A number of RyR2 (cardiac ryanodine receptor) mutations linked to ventricular arrhythmia and sudden death are located within the last C-terminal ∼500 amino acid residues, which is believed to constitute the ion-conducting pore and gating domain of the channel. We have previously shown that mutations located near the C-terminal end of the predicted TM (transmembrane) segment 10, the inner pore helix, can either increase or decrease the propensity for SOICR (store-overload-induced Ca2+ release), also known as spontaneous Ca2+ release. In the present study, we have characterized an RyR2 mutation, V4653F, located in the loop between the predicted TM 6 and TM 7a, using an ER (endoplasmic reticulum)-targeted Ca2+-indicator protein (D1ER). We directly demonstrated that SOICR occurs at a reduced luminal Ca2+ threshold in HEK-293 cells (human embryonic kidney cells) expressing the V4653F mutant as compared with cells expressing the RyR2 wild-type. Single-channel analyses revealed that the V4653F mutation increased the sensitivity of RyR2 to activation by luminal Ca2+. In contrast with previous reports, the V4653 mutation did not alter FKBP12.6 (FK506-binding protein 12.6 kDa; F506 is an immunosuppressant macrolide)–RyR2 interaction. Luminal Ca2+ measurements also showed that the mutations R176Q/T2504M, S2246L and Q4201R, located in different regions of the channel, reduced the threshold for SOICR, whereas the A4860G mutation, located within the inner pore helix, increased the SOICR threshold. We conclude that the cytosolic loop between TM 6 and TM 7a plays an important role in determining the SOICR threshold and that the alteration of the threshold for SOICR is a common mechanism for RyR2-associated ventricular arrhythmia.

INTRODUCTION

In heart failure, the leading cause of death is VT (ventricular tachycardia). It is becoming apparent that a major cause of VT is DADs (delayed-after-depolarizations) [1,2], which are produced by spontaneous Ca2+ release from the SR (sarcoplasmic reticulum) via the RyR2 (cardiac ryanodine receptor) during SR Ca2+ overload [37], a process that we have referred to as SOICR (store-overload-induced Ca2+ release) [8,9]. There are several inheritable diseases that result in VT and are caused by mutations in RyR2 [812], including CPVT (catecholaminergic polymorphic VT) and ARVD2 (arrhythmogenic right ventricular cardiomyopathy type 2). Under conditions of physical or emotional stress, patients carrying such mutations can experience DADs, VT and sudden death [1321]. Although SOICR is known to be an underlying cause of these effects, exactly how these mutations increase the propensity for SOICR is unclear.

RyRs are homotetrameric ion channels, with each subunit composed of a cytoplasmic and TM (transmembrane) domain. The TM domain is located in the C-terminal region (∼1/10 of the molecule) and is thought to comprise the ion-conducting pore and the gate of the channel. On the other hand, the N-terminal region (9/10 of the molecule) forms the cytoplasmic domain that is believed to contain sites for a number of modulators, including the FKBP12.6 (FK506-binding protein 12.6 kDa; F506 is an immunosuppressant macrolide) [2224]. The disease-causing mutations of RyR2 are located in three distinct regions within the sequence of the channel at the N-terminus, central region and C-terminus. Many of those mutations lying within the N-terminal or central cytosolic regions of the channel have previously been characterized as increasing the activity of the channel [818,21]. However, the effect of those mutations occurring in the C-terminus of the channel is less clear, as they have been reported to both increase and decrease channel activity [8,9,14,25]. The impact of mutations located within this region is likely to be determined by which part of the channel pore they are found in. The channel pore comprises a selectivity filter (Gly4826-Asp4831), a pore helix (Met4806-Arg4824), an inner helix (Ile4850-Lys4883) and an outer helix (Gln4768-Arg4792) [26]. The previously characterized mutations I4867M and N4895D, which are located within the inner helix and the following loop structure, both lead to increases in channel activity and luminal Ca2+ activation, whereas A4860G, also located in the inner helix, leads to a decrease in channel activity and a loss of luminal Ca2+ activation [8,9,25]. The proximity of these mutations to one another and the diversity of their effects on channel function raise the question of how other mutations located within the channel pore, helices or loops affect channel function.

