Loss of luminal Ca2� activation in the cardiacryanodine receptor is associated with ventricularfibrillation and sudden deathDawei Jiang, Wenqian Chen, Ruiwu Wang, Lin Zhang, and S. R. Wayne Chen*
Department of Physiology and Biophysics, University of Calgary, Calgary, AB, Canada T2N 4N1
Edited by David H. MacLennan, University of Toronto, Toronto, Canada, and approved September 19, 2007 (received for review July 13, 2007)
Different forms of ventricular arrhythmias have been linked tomutations in the cardiac ryanodine receptor (RyR)2, but the mo-lecular basis for this phenotypic heterogeneity is unknown. Wehave recently demonstrated that an enhanced sensitivity to lumi-nal Ca2� and an increased propensity for spontaneous Ca2� releaseor store-overload-induced Ca2� release (SOICR) are common de-fects of RyR2 mutations associated with catecholaminergic poly-morphic or bidirectional ventricular tachycardia. Here, we investi-gated the properties of a unique RyR2 mutation associated withcatecholaminergic idiopathic ventricular fibrillation, A4860G. Single-channel analyses revealed that, unlike all other disease-linked RyR2mutations characterized previously, the A4860G mutation dimin-ished the response of RyR2 to activation by luminal Ca2�, but hadlittle effect on the sensitivity of the channel to activation bycytosolic Ca2�. This specific impact of the A4860G mutation indi-cates that the luminal Ca2� activation of RyR2 is distinct from itscytosolic Ca2� activation. Stable, inducible HEK293 cells expressingthe A4860G mutant showed caffeine-induced Ca2� release butexhibited no SOICR. Importantly, HL-1 cardiac cells transfected withthe A4860G mutant displayed attenuated SOICR activity comparedwith cells transfected with RyR2 WT. These observations providethe first evidence that a loss of luminal Ca2� activation and SOICRactivity can cause ventricular fibrillation and sudden death. Thesefindings also indicate that although suppressing enhanced SOICRis a promising antiarrhythmic strategy, its oversuppression can alsolead to arrhythmias.
spontaneous Ca2� release � sudden cardiac death � ventricular arrhythmia
The cardiac ryanodine receptor (RyR)2 is an intracellular Ca2�
release channel located in the sarcoplasmic reticulum (SR) thatplays an essential role in excitation–contraction coupling and SRCa2� homeostasis in cardiac muscle (1). Mutations in RyR2 causeseveral ventricular arrhythmias, including catecholaminergic poly-morphic ventricular tachycardia (CPVT) or bidirectional ventricu-lar tachycardia (VT), catecholaminergic idiopathic ventricular fi-brillation, and arrhythmogenic right ventricular dysplasia type 2(2–6). To date, more than 60 disease-associated RyR2 mutationshave been identified (7–9). An important unresolved question ishow each of these mutations alters the function of RyR2 and causesventricular arrhythmia.
The impact of disease-associated RyR2 mutations likely involvesthe physiological function of RyR2. SR Ca2� release and excita-tion–contraction coupling, both of which are believed to take placevia a mechanism known as Ca2�-induced Ca2� release (1). In thisprocess, a small Ca2� influx through the voltage-dependent L-typeCa2� channel during membrane depolarization opens the RyR2channel, leading to a large Ca2� release from the SR and subse-quent muscle contraction. Besides this depolarization-inducedCa2� release, spontaneous SR Ca2� release can occur in theabsence of membrane depolarization during SR Ca2� overload(10–14). Considering its dependence on the SR Ca2� store, we referto this spontaneous SR Ca2� release as store-overload-inducedCa2� release (SOICR) (15, 16). It is well known that SOICR canresult in delayed afterdepolarizations (DADs), which, in turn, can
lead to triggered arrhythmias (17). It is also known that CPVT istriggered by emotional and physical stresses, conditions that caninduce SR Ca2� overload. These observations suggest that CPVTis likely to be caused by SR Ca2� overload-induced DADs (9, 18).In support of this view, we have recently shown that a number ofRyR2 CPVT mutations enhance the sensitivity of the channel toactivation by luminal Ca2� and increase the propensity for SOICR(15, 16). Furthermore, DADs were readily observed in cardiacmyocytes isolated from mice harboring an RyR2 CPVT mutation,R4496C. These R4496C knockin mice, like patients with CPVT,display typical polymorphic or bidirectional VT in response tocatecholaminergic stimulation (19, 20). Some RyR2 mutations havealso been shown to alter the sensitivity of the channel to caffeine,cytosolic Ca2�, or �-adrenergic stimulation (21–24). Taken to-gether, these findings indicate that enhanced sensitivity to variousstimuli is a common defect of CPVT RyR2 mutations.
