake incardiomyocytes (Inagaki et al., 2000; Loughrey et al., 2007).The cause of this discrepancy is unknown, but the pH5 7.5 inAlmassy et al. (2008) was much higher than in our experi-ments and in the cytosol of cells (pH ∼7.0), which may affectthe action of K201.K201 Modulation of RyR1 and RyR2: A Review of the

Literature. Studies with SkM SR microsomes suggest thatK201 increases RyR1-mediated SR leak, which was prevented

tc1 = 13.9 6 0.1 millisecond (63%6 4%) and tc2 = 71.6 6 0.2 millisecond (37% 6 4%). For 40 mMK201, dwell open times were similar but had differentprobability: to1 = 4.15 6 0.11 millisecond (38% 6 3%) and to2 = 27.6 6 0.2 millisecond (62% 6 3%). In contrast, dwell closed times were significantlyshorter and distributed with tc1 = 3.11 6 0.13 millisecond (51% 6 3%) and tc2 = 22.8% 6 1.2 milliseconds (49% 6 4%).

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by RyR1 blockers (RR and tetracaine). K201 is clearly a lesspotent agonist of RyRs than caffeine, and its action was notsignificantly affected by the addition of calmodulin. Ourbilayer data indicate that K201 preferentially acts on chan-nels that are moderately active (RyR2 channels incubatedwith diastolic Ca21 levels or HA RyR1 channels). In contrast,K201 does not affect channels that are in the closed state(RyR2 under resting Ca21 conditions or LA RyR1). Our dataalso indicate that K201 would have no direct effects on RyR2at doses that have been commonly used in cellular studies(0.3–2 mM). Nonetheless, K201 is a positively charged and cellpermeablemolecule. Because of more negative potential of thecytosol (280 to 290 mV), K201 could accumulate there at10–20 times higher concentrations than those added to thebathing solution.

Some aspects of K201-mediated RyR2 stabilization andprevention of SR leak are controversial, including the ideasthat K201 promotes FKBP12.6 binding to the channels and/ormodulates phosphorylation status (Yano et al., 2003;Wehrenset al., 2005; Xiao et al., 2007; Blayney et al., 2010; Sachereret al., 2012). In our hands, the effects of K201 on RyR2 openprobability were not directly affected by adding FKBP12.6,which is in line with our previous reports of FKBP12.6 notbeing a direct modulator of RyR2 (Barg et al., 1997; Xiao et al.,2007). Still, there are various conflicting opinions on the directmodulation of RyRs by FKBPs (Fill and Copello, 2002).Furthermore, our results do not rule out the possibility of anindirect modulation of RyR-FKBP interactions by K201.A previous study conducted on reconstituted purified

RyR1 under full activation conditions (which allows only

Fig. 4. K201 activates cardiac RyR2. (A and B) Single-channel recordings of pig cardiac RyR2 at a SRmembrane voltage (Vm) of 0 mV. Luminal solutionand cytosolic solutions are similar to those described in Fig. 3. Cytosolic solutions contained 5 mM ATP-Mg2+ (0.5 mM free Mg2+) and were buffered withBAPTA/dibromoBAPTA plus variable [Ca2+] to attain a final free [Ca2+] of either 5 mM (A) or 100 nM (B). Top panels show 2-second segments of channelactivity and Po values estimated from 4-minute recordings under control conditions. Middle panels show the effect of the addition of 80 mM K201. Thebottom panels show the effect of the addition of caffeine (10 mM). (C) Dose responses of RyR2 channel Po to K201 (1.5–80 mM) in the presence of 5 mM(triangles) or 100 nM Ca2+ (circles). Recordings and summary data show that K201 activates RyR2 when channels are incubated with 5 mM Ca2+ but isineffective when channels are closed. In contrast, caffeine (10 mM) was highly effective under both conditions. (D) Example of K201 (20 mM)-inducedactivation of RyR2 at 5 mM Ca2+ and lack of effect of FKBP12.6 (20 mM). Po values were obtained from a 16-minute recording in each condition. Thisexperiment is representative of n = 4 experiments. (E) Effects of K201 on RyR2 recorded at 0, 215, and 260 mV. Traces are examples of 1-secondrecordings of RyR2 under control conditions and in the presence of 80 mMK201. On the right side of the traces, we show current amplitude distributionsobtained from 2-minute recordings. In most cases, except K201 at260 mV, the distributions can be fit with two Gaussian distributions (one for the openand one for the closed state). For K201 at 260 mV, however, there is a fraction of intermediate events that represent the flicker closures and substatesthat reached an average probability of 10.5% 6 2.9% (n = 4 experiments).