The CPVT mutation V4653F is unique in that it is proposed to be cytoplasmically located within a loop between TM-spanning helices 6 and 7a (TM 6 and TM 7a) (we assume that the RyR2 channel has six TM segments, as described in the recently proposed model by MacLennan's group, and number them according to their original convention (see Figure 5 in [27]), a structure not previously studied. This location of V4653F makes the characterization of the mutated channel intriguing as it may shed light on how mutations in the cytosolic domain affect the TM domain. Studies by others have shown V4653F to increase the activity of the channel through the dissociation of FKBP12.6 from the RyR2 macromolecular complex [28]. However, studies performed using a series of C-terminal truncated forms of RyR2 (missing the TM domain) still exhibit FKBP12.6 binding [29]. Other theories have been proposed to explain how CPVT mutations may alter channel function through changes in intra- and inter-domain interactions, including domain unzipping and I-domain signal transduction [30,31]. We have shown that a number of mutations of RyR2 increase its luminal Ca2+-sensitivity and propensity for SOICR [8,9].

In the present study, we aimed to directly determine the effect of CPVT mutations on the threshold for SOICR. Through measurement of the luminal Ca2+ threshold, for the first time, we show that the CPVT mutation V4653F reduces the threshold at which SOICR occurs. Further characterization of V4653F shows it to share similar properties with other gain-of-function mutations. By extending the measurement of the luminal Ca2+ threshold to other CPVT mutations, we show that a reduced threshold for SOICR as a result of an increased sensitivity to luminal Ca2+, or an increased threshold for SOICR as a result of a decreased sensitivity to luminal Ca2+, underlies a common mechanism of CPVT mutations.

EXPERIMENTAL

Materials

Restriction endonucleases and DNA-modifying enzymes were purchased from New England Biolabs (Ipswich, MA, U.S.A.). The anti-RyR and anti-FKBP antibodies were obtained from Affinity BioReagents (Golden, CO, U.S.A.). Soya-bean phosphatidylcholine was obtained from Avanti Polar Lipids (Alabaster, AL, U.S.A.). CHAPS and other reagents were purchased from Sigma.

Site-directed mutagenesis and generation of inducible HEK-293 cell lines (human embryonic kidney cell lines)

The point mutations R176Q/T2504M, S2246L, Q4201R, V4653F and A4860G in the mouse RyR2 were made using the overlap extension method and inserted into Flp-In TREx-293 cells (Invitrogen) as previously described [9]. Briefly, Flp-In T-REx-293 cells were co-transfected with the inducible expression vector pcDNA5/FRT/TO containing the mutant cDNAs and the pOG44 vector encoding the Flp recombinase in 1:5 ratios using the calcium phosphate precipitation method. The transfected cells were washed with PBS (137 mM NaCl, 8 mM Na2HPO4, 1.5 mM KH2PO4 and 2.7 mM KCl) 24 h after transfection followed by a change to fresh medium for 24 h. The cells were then washed again with PBS, harvested and plated on to new dishes. After the cells had attached (∼4 h), the growth medium was replaced with a selection medium containing 200 μg/ml hygromycin (Invitrogen). The selection medium was changed every 3–4 days until the desired number of cells was grown. The hygromycin-resistant cells were pooled, divided into aliquots and stored at −80 °C. These positive cells are believed to be isogenic, because the integration of RyR2 cDNA is mediated by the Flp recombinase at a single FRT site. Each HEK-293 cell line was tested for RyR2 expression using Western-blot analysis and immunocytofluorescent staining (results not shown).

Single-cell Ca2+ imaging (luminal Ca2+)

Luminal Ca2+ transients in HEK-293 cells expressing wt (wild-type) or mutant channels were measured using single-cell Ca2+ imaging and the Ca2+-sensitive FRET (fluorescence resonance energy transfer)-based cameleon protein D1ER [32]. Stable, inducible HEK-293 cells expressing wt or mutant channels as described above were used with the addition of transfection, using the calcium phosphate precipitation method, with D1ER cDNA 24 h before induction of RyR2 expression. The cells were perfused continuously with KRH (Krebs–Ringer Hepes) buffer (125 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 6 mM glucose, 1.2 mM MgCl2 and 25 mM Hepes, pH 7.4) containing various concentrations of CaCl2 (0 or 1 mM) and tetracaine (500 μM) or caffeine (10 mM) at room temperature (22 °C). Images were captured with Simple PCI 6 software (Compix Inc.) at λemission=470 and 535 nm and λexcitation=430 nm every 2 s by using an inverted microscope (Nikon TE2000-S) equipped with an S-Fluor ×20/0.75 objective. The amount of FRET was determined from the ratio of the emissions at 535 and 470 nm.

Single-channel recordings

Single-channel analyses were carried out as described previously [9]. Recombinant wt and RyR2 mutant proteins were partially purified from cell lysate by sucrose-density-gradient centrifugation. Heart phosphatidylethanolamine and brain phosphatidylserine (Avanti Polar Lipid), dissolved in chloroform, were combined in a 1:1 ratio (w/w), dried under nitrogen gas and suspended in 30 μl of n-decane at a concentration of 12 mg of lipid/ml. Bilayers were formed across a 250-μm hole in a Delrin partition separating two chambers. The cis-chamber (800 μl) was connected to the head stage input of an Axopatch 200A amplifier (Axon Instruments). The trans-chamber (1.2 ml) was held at virtual ground. A symmetrical solution containing 250 mM KCl and 25 mM Hepes (pH 7.4) was used for all recordings, unless indicated otherwise. A 4 μl aliquot (∼1 μg of protein) of the sucrose-density-gradient-purified recombinant wt or mutant RyR2 protein was added to the trans-chamber. Spontaneous channel activity was always tested for sensitivity to EGTA and Ca2+. The chamber to which the addition of EGTA inhibited the activity of the incorporated channel was presumed to correspond to the cytoplasmic side of the channel. The direction of single-channel currents was always measured from the luminal to the cytoplasmic side of the channel, unless mentioned otherwise. Recordings were filtered at 2500 Hz. Free Ca2+ concentrations were calculated using the computer program described by Fabiato [32a]. Data analyses were carried out using the pClamp 8.1 software (Axon Instruments).

Preparation of cell lysate and [3H]ryanodine binding

Preparation of cell lysate and equilibrium [3H]ryanodine binding were carried out as described previously [9] with KCl reduced to 100 mM.

RyR2–FKBP12.6 co-immunoprecipitation assays and immunoblotting

A 350 μl portion of HEK-293 cell lysate (wt or V4653F) was incubated with various amounts of FKBP12.6-transiently-transfected HEK-293 cell lysate (0, 0.3, 1, 3, 10, 30 and 100%) and 20 μl of Protein G–Sepharose prebound with 1 μl of anti-RyR antibody (34C) at 4 °C for 17–19 h. The Protein G–34C precipitates were washed with ice-cold lysis buffer (25 mM Tris/50 mM Hepes, pH 7.4, 137 mM NaCl, 1% CHAPS, 0.5% soya-bean phosphatidylcholine and 5 mM MgCl2) containing a protease inhibitor mix (1 mM benzamidine, 2 μg/ml leupeptin, 2 μg/ml pepstatin A, 2 μg/ml aprotinin, 0.5 mM PMSF and 2.5 mM dithiothreitol) three times, each time for 2 min. The proteins bound to the Sepharose beads were then solubilized by the addition of 40 μl of Laemmli's sample buffer [28] plus 5% (v/v) 2-mercaptoethanol and boiled for 5 min. The samples were then separated by SDS/PAGE (6% for RyR2 or 16% for FKBP12.6) [33]. The SDS/PAGE-resolved proteins were transferred on to PVDF membranes at 45 V for 18–20 h (for RyR2) or 100 V for 2 h (for FKBP12.6) at 4 °C in the presence of 0.01% SDS by the method of Towbin et al. [34]. The PVDF membranes containing the transferred proteins were blocked for 30 min with PBS containing 0.5% Tween 20 and 5% (w/v) non-fat dried skimmed milk powder. The blocked membranes were then incubated with anti-RyR (34C) or anti-FKBP antibodies (both 1:1000) for 3 h and washed three times for 5 min in PBS containing 0.5% Tween 20. The membrane was then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (1:20000) for 30 min. After washing three times for 5 min in PBS containing 0.5% Tween 20, the RyR2 or FKBP12.6 proteins were detected by enhanced chemiluminescence (SuperSignal; Pierce).