Electrophysiological studies have revealed that not all patientsharboring RyR2 mutations show the characteristic, stress-inducedpolymorphic/bidirectional VT. Some patients, such as those withthe A4860G mutation, display catecholaminergic idiopathic ven-tricular fibrillation (4), suggesting that the A4860G mutation maypossess properties different from those of CPVT mutations that arelinked to polymorphic/bidirectional VT. To test this possibility, inthe present study, we systematically characterized the A4860Gmutation at the molecular and cellular levels. We found that theA4860G mutant diminished the luminal Ca2� activation of RyR2and SOICR but retained a normal sensitivity to activation bycytosolic Ca2�. Our data provide insight into the mechanism ofactivation of RyR2 by luminal Ca2� and demonstrate that ventric-ular arrhythmia can result not only from gain-of-function mutationsbut also from a loss-of-function mutation in RyR2.
ResultsThe A4860G Mutation Diminishes the Response of Single RyR2 Chan-nels to Luminal Ca2�. The A4860G mutation is located in the poreinner helix, which is thought to play an essential role in theactivation and gating of RyR2 (25). To assess whether the A4860Gmutation alters the Ca2�-dependent activation of the channel, weincorporated single RyR2 WT and A4860G mutant channels intoplanar lipid bilayers and examined their responses to luminal andcytosolic Ca2�. Fig. 1 shows the response of single RyR2 WT and
Author contributions: D.J. and W.C. contributed equally to this work; D.J., W.C., R.W., andS.R.W.C. designed research; D.J., W.C., R.W., and L.Z. performed research; D.J., W.C., R.W.,L.Z., and S.R.W.C. analyzed data; and S.R.W.C. wrote the paper.
A4860G mutant channels to luminal Ca2�. A single RyR2 WTchannel exhibited little activity in the presence of 45 nM cytosolicCa2�, 2.5 mM cytosolic ATP, and 45 nM luminal Ca2� (Fig. 1Aa).Raising the luminal Ca2� concentration from 45 nM to 300 �Mmarkedly increased the open probability (Po) of the RyR2 WTchannel (Fig. 1A b–d). For instance, at 100 �M luminal Ca2�, theaverage Po of single RyR2 WT channels was 0.28 � 0.08 (mean �SEM, n � 9) (Fig. 1C). We have recently shown that a number ofdisease-causing RyR2 mutations enhance the luminal Ca2� re-sponse of the channel (16). Surprisingly and in contrast, singleA4860G mutant channels hardly responded to increasing luminalCa2� concentrations (Fig. 1B). The average Po of single A4860Gmutant channels remained low (� 0.02) at luminal Ca2� concen-trations ranging from �100 nM to 50 mM (n � 10) (Fig. 1D). Theseobservations demonstrate that the A4860G mutation dramaticallydiminishes the sensitivity of the RyR2 channel to activation byluminal Ca2�.
Single A4860G Mutant Channels Retain a Normal Sensitivity to Cyto-solic Ca2� Activation. The response of single RyR2 WT and A4860Gmutant channels to cytosolic Ca2� is shown in Fig. 2. Both the WTand mutant channels were activated by cytosolic Ca2� with athreshold of �100 nM. Further increases in cytosolic Ca2� mark-edly increased the Po of the WT channel to near unity and that ofthe mutant channel to �0.8. Analysis of all data points from anumber of single WT and mutant channels by using the Hillequation yielded an EC50 value of 0.31 �M (n � 16) for RyR2 WTand 0.35 �M for the A4860G mutant (n � 12). Hence, despite anearly complete inhibition of luminal Ca2� activation, the A4860Gmutation had little effect on the sensitivity of the channel toactivation by cytosolic Ca2�. However, the A4860G mutation did
alter the sensitivity of the channel to inactivation by high concen-trations of cytosolic Ca2�. As shown in Fig. 2C, single A4860Gmutant channels were inactivated by cytosolic Ca2� with an IC50 of0.97 mM (n � 13), which was �4-fold lower than the IC50 value (3.6mM, n � 17) of single RyR2 WT channels.
The A4860G Mutation Alters the Gating Properties of Single RyR2Channels. Single RyR2 WT and A4860G mutant channels alsodiffered markedly in their gating kinetics. Long opening eventswere readily observed in single RyR2 WT channels but not in singleA4860G mutant channels, especially at activating Ca2� concentra-tions between 1 and 100 �M (compare Fig. 2A b–d with Fig. 2Bb–d). Further kinetic analysis revealed that single A4860G mutantchannels differed from single RyR2 WT channels mainly in theirmean open times. Although both RyR2 WT and the A4860Gmutant channels displayed a bell-shaped relationship betweenmean open times and cytosolic Ca2� concentrations (Fig. 3A), theaverage maximum mean open time (�1 ms) of single A4860Gmutant channels was �10-fold shorter than the average maximummean open time of single RyR2 WT channels (�10 ms) (Fig. 3A).However, the mean closed times of single WT and mutant channelsat different cytosolic Ca2� concentrations were comparable, withboth displaying an inverted bell-shaped relationship (Fig. 3B).These observations indicate that the A4860G mutation markedlyshortens the duration of channel openings.