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measurement of the effect of antagonist/blockers) reportedthatK-201 induced conductance substates in a voltage-dependentmanner and at negative but not at positive SR voltages (Almassyet al., 2008). Here, we used a different model (RyRs reconstitutedfrom native SR microsomes) and experimental conditions whereRyR1 and RyR2 had low or moderate activity (which alloweddetecting agonistic effects).Under these conditions,K201 acted asa mild agonist, and we did not distinguish any blocking events orsubstates within the range of140 to215 mV. However, at a SRvoltage of 260 mV (similar as in Almassy et al., 2008), weobserved flicker block and intermediate states of conductance(which could be attributed to short-lived substates) in RyR2inducedbyK201. The blocking effect ofK201 is intriguing, but therole of voltage dependence in RyRs has unknown physiologicsignificance as channels are thought to operate at a nearlyconstant SR voltage of ∼0 mV (Fill and Copello, 2002;Diaz-Sylvester et al., 2011).Only a few studies tested the direct effects of K201 on RyR2.

Two articles reported a minor decrease in [3H] ryanodinebinding by SR microsomes (which is used as an index of RyRsactivity) induced by 1 or 50 mM K201 (Hunt et al., 2007;Blayney et al., 2010). In principle, these differences with ourresults may relate to the lack of ATP/Mg21 and the very lowluminal Ca21 in the SR in [3H] ryanodine binding studies,which made RyRs less sensitive to agonists (Sitsapesan andWilliams, 1990; Ogawa, 1994; Fill and Copello, 2002). It ispossible that the experimental conditions used in the[3H] ryanodine binding studies are similar to those in ourlow cytosolic Ca21 conditions, where K201 is not effective asan agonist.A SERCA Role for the Cell-Protective Action of

K201? There is consensus that the “normalization” ofEC-coupling induced by K201 correlates with improvedcardiovascular cell function in pathologies, including arrhyth-mia and heart failure (Yano et al., 2003; Wehrens et al., 2005;Loughrey et al., 2007; Toischer et al., 2010; Driessen et al.,2014; Sedej et al., 2014; Kim et al., 2015). K201 also benefitsischemic SkM (Wehrens et al., 2005). Still, K201mechanism ofaction is unclear, as it is apparent that there aremany targets,including Ca21 and K1 channels, that may contribute to thecellular actions of the drug (Driessen et al., 2014).We think K201 stabilizes RyR2 through modulation of

SERCA. It is well known that RyRs activity heavily dependson luminal Ca21 levels (Sitsapesan andWilliams, 1998; Fill andCopello, 2002; Terentyev et al., 2002; Diaz-Sylvester et al., 2011;Porta et al., 2012). Although the effects of K201 on SR Ca21

content appear to be mild under control conditions (Loughreyet al., 2007), the drug could have more significant effects underpathologic situations as in ischemia. During ischemia, there isslow Ca21 accumulation in the SR, which after many cycles(minutes) produces significant SR overload (Murphy andSteenbergen, 2008; Said et al., 2008; Valverde et al., 2010).Mild K201-induced SERCA block, largely innocuous undernormal conditions, may be enough to help prevent SR overloadand the abnormal events (sparks, waves, and massive Ca21

release) observed in early reperfusion, which are associatedwith cardiac tissue damage (Talukder et al., 2009; Valverdeet al., 2010; Garcia-Dorado et al., 2012; Mattiazzi et al., 2015).Supporting this hypothesis, K201 is capable to stabilize RyR2only if added before (not after) ouabain-induced SR overload(Sedej et al., 2014); this would be expected if the target isSERCA, not the RyR2.

In conclusion, K201 is a novel Ca21 dependent inhibitor ofSERCA that may be highly potent under cell resting condi-tions and may play an important role in preventing restingRyR-mediated SR Ca21 leak in muscle and heart pathologies.K201 also modulates RyRs, acting as an agonist at theputative SR voltage, but with much lower potency under cellresting conditions.

Acknowledgments

The authors thank Dr. Sidney Fleischer for kindly providingpurified FKBPs and SkM SRmicrosomes; V. A. Copello for comments;L. Moss, Pharmacology Graduate Student Representative; and G.Stauffer, Office Administrator, SIU-SOM, for proofreading themanuscript.

Authorship Contributions

Participated in research design: Darcy, Diaz-Sylvester, Copello.Conducted experiments: Darcy, Diaz-Sylvester, Copello.Contributed new reagents or analytic tools: Copello.Performed data analysis: Darcy, Diaz-Sylvester, Copello.Wrote or contributed to the writing of the manuscript: Darcy,

Diaz-Sylvester, Copello.

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