RESULTS

V4653F reduces the luminal Ca2+ threshold at which SOICR occurs

To measure the luminal Ca2+ threshold at which SOICR occurs, we expressed a luminal Ca2+-indicator protein (D1ER) in stable-inducible HEK-293 cells expressing either wt or V4653F. D1ER is a soluble FRET-based Ca2+-indicator protein that is expressed within the lumen of the SR/ER (endoplasmic reticulum) due to a KDEL retention motif [32]. In the absence of Ca2+ binding, no FRET occurs; however, upon Ca2+ binding a conformational change takes place, allowing FRET to be observed. Therefore, by monitoring the incidence of FRET, the relative luminal Ca2+ level can be estimated. Figure 1(A) shows the relative luminal Ca2+ levels of HEK-293 cells expressing either wt or V4653F under various conditions. As the external Ca2+ concentration was increased from 0 to 1 mM, the luminal Ca2+ level increased, as shown by an increase in the FRET signal. As the luminal Ca2+ level increased, SOICR occurred, which was reflected by downward deflections in the FRET signal. SOICR was then suppressed by blocking the channel through the addition of 500 μM tetracaine. This suppression of SOICR allowed the luminal Ca2+ level to increase to its maximum capacity (when SR Ca2+ uptake balances passive Ca2+ leak) before the lumen was emptied of Ca2+ by the addition of 10 mM caffeine. Figure 1(B) shows the luminal threshold (percentage of maximum luminal Ca2+ store capacity) at which SOICR occurs in a large number of cells expressing either wt (n=111) or V4653F (n=135) and confirms that SOICR occurs at a significantly lower luminal Ca2+ threshold in cells expressing V4653F (82.9±0.8%) than in cells expressing wt (93.8±0.6%) (P<0.0001) (Figure 1Ba). It should be noted that there was no significant difference in the maximum luminal Ca2+ store capacity, the maximum luminal Ca2+ level (Fmax) or the minimum luminal Ca2+ level (Fmin) between HEK-293 cells expressing wt or V4653F. The maximum luminal Ca2+ store capacity of V4653F-expressing cells was 103.4±1.6% of that of wt-expressing cells (P=0.12) (Figure 1Bb). The maximum (in the presence of tetracaine) and minimum (in the presence of caffeine) luminal Ca2+ levels were 100.7±0.6% (P=0.4) and 99.7±0.6% (P=0.7) respectively of that of wt-expressing cells (Figures 1Bc and 1Bd).

V4653F decreases the threshold for SOICR

Figure 1
V4653F decreases the threshold for SOICR

HEK-293 cells expressing wt or V4653F were grown on glass coverslips. Cells were transfected with D1ER cDNA 48 h before imaging and RyR expression was induced 24 h before imaging. Cells were perfused with KRH buffer with either 0 or 1 mM Ca2+ and either 500 μM tetracaine or 10 mM caffeine. (A) Representative traces from HEK-293 cells expressing either wt or V4653F were captured using single-cell imaging. (Ba) The threshold for SOICR, (Bb) maximum luminal Ca2+ store capacity and (Bc) maximum and (Bd) minimum luminal Ca2+ levels in cells expressing wt or V4653F. The threshold for SOICR is expressed as a percentage of the maximum luminal Ca2+ store capacity, which is determined by subtracting the Fmin (Ca2+ level after caffeine) from Fmax (Ca2+ level after tetracaine). Values were normalized to the wt level (100%). Results shown are means±S.E.M. for 111 or 135 cells recorded from six separate experiments.

Figure 1
V4653F decreases the threshold for SOICR

HEK-293 cells expressing wt or V4653F were grown on glass coverslips. Cells were transfected with D1ER cDNA 48 h before imaging and RyR expression was induced 24 h before imaging. Cells were perfused with KRH buffer with either 0 or 1 mM Ca2+ and either 500 μM tetracaine or 10 mM caffeine. (A) Representative traces from HEK-293 cells expressing either wt or V4653F were captured using single-cell imaging. (Ba) The threshold for SOICR, (Bb) maximum luminal Ca2+ store capacity and (Bc) maximum and (Bd) minimum luminal Ca2+ levels in cells expressing wt or V4653F. The threshold for SOICR is expressed as a percentage of the maximum luminal Ca2+ store capacity, which is determined by subtracting the Fmin (Ca2+ level after caffeine) from Fmax (Ca2+ level after tetracaine). Values were normalized to the wt level (100%). Results shown are means±S.E.M. for 111 or 135 cells recorded from six separate experiments.