The A4860G Mutation Inhibits the Basal Activity of RyR2. We havedemonstrated that disease-causing RyR2 mutations located in theCOOH-terminal region enhance both the luminal Ca2� activationand basal activity of RyR2 (15). To test whether the basal activityof RyR2 is altered by the A4860G mutation, we carried out
Fig. 1. The A4860G RyR2 mutation abolishes theluminal Ca2� activation of single RyR2 channels. (A andB) Single-channel activities of RyR2 WT (A) and theA4860G mutant (B) were recorded in a symmetricalrecording solution containing 250 mM KCl and 25 mMHepes (pH 7.4) at a holding potential of �20 mV. EGTAwas added to either the cis or trans chamber to deter-mine the orientation of the incorporated channel. Theside of the channel to which the addition of EGTAinhibited the activity of the incorporated channel pre-sumably corresponds to the cytosolic face of the chan-nel. The Ca2� and ATP concentrations on the cytosolicface of the channel were adjusted to �45–62 nM and2.5 mM, respectively. The luminal Ca2� concentrationwas increased to various levels by the addition of ali-quots of CaCl2 solution. Single-channel current tracesof RyR2 WT at 45 nM (a), 3.5 �M (b), 50 �M (c), and 300�M (d) luminal Ca2� are shown in A. Single-channelcurrent traces of the A4860G mutant at 300 �M (a), 2.5mM (b), 10 mM (c), and 30 mM (d) luminal Ca2� areshown in B. Openings are downward. Po, arithmeticmean open time (To), and arithmetic mean closed time(Tc) are indicated at the top of the traces. A short lineto the right of each current trace indicates the baseline.(C and D) The relationship between Po and luminalCa2� concentration is shown for RyR2 WT (C) and theA4860G mutant (D). The data points shown aremeans � SEM from nine single RyR2 WT (C) and 10single A4860G mutant channels.
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[3H]ryanodine binding to RyR2 WT and the A4860G mutant in thepresence of very low concentrations of Ca2� (�3 nM; pCa � 8.49)and increasing concentrations of KCl. As shown in Fig. 3C, elevat-ing the concentration of KCl in the binding solution from 50 to 1,000mM increased the basal level of [3H]ryanodine binding to RyR2WT to 13.2 � 1.3% (mean � SEM, n � 7) of the maximum binding,whereas the basal level of [3H]ryanodine binding to the A4860Gmutant was 3.1 � 0.8% (n � 3) at 1 M KCl, significantly lower thanthat of RyR2 WT (P � 0.002). Similarly, elevating the concentra-tion of sucrose in the binding solution from 200 mM to 1.5 Msubstantially increased the basal level of [3H]ryanodine binding toRyR2 WT to 54.8 � 0.5% (mean � SEM, n � 3), whereas the basallevel of [3H]ryanodine binding to the A4860G mutant at 1.5 Msucrose was 15.6 � 0.3% (n � 3), significantly lower than that ofRyR2 WT (P � 0.0001) (Fig. 3D). These results indicate that theA4860G mutation, unlike other CPVT mutations, inhibits the basalactivity of RyR2.
The A4860G Mutation Abolishes SOICR in HEK293 Cells. Given theclose link between SOICR and the luminal Ca2� activation of
RyR2, diminished luminal Ca2� activation would be expected toabolish SOICR. To test this hypothesis, we generated stable,tetracycline-inducible HEK293 cell lines expressing either RyR2WT or the A4860G mutant and assessed their SOICR activity. Thecells were perfused with increasing extracellular Ca2� ([Ca2�]o) (0to 1.0 mM) to induce Ca2� overload. The resultant SOICR wasmonitored by using a fluorescent Ca2� dye, fura-2 acetoxymethylester, and single-cell Ca2� imaging. As shown in Fig. 4, Ca2�
oscillations occurred in RyR2 WT cells at �0.3 mM [Ca2�]o (Fig.4 A and B), and the number of Ca2� oscillating cells increased withincreasing [Ca2�]o (Fig. 4C). In contrast, HEK293 cells expressingthe A4860G mutant displayed no Ca2� oscillations (Fig. 4 A–C).However, despite their lack of SOICR, the A4860G mutant cellsretained caffeine-induced Ca2� release (Fig. 4B) and showed anincreased store Ca2� content compared with the RyR2 WT cells(152.1 � 6.4%, P � 0.0001) (Fig. 4D), suggesting an increasedthreshold for SOICR. It should be noted that the expression levelsof RyR2 WT and the A4860G mutant were comparable, asrevealed by Western blot analysis (data not shown).