V4653F increases the sensitivity of single RyR2 channels to activation by luminal Ca2+

The fact that the V4653F mutation reduces the threshold for SOICR suggests that it may sensitize the channel to luminal Ca2+, thereby facilitating SOICR at a lower luminal Ca2+ level. To test this hypothesis, we assessed the luminal Ca2+ response of the channel. Figure 2(Aa) shows that V4653F exhibits very little activity at low luminal (45 nM) and cytosolic (45 nM) Ca2+. However, increasing the luminal Ca2+ to 300 μM has a marked effect on the activity of the channel, increasing the open probability (Po) from 0.0006 to 0.281 (Figure 2Ab). The average Po of V4653F and wt at a range of luminal Ca2+ concentrations are shown in Figure 2(B), illustrating that V4653F is much more sensitive to luminal Ca2+ than wt.

V4653F increases luminal Ca2+ activation

Figure 2
V4653F increases luminal Ca2+ activation

The single-channel activity of V4653F was recorded in a symmetrical recording solution containing 250 mM KCl and 25 mM Hepes (pH 7.4). The Ca2+ concentrations on both the cytosolic and luminal faces of the channel were adjusted to 45 nM. The luminal Ca2+ concentration was then increased to various levels by the addition of aliquots of CaCl2 solution. The control trace is shown in (Aa) and the single current trace, recorded at 300 μM luminal Ca2+, is shown in (Ab); the holding potential was −20 mV. Openings are downwards. The open probability (Po), arithmetic mean open time (To) and the arithmetic closed time (Tc) are indicated at the top of each panel. (B) The relationship between Po and luminal Ca2+ concentration of wt and V4653F. Data points are means±S.E.M. for seven V4653F and 22 wt single channels.

Figure 2
V4653F increases luminal Ca2+ activation

The single-channel activity of V4653F was recorded in a symmetrical recording solution containing 250 mM KCl and 25 mM Hepes (pH 7.4). The Ca2+ concentrations on both the cytosolic and luminal faces of the channel were adjusted to 45 nM. The luminal Ca2+ concentration was then increased to various levels by the addition of aliquots of CaCl2 solution. The control trace is shown in (Aa) and the single current trace, recorded at 300 μM luminal Ca2+, is shown in (Ab); the holding potential was −20 mV. Openings are downwards. The open probability (Po), arithmetic mean open time (To) and the arithmetic closed time (Tc) are indicated at the top of each panel. (B) The relationship between Po and luminal Ca2+ concentration of wt and V4653F. Data points are means±S.E.M. for seven V4653F and 22 wt single channels.

Effect of V4653F on Ca2+-dependent [3H]ryanodine binding

In addition to luminal Ca2+ activation, RyR2 can also be activated by cytosolic Ca2+ [the basis of CICR (Ca2+-induced Ca2+ release)]. In order to determine the impact of V4653F on the sensitivity of cytosolic Ca2+ activation, we assessed the [3H]ryanodine-binding response of the channel to Ca2+. [3H]Ryanodine binding to RyR2 in response to Ca2+ is a good indication of the activation of the channel as [3H]ryanodine can only bind to the active form of the channel. Previous experiments have shown that the EC50 of Ca2+ activation of [3H]ryanodine binding is similar to that of the cytosolic Ca2+ activation of single RyR2 channels [8,35]. Figure 3 shows the overall Ca2+ response of [3H]ryanodine binding to wt and V4653F. It shows that there is a notable difference in [3H]ryanodine binding at low Ca2+ concentrations {pCa (=−log [Ca2+])>7}, where V4653F exhibits an increased basal activity, but that the EC50 for activation of [3H]ryanodine binding is not dramatically altered by the V4653F (wt, EC50=0.33±0.021 μM, n=7; V4653F, EC50=0.2±0.015 μM, n=5). The high basal activity probably reflects the instability of the closed state of the channel, which becomes apparent during [3H]ryanodine binding assays due to the slow off-rate of [3H]ryanodine binding and the long duration (2–3 h) of the binding assays.

Effect of V4653F on the Ca2+ activation of the channel

Figure 3
Effect of V4653F on the Ca2+ activation of the channel

[3H]Ryanodine binding to a cell lysate prepared from HEK-293 cells transfected with wt or V4653F cDNA was performed at various Ca2+ concentrations (0.2 nM to 0.1 mM), 100 mM KCl and 5 nM [3H]ryanodine. Amounts of [3H]ryanodine binding at various Ca2+ concentrations were normalized to the maximal binding. Data points shown are means±S.E.M. for five (for V4653F) or seven (for wt) separate experiments.

Figure 3
Effect of V4653F on the Ca2+ activation of the channel

[3H]Ryanodine binding to a cell lysate prepared from HEK-293 cells transfected with wt or V4653F cDNA was performed at various Ca2+ concentrations (0.2 nM to 0.1 mM), 100 mM KCl and 5 nM [3H]ryanodine. Amounts of [3H]ryanodine binding at various Ca2+ concentrations were normalized to the maximal binding. Data points shown are means±S.E.M. for five (for V4653F) or seven (for wt) separate experiments.

V4653F does not alter FKBP12.6–RyR2 interaction

The V4653F mutation has previously been shown to reduce the affinity of the channel for FKBP12.6; however, many of the other CPVT mutations initially shown to alter the affinity of the channel for FKBP12.6 have since then been otherwise characterized. In order to determine whether V4653F alters FKBP12.6–RyR2 interaction, we expressed wt, V4653F and FKBP12.6 in HEK-293 cells. Varying amounts of cell lysate from cells expressing FKBP12.6 were incubated with either wt or V4653F cell lysate in the presence of beads coupled with an anti-RyR antibody (34C). Figures 4(A) and 4(B) show that there was comparable binding of FKBP12.6 to wt and V4653F over a wide range of FKBP12.6 levels, suggesting that the V4653F mutation does not affect the binding of FKBP12.6 to the channel (n=3, P>0.4). In a similar assay without the addition of wt or V4653F cell lysate, no FKBP12.6 could be detected (results not shown).

Effect of V4653F on FKBP12.6–RyR2 interaction

Figure 4
Effect of V4653F on FKBP12.6–RyR2 interaction

(A) Western blots showing the interaction between wt or V4653F and various amounts of FKBP12.6 (values indicate percentage of maximal amount used). The RyR2–FKBP12.6 complex was co-immunoprecipitated using an anti-RyR antibody followed by immunoblotting with anti-RyR (upper panel) and anti-FKBP12/12.6 (lower panel) antibodies. Results shown are representative of three separate experiments. (B) Densitometry of the protein bands showed that there are no significant differences in the amount of protein co-immunoprecipitated at any given FKBP12.6 level (P>0.4).

Figure 4
Effect of V4653F on FKBP12.6–RyR2 interaction

(A) Western blots showing the interaction between wt or V4653F and various amounts of FKBP12.6 (values indicate percentage of maximal amount used). The RyR2–FKBP12.6 complex was co-immunoprecipitated using an anti-RyR antibody followed by immunoblotting with anti-RyR (upper panel) and anti-FKBP12/12.6 (lower panel) antibodies. Results shown are representative of three separate experiments. (B) Densitometry of the protein bands showed that there are no significant differences in the amount of protein co-immunoprecipitated at any given FKBP12.6 level (P>0.4).

An altered luminal Ca2+ threshold at which SOICR occurs is common to CPVT mutations

We have previously characterized many other CPVT mutations throughout the sequence of RyR2 and have consistently observed an alteration in the luminal Ca2+-sensitivity of the channel. We have also indirectly shown, using cytosolic Ca2+ measurements, that the mutations appear to alter the threshold for SOICR. To directly determine whether other, previously characterized, mutations of RyR2 also reduce the threshold for SOICR, we studied a further four CPVT RyR2 mutations. Three gain-of-function mutations were chosen, one from each of the three identified CPVT hotspots: R176Q/T2504M from the N-terminal region, S2246L from the central region and Q4201R from the C-terminal region. We also chose a loss-of-function mutation, A4860G, which displays no luminal Ca2+ activation, but retains normal cytosolic Ca2+ activation [25]. Figure 5(A) shows the typical response of stable, inducible HEK-293 cells expressing the four mutations to the previously described regime. It shows that the A4680G-expressing cells display no discernible SOICR and that, in these cells, the addition of tetracaine does not cause an increase in the luminal Ca2+ level. These results demonstrate that A4860G increases the threshold for SOICR to such an extent that maximum luminal Ca2+ store capacity of the ER can be achieved without the addition of tetracaine.