Fig. 2. The cytosolic Ca2� response of single RyR2 WTand A4860G mutant channels. (A and B) Single-channel activities of the RyR2 WT (A) and the A4860Gmutant (B) were recorded as described in the legend ofFig. 1. Single-channel current traces for RyR2 WT at 191nM (a), 223 nM (b), 317 nM (c), 500 nM (d), 6.2 mM (e),and 21.2 mM ( f) cytosolic Ca2� are depicted in A.Single-channel current traces for the A4860G mutantat 191 nM (a), 275 nM (b), 440 nM (c), 3.5 �M (d), 2.5mM (e), and 5.0 mM ( f) cytosolic Ca2� are shown in B.The luminal Ca2� concentration under various condi-tions in A and B was 45 nM. The holding potential was�20 mV, except for that shown in Af (�30 mV). Open-ings are downward, and the baselines are indicated. Po,To, and Tc are shown at the top of the traces. (C) Therelationship between Po and cytosolic Ca2� concentra-tion of single RyR2 WT (filled circles) and single A4860Gmutant (open circles) channels is shown. The datapoints shown are from 17 single RyR2 WT channels and13 single A4860G mutant channels.
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HL-1 Cardiac Cells Transfected with A4860G Display Attenuated SOICRActivity. To investigate whether the A4860G mutation exerts itseffect on SOICR in the context of cardiac myocytes, we transfectedHL-1 cardiac cells, a mouse atrial cell line, with a GFP-tagged RyR2WT or GFP-tagged A4860G mutant and assessed their SOICRactivity by using single-cell Ca2� imaging. Fig. 5 shows that HL-1cells transfected with the GFP-tagged A4860G mutant (Fig. 5 B andC) exhibited a reduced propensity for SOICR compared with HL-1cells transfected with the GFP-tagged RyR2 WT (P � 0.005) (Fig.5 A and C). These results indicate that the A4860G mutation canalso suppress SOICR in cardiac cells.
DiscussionIt is generally believed that RyR2-associated CPVT is triggered byDADs induced by SR Ca2� overload because of enhanced RyR2and SOICR activity. In line with this belief, all of the disease-linkedRyR2 mutants characterized previously display an increased sen-sitivity to activation by various stimuli (7–9). Thus, it has hithertobeen assumed that RyR2-associated CPVT is associated withgain-of-function mutations. However, different forms of ventriculararrhythmias have been observed in patients with different RyR2mutations (4), suggesting that multiple mechanisms for RyR2-associated ventricular arrhythmias may exist. In the present study,we provide the first evidence that a loss-of-function RyR2 mutationis associated with ventricular fibrillation and sudden death. Wedemonstrated that the A4860G mutation eliminated luminal Ca2�
activation but had little impact on the sensitivity of the channel tocytosolic Ca2� activation. We further showed that the A4860Gmutation markedly attenuated SOICR but maintained caffeine-induced Ca2� release. The specific effect of the A4860G mutationon luminal Ca2� activation indicates that luminal Ca2� activation isdistinct from the cytosolic Ca2� activation of RyR2. Our results alsoindicate that, like enhanced SOICR, attenuated SOICR is also
arrhythmogenic. Therefore, oversuppression of SOICR can lead tocardiac arrhythmias, and an appropriate approach to the treatmentof RyR2-associated ventricular arrhythmias would be to normalizethe SOICR activity, rather than to eliminate it.
How Do Depressed Luminal Ca2� Activation and SOICR Lead toVentricular Arrhythmia? Based on the close link between SOICRand DADs, it has been proposed that gain-of-function RyR2mutations cause CPVT by enhancing SR Ca2� leak or SOICR,consequently increasing the occurrence of DADs and triggeredarrhythmia (7–9). A loss-of-function RyR2 mutation would beexpected to suppress SR Ca2� leak and SOICR, thus decreasing theoccurrence of DADs. How then could a lack of luminal Ca2�
activation and SOICR lead to ventricular arrhythmia and suddendeath?