CPVT mutations alter the threshold for SOICR

Figure 5
CPVT mutations alter the threshold for SOICR

HEK-293 cells expressing wt or mutant RyR2 were grown on glass coverslips. Cells were transfected with D1ER cDNA 48 h before imaging and RyR expression was induced 24 h before imaging. Cells were perfused with KRH buffer with either 0 or 1 mM Ca2+ and either 500 μM tetracaine or 10 mM caffeine. (A) Representative traces from HEK-293 cells expressing either wt or mutant RyR2 captured using single-cell imaging [trace for wt is the same as shown in Figure 1(A), as wt cell data were pooled from both assays]. (Ba) The threshold for SOICR, (Bb) maximum luminal Ca2+ store capacity and (Bc) maximum and (Bd) minimum luminal Ca2+ levels in cells expressing wt or mutant RyR2. The threshold for SOICR is expressed as a percentage of the maximum luminal Ca2+ store capacity, which is determined by subtracting the Fmin (Ca2+ level after caffeine) from Fmax (Ca2+ level after tetracaine). Values were normalized to the wt level (100%). Results shown are the means±S.E.M. for 78–150 cells recorded over three to six separate experiments.

Figure 5
CPVT mutations alter the threshold for SOICR

HEK-293 cells expressing wt or mutant RyR2 were grown on glass coverslips. Cells were transfected with D1ER cDNA 48 h before imaging and RyR expression was induced 24 h before imaging. Cells were perfused with KRH buffer with either 0 or 1 mM Ca2+ and either 500 μM tetracaine or 10 mM caffeine. (A) Representative traces from HEK-293 cells expressing either wt or mutant RyR2 captured using single-cell imaging [trace for wt is the same as shown in Figure 1(A), as wt cell data were pooled from both assays]. (Ba) The threshold for SOICR, (Bb) maximum luminal Ca2+ store capacity and (Bc) maximum and (Bd) minimum luminal Ca2+ levels in cells expressing wt or mutant RyR2. The threshold for SOICR is expressed as a percentage of the maximum luminal Ca2+ store capacity, which is determined by subtracting the Fmin (Ca2+ level after caffeine) from Fmax (Ca2+ level after tetracaine). Values were normalized to the wt level (100%). Results shown are the means±S.E.M. for 78–150 cells recorded over three to six separate experiments.

Figure 5(Ba) shows that all three gain-of-function mutations result in Ca2+ oscillations occurring at a significantly reduced luminal Ca2+ threshold compared with wt (wt=93.8±0.6%, R176Q/T2504M=80.6±0.7%, S2246L=77.2±1.1% and Q4201R=78.3±1.0%; all values are percentage of maximum luminal Ca2+ store capacity, P<0.0001), which are similar to the results obtained with V4653F. Again, as with V4653F, there was a comparable maximum luminal Ca2+ store capacity between the cells expressing wt and the CPVT mutations (R176Q/T2504M=103.7±1.2%, S2246L=101.6±1.6% and Q4201R=100.6±1.8%; all values are percentage of wt maximum luminal Ca2+ store capacity) (Figure 5Bb). Additionally, the mutations did not markedly alter the maximum (Fmax) or minimum (Fmin) luminal Ca2+ levels induced by tetracaine or caffeine (Figures 5Bc and 5Bd). The results for both the gain- and loss-of-function mutations are consistent with the notion that CPVT mutations in RyR2 alter the threshold at which SOICR occurs.

DISCUSSION

Although our previous studies have suggested that CPVT RyR2 mutations reduce the threshold for SOICR [8,9], this conclusion is inferred from measurements of cytoplasmic Ca2+ and not direct luminal Ca2+ recording. To overcome this deficiency, in the present study we used a luminal Ca2+-sensitive indicator protein (D1ER) to monitor luminal Ca2+ directly. The use of this probe allowed us to definitively determine the effect of RyR2 mutations on the threshold for SOICR. We found that V4653F has a reduced threshold for SOICR. Further studies showed that this mutation also increased the sensitivity of the channel to luminal Ca2+ activation. By extending the study to encompass mutations throughout the sequence of RyR2, we determined that an alteration of the threshold for SOICR is an underlying mechanism for both gain- and loss-of-function CPVT RyR2 mutations.

Characterization of the CPVT-linked mutation V4653F

Many of the CPVT mutations located within the cytoplasmic domain of RyR2 have been characterized and all appear to share a common phenotype − an increase in channel activity in response to luminal Ca2+ and an increased propensity for SOICR, and are accordingly referred to as gain-of-function mutations. Currently, fewer of the CPVT mutations located within the TM domain of RyR2 have been studied, but those that have been studied do not share a consistent phenotype. Most of the mutations located within this domain are gain-of-function mutations, but there is also a mutation (A4860G) that leads to a loss of luminal Ca2+ activation and a suppression of SOICR [25], but still retains cytosolic Ca2+ activation. In light of these studies, it seems apparent that mutations located within or near the TM domain require further study to determine the effect of each individual mutation and how it causes channel dysfunction.