A growing body of evidence indicates that SR Ca2� release plays
Fig. 3. Effect of the A4860G mutation on channel gating and [3H]ryanodinebinding. (A and B) The arithmetic mean open times (A) and the arithmeticmean closed times (B) for RyR2 WT (filled circles) and the A4860G mutant(open circles) channels at various cytosolic Ca2� concentrations are shown. Themean open and closed times for the RyR2 WT and the A4860G mutantchannels were obtained from analyses of the single-channel data shown inFig. 2. (C and D) [3H]ryanodine binding to cell lysate prepared from HEK293cells expressing RyR2 WT (filled circles) and the A4860G mutant (open circles)was carried out at �3 nM Ca2� (pCa � 8.49), 5 nM [3H]ryanodine, and variousconcentrations of KCl (50–1,000 mM) (C) or at �3 nM Ca2� (pCa � 8.49), 100mM KCl, 5 nM [3H]ryanodine, and various concentrations of sucrose (200–1,500 mM) (D). The [3H]ryanodine binding shown in C was normalized to thebinding measured in the presence of 800 mM KCl and 100 mM Ca2�, whereasthe [3H]ryanodine binding shown in D was normalized to the binding ob-tained at 100 mM KCl, 1.2 M sucrose, and 100 �M Ca2�. The data points shownare means � SEM from three separate experiments. Fig. 4. HEK293 cells expressing the A4860G mutant display no SOICR activity.
Stable, inducible HEK293 cells expressing RyR2 WT or the A4860G mutant wereinduced with tetracycline and loaded with 5 �M fura-2 acetoxymethyl ester inKrebs–Ringer–Hepes buffer for 20 min at room temperature. The cells werecontinuously perfused with Krebs–Ringer–Hepes buffer without (0 mM) or with0.1, 0.2, 0.3, 0.5, or 1.0 mM CaCl2 or with 1.0 mM CaCl2 plus 5 mM caffeine (caff).(A) Single-cell fluorescent Ca2� images of HEK293 cells expressing RyR2 WT(Upper) or the A4860G mutant (Lower) at various [Ca2�]o (0–1.0 mM). Fura-2ratios of representative RyR2 WT and A4860G mutant cells are shown in B. (C) Thefraction (%, mean � SEM) of RyR2 WT (open circles) and A4860G mutant (filledtriangles) cells that displayed Ca2� oscillations at various [Ca2�]o. The total num-bersofcellsanalyzedforCa2� oscillationswere150forA4860Gand1,420forRyR2WT from 3 to 16 separate experiments. (D) The frequency of Ca2� oscillations andthe store Ca2� level in HEK293 cells expressing RyR2 WT or the A4860G mutant.The frequency of oscillations was estimated from the number of peaks, whereasthe store Ca2� content was determined by measuring the amplitude of caffeine(5 mM) induced Ca2� release in the presence of 1.0 mM [Ca2�]o. Values werenormalized to the RyR2 WT level (100%). The data shown are means � SEM from3 to 16 separate experiments.
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an important role in the genesis of Ca2� alternans, which is theprimary cause of electromechanical alternans that can lead toventricular fibrillation and sudden death (26, 27). For example,Huser et al. showed that inhibition of glycolysis potentiated fre-quency-induced Ca2� alternans and electromechanical alternans incardiac myocytes (28). This is thought to result from the inhibitionof RyR2 by glycolytic metabolites and/or because of a reduction inthe concentration of ATP (26). Diaz and colleagues demonstratethat subcellular Ca2� alternans could be induced in ventricularmyocytes by tetracaine and acidosis or by reducing the opening ofthe L-type Ca2� channel (29, 30). It is known that the Po of RyR2is reduced under these conditions (tetracaine, low pH, and reducedL-type Ca2� channel current). The depressed RyR2 activity isbelieved to result in spatially and temporally desynchronized SRCa2� release in the form of subcellular Ca2� alternans (29, 30).Recently, Picht et al. have shown that although SR Ca2� contentfluctuation is an important modulator of Ca2� alternans, it is notrequired for the initiation of frequency-induced Ca2� alternans(31). Instead, beat-to-beat alternation in the availability of activeRyR channels or in the recovery of RyR2 from inactivation isbelieved to be the key to the initiation of frequency-induced Ca2�
alternans. In this regard, it has been proposed that cardiac alternansis a ‘‘disease’’ of the RyR2 channel (32).
Given the link between depressed RyR2 activity and Ca2�
alternans, we hypothesize that the A4860G RyR2 mutation, whichdepresses the activity of the channel, is likely to cause ventricularfibrillation and sudden death by increasing the propensity for Ca2�
alternans and thus electromechanical alternans. Hence, the resultsof the present study and those of previous studies suggest that thereare at least two distinct mechanisms by which RyR2 mutations cancause ventricular arrhythmias. The gain-of-function RyR2 muta-tions enhance diastolic spontaneous Ca2� release or SOICR,leading to DAD-based ventricular arrhythmias. However, the de-pression-of-function RyR2 mutations, such as A4860G, alter sys-tolic Ca2� transients, resulting in alternans-associated ventricularfibrillation. However, exactly how the A4860G mutation causesventricular fibrillation is unknown. The generation and character-ization of knockin mice harboring the A4860G mutation will shedsome definitive insights into the cellular mechanism by whichA4860G causes ventricular fibrillation and sudden death.
The Activation of RyR2 by Luminal Ca2� Is Not Mediated by Luminal-to-Cytosolic Ca2� Flux. Despite its important role in the pathogenesisof cardiac arrhythmias, the precise mechanism underlying theluminal Ca2� activation of RyR2 is unclear and controversial. It hasbeen proposed that luminal Ca2� activates RyRs by passing throughthe open channel and acting on the cytosolic Ca2� activation site (a‘‘feed-through’’ hypothesis) (33, 34). However, Gyorke and Gyorke(35) and Ching et al. (36) found that RyR2 could still be activatedby luminal Ca2� in the absence of luminal-to-cytosolic Ca2� flux.Furthermore, the application of trypsin to the luminal side of theRyR2 channel diminishes luminal Ca2� activation but not Ca2�
fluxes, arguing against the feed-through mechanism and suggestingthe existence of a luminal Ca2� activation site distinct from thecytosolic Ca2� activation site (36). Recently, a third model thatincorporates both the feed-through and true luminal Ca2� activa-tion mechanisms, called the luminal-triggered Ca2� feed-throughmechanism, has been proposed. In this model, luminal-to-cytosolicCa2� flux is required for a full activation of the channel by luminalCa2� (37). However, in the present study, we found that elevat-ing the luminal Ca2� concentration to 50 mM did not activatethe A4860G mutant channel, despite the presence of luminal-to-cytosolic Ca2� flux and the normal activation of the channelby cytosolic Ca2�. These observations indicate that luminal-to-cytosolic Ca2� flux does not activate the RyR2 channel and stronglysuggest that the activation of RyR2 by luminal Ca2� is not mediatedby the luminal-to-cytosolic Ca2� flux but by a luminal Ca2� sensor.
Molecular Determinants of the Luminal Ca2� Activation of RyR2. Themolecular identity of this putative luminal Ca2� sensor has yet to bedefined. It has been proposed that the SR Ca2� buffering proteincalsequestrin acts as a luminal Ca2� sensor and is responsible for theactivation of RyR2 by luminal Ca2� (38). However, recent studieson calsequestrin knockout mice have shown that the RyR2 channelcan still sense luminal Ca2� in the absence of calsequestrin. Theseobservations indicate that although calsequestrin can modulate SRCa2� release, it is not required for luminal Ca2� sensing (39).Consistent with this view, purified native and recombinant RyRsremain sensitive to luminal Ca2� (34, 41). The putative luminalCa2� sensor is likely formed by negatively charged amino acidresidues that are accessible to luminal Ca2�. Hence, it is alsounlikely that the nonpolar residue A4860, located near the middleof the pore inner helix, is directly involved in luminal Ca2� sensing,although mutating this residue abolishes luminal Ca2� activation.The A4860 residue, however, is thought to be essential for channelgating (25). In support of this view, we found that in addition toabolishing luminal Ca2� dependent gating, the A4860G mutationalso reduces the mean open time of the channel activated bycytosolic Ca2� by �10-fold and diminishes the basal activity of
Fig. 5. HL-1 cardiac cells transfected with the A4860G mutant exhibitattenuated SOICR. HL-1 cardiac cells were transfected with a GFP-tagged RyR2WT or a GFP-tagged A4860G mutant by using the Nucleofection method(Amaxa). Transfected cells were loaded with 5 �M fura-2 acetoxymethyl esterand identified based on their GFP fluorescence. Their SOICR activities werethen monitored by using single-cell Ca2� imaging. Fura-2 ratios of represen-tative HL-1 cells expressing the GFP-tagged RyR2 WT (A) and the GFP-taggedA4860G mutant (B) are shown. (C) The fraction (%, mean � SEM) of RyR2 WT(filled circles) and the A4860G mutant (open circles) cells that displayed Ca2�
oscillations at various [Ca2�]o. The total numbers of HL-1 cells analyzed were79 for RyR2 WT and 73 for the A4860G mutant. The data shown are means �SEM from four to five separate experiments.
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RyR2 induced by hyperosmotic conditions (e.g., high concentra-tions of KCl or sucrose). These observations suggest that theluminal and cytosolic Ca2� activation of RyR2 are mediated bydifferent gating mechanisms and that the A4860 residue is essentialfor luminal Ca2� dependent gating.