The V4653F mutation is located within a previously uncharacterized structure between TM 6 and TM 7a of RyR2 (see Figure 5 in [27]). Although it is located within the cytoplasmic domain of the channel, its position in the amino acid sequence is very close to the TM helices that form the channel pore [27]. Its cytosolic location, so close to the pore-forming TM structure, makes the characterization of this mutation intriguing as it may offer a mechanistic basis for explaining how other cytosolic mutations are able to alter the function of the channel. Using luminal Ca2+ recordings, we found that V4653F decreased the threshold at which SOICR occurred. Owing to the inherent difficulties associated with quantitative measurements of luminal Ca2+ concentration, we determined the relative luminal Ca2+ levels at which SOICR occurs as compared with the maximum luminal Ca2+ store capacity, using tetracaine to block Ca2+ leak/spontaneous release in an assay similar to that used by others [36,37]. Single-channel recording also showed that V4653F increased the luminal Ca2+-sensitivity of the channel. These results suggest that an increase in the luminal Ca2+-sensitivity of the channel is the mechanism by which V4653F reduces the threshold at which SOICR occurs. This mechanism is consistent with that previously proposed for other CPVT mutations [8,9]. However, until the present study, there has only been indirect evidence, cytosolic Ca2+ measurements, to support the claim.

Although our results indicate that the V4653F mutation increases the luminal Ca2+-sensitivity of the channel and inversely affects the threshold for SOICR, they do not offer a molecular basis for these effects. The location of V4653F within a loop between TM 6 and TM 7a suggests that it may be involved in the interaction between this loop and the C-terminus of the channel. It has been predicted that both structures protrude out of the TM domain into the cytosolic domain. If these two structures (the loop between TM 6 and TM 7a and the C-terminus) do interact within the cytosolic domain, one may expect mutations in either region to yield similar effects on the channel. Indeed, the CPVT-linked mutation, N4895D, located in the C-terminus of the channel, has previously been characterized as increasing its luminal Ca2+-sensitivity and increasing the propensity for SOICR [8]. Hence, it is possible that mutations in the loop TM 6–TM 7a and the C-terminus may alter domain–domain interactions within the C-terminal region that is thought to constitute the gating domain of the channel.

An altered threshold for store-overload-induced SR/ER Ca2+ release is the underling mechanism for CPVT/ARVD2-linked RyR2 mutations

We have previously proposed that CPVT/ARVD2 mutations reduce the threshold for SOICR by increasing the sensitivity of the channel to activation by luminal Ca2+ and thus increasing the propensity for DADs and triggered arrhythmias under conditions of SR Ca2+ overload [8,9]. These previous studies also showed that CPVT/ARVD2 mutations do not alter the sensitivity of the channel to cytosolic Ca2+ nor do they alter the resting cytosolic Ca2+ [8,9]. Based on the results presented in the present study, we can now link a decrease/increase in luminal Ca2+-sensitivity to a corresponding increase/decrease in the luminal Ca2+ threshold for SOICR. The gain-of-function mutations Q4201R, S2246L, R176Q/T2504M and V4653F all showed a decrease in the threshold for SOICR compared with wt. The loss-of-function mutation A4860G had an increased threshold for SOICR (to such an extent that no SOICR occurred under the experimental conditions used in the present study) and has a corresponding lack of luminal Ca2+-sensitivity [25]. These results suggest that all of the studied CPVT mutations share a common causal mechanism, altering the sensitivity of the channel to luminal Ca2+ and subsequently causing an inverse change in the threshold for SOICR. The increased luminal Ca2+-sensitivity and reduced threshold for SOICR are also consistent with the fact that susceptible patients only experience impaired Ca2+ release under conditions of stress, when the SR Ca2+ load would be increased. This increased SR Ca2+ load would be more likely to exceed the lowered threshold for SOICR in patients with CPVT, leading to DADs and arrhythmic activities.

Role of FKBP12.6 in CPVT/ARVD2

Alteration of FKBP12.6–RyR2 interaction has been proposed to be an important factor in how CPVT mutations alter RyR2 channel function. Much of this evidence comes from work performed by Lehnart et al. [28]. They have shown that a number of RyR2 CPVT mutations, R2474S, S2246L, P2328S, Q