The Role of Luminal Ca2� Activation of RyR2 in Excitation–ContractionCoupling and Arrhythmogenesis. Despite the essential role thatRyR2 plays in fura-2 coupling, SR Ca2� handling, and cardiac celldevelopment, neither gain- nor loss-of-function mutations in RyR2seem to alter normal cardiac function because patients with RyR2mutations do not normally show abnormalities in cardiac structureand function at rest (9). This is probably attributable to the uniqueproperties of these mutations. For instance, although the A4860Gmutation diminishes luminal Ca2� activation, it retains a normalsensitivity to cytosolic Ca2� activation. Similarly, a number ofgain-of-function RyR2 mutations enhance luminal Ca2� activationbut have no effect on the cytosolic Ca2� activation of RyR2 (15, 16).Hence, the preferential impact of RyR2 mutations on luminal, butnot cytosolic, Ca2� activation may account for the lack of effect ofthe RyR2 mutations on cardiac function because the cytosolic Ca2�
activation of RyR2 is thought to underlie the normal physiologicalmechanism of cardiac muscle contraction. Furthermore, defectiveluminal Ca2� activation may be compensated for by the SR Ca2�
content. This is because an enhanced sensitivity to luminal Ca2�
activation would lead to increased SR Ca2� release and a reducedSR Ca2� content, which in turn would decrease luminal Ca2�
activation. Similarly, a decreased sensitivity of the channel toluminal Ca2� activation would result in decreased SR Ca2� releaseand an increased SR Ca2� content, which in turn would increaseluminal Ca2� activation (42). Hence, because of the autoregulationof SR Ca2� release, altered luminal Ca2� activation, under normalconditions, may not have a major impact on cardiac function.However, under conditions of SR Ca2� overload, SR autoregula-tion becomes ineffective, resulting in SOICR or desynchronized SRCa2� release and consequently arrhythmias. Therefore, alteredluminal Ca2� activation of RyR2 can exert a major impact onSOICR and cardiac arrhythmias.
ConclusionIn summary, we demonstrate that depressed luminal Ca2� activa-tion of RyR2 and attenuated SOICR activity is associated withcatecholaminergic idiopathic ventricular fibrillation and suddendeath. The finding that the A4860G mutation diminishes luminal,but not cytosolic, Ca2� activation indicates that the activation ofRyR2 by luminal Ca2� is distinct from its cytosolic Ca2� activation.Because altered luminal Ca2� regulation and SOICR are common
defects of RyR2 mutations, targeting the luminal Ca2� activation ofRyR2 and SOICR represents a promising therapeutic strategy forthe treatment of Ca2�-associated ventricular arrhythmias.
Materials and MethodsAdditional information regarding the materials and methodsused is available in supporting information (SI) Materials andMethods.
Site-Directed Mutagenesis and DNA Transfection. The point mutationA4860G was generated by using the overlap extension method andused to transfect HEK293 cells by using Ca2� phosphate precipi-tation as described in ref. 16. HL-1 cardiac cells were transfectedwith RyR2 WT or A4860G cDNA by using the Nucleofector kitaccording to the instructions of the manufacturer (Amaxa, Gaith-ersburg, MD).
Generation of Stable, Inducible HEK293 Cell Lines Expressing RyR2 WTand the A4860G Mutant. Stable, inducible HEK293 cell lines ex-pressing RyR2 WT or the A4860G mutant were generated by usingthe Flp-In T-REx Core kit according to the instructions of themanufacturer (Invitrogen Life Technologies, Carlsbad, CA).
Single-Channel Recordings in Planar Lipid Bilayers. Single-channelanalyses were carried out as described in ref. 16. Free Ca2�
concentrations were calculated by using the computer program ofFabiato and Fabiato (43).
[3H]Ryanodine Binding. Equilibrium [3H]ryanodine (NEN Life Sci-ence, Boston, MA) binding to cell lysate was performed as de-scribed in ref. 16.
Single-Cell Ca2� Imaging. Intracellular Ca2� transients in HEK293cells expressing RyR2 WT or the A4860G mutant or HL-1 cellstransfected with RyR2 WT or A4860G were measured by usingsingle-cell Ca2� imaging as described in ref. 16.
We thank Dr. Jonathan Lytton for the use of the single-cell Ca2� imagingfacility at the University of Calgary, Dr. William C. Claycomb (LouisianaState University Health Sciences Center, New Orleans, LA) for provid-ing HL-1 cardiac cells, and Jeff Bolstad for critical reading of themanuscript. This work was supported by research grants from theNational Institutes of Health and the Heart and Stroke Foundation ofAlberta, Northwest Territories and Nunavut (S.R.W.C.). D.J. and W.C.are recipients of an Alberta Heritage Foundation for Medical ResearchStudentship award. S.R.W.C. is an Alberta Heritage Foundation forMedical Research Scientist.
GA (2001) Circulation 103:196–200.3. Tiso N, Stephan DA, Nava A, Bagattin A, Devaney JM, Stanchi F, Larderet G, Brahmbhatt
B, Brown K, Bauce B, et al. (2001) Hum Mol Genet 10:189–194.4. Priori SG, Napolitano C, Memmi M, Colombi B, Drago F, Gasparini M, DeSimone L,
Coltorti F, Bloise R, Keegan R, et al. (2002) Circulation 106:69–74.5. Laitinen PJ, Brown KM, Piippo K, Swan H, Devaney JM, Brahmbhatt B, Donarum EA,
Marino M, Tiso N, Viitasalo M, et al. (2001) Circulation 103:485–490.6. Tester DJ, Kopplin LJ, Will ML, Ackerman MJ (2005) Heart Rhythm 2:1099–1105.7. Ter Keurs HE, Boyden PA (2007) Physiol Rev 87:457–506.8. George CH, Jundi H, Thomas NL, Fry DL, Lai FA (2007) J Mol Cell Cardiol 42:34–50.9. Mohamed U, Napolitano C, Priori SG (2007) J Cardiovasc Electrophysiol 18:791–797.
10. Kass RS, Tsien RW (1982) Biophys J 38:259–269.11. Orchard C, Eisner D, Allen D (1983) Nature 304:735–738.12. Stern M, Kort A, Bhatnagar G, Lakatta E (1983) J Gen Physiol 82:119–153.13. Wier W, Kort A, Stern M, Lakatta E, Marban E (1983) Proc Natl Acad Sci USA 80:7367–7371.14. Marban E, Robinson SW, Wier WG (1986) J Clin Invest 78:1185–1192.15. Jiang D, Xiao B, Yang D, Wang R, Choi P, Zhang L, Cheng H, Chen SRW (2004) Proc Natl
Acad Sci USA 101:13062–13067.16. Jiang D, Wang R, Xiao B, Kong H, Hunt DJ, Choi P, Zhang L, Chen SRW (2005) Circ Res
97:1173–1181.17. Pogwizd SM, Bers DM (2004) Trends Cardiovasc Med 14:61–66.18. Lakatta EG (1992) Cardiovasc Res 26:193–214.19. Cerrone M, Colombi B, Santoro M, di Barletta MR, Scelsi M, Villani L, Napolitano C, Priori
SG (2005) Circ Res 96:e77–e82.
20. Liu N, Colombi B, Memmi M, Zissimopoulos S, Rizzi N, Negri S, Imbriani M, NapolitanoC, Lai FA, Priori SG (2006) Circ Res 99:292–298.
21. Jiang D, Xiao B, Zhang L, Chen SR (2002) Circ Res 91:218–225.22. George CH, Higgs GV, Lai FA (2003) Circ Res 93:531–540.23. Thomas NL, George CH, Lai FA (2004) Cardiovasc Res 64:52–60.24. George CH, Jundi H, Walters N, Thomas NL, West RR, Lai FA (2006) Circ Res 98:88–97.25. Wang R, Bolstad J, Kong H, Zhang L, Brown C, Chen SRW (2004) J Biol Chem 279:3635–3642.26. Blatter LA, Kockskamper J, Sheehan KA, Zima AV, Huser J, Lipsius SL (2003) J Physiol
(London) 546:19–31.27. Walker ML, Rosenbaum DS (2003) Cardiovasc Res 57:599–614.28. Huser J, Wang YG, Sheehan KA, Cifuentes F, Lipsius SL, Blatter LA (2000) J Physiol
(London) 524(Pt 3):795–806.29. Diaz ME, Eisner DA, O�Neill SC (2002) Circ Res 91:585–593.30. Diaz ME, O’Neill SC, Eisner DA (2004) Circ Res 94:650–656.31. Picht E, DeSantiago J, Blatter LA, Bers DM (2006) Circ Res 99:740–748.32. Pieske B, Kockskamper J (2002) Circ Res 91:553–555.33. Tripathy A, Meissner G (1996) Biophys J 70:2600–2615.34. Xu L, Meissner G (1998) Biophys J 75:2302–2312.35. Gyorke I, Gyorke S (1998) Biophys J 75:2801–2810.36. Ching LL, Williams AJ, Sitsapesan R (2000) Circ Res 87:201–226.37. Laver DR (2007) Biophys J 92:3541–3555.38. Gyorke I, Hester N, Jones LR, Gyorke S (2004) Biophys J 86:2121–2128.39. Knollmann BC, Chopra N, Hlaing T, Akin B, Yang T, Ettensohn K, Knollmann BE, Horton
KD, Weissman NJ, Holinstat I, et al. (2006) J Clin Invest 116:2510–2520.41. Sitsapesan R, Williams AJ (1997) J Membr Biol 159:179–185.42. Trafford AW, Diaz ME, Sibbring GC, Eisner DA (2000) J Physiol (London) 522(Pt 2):259–270.43. Fabiato A, Fabiato F (1979) J Physiol (Paris) 75:463–505.
18314 � www.pnas.org�cgi�doi�10.1073�pnas.0706573104 Jiang et al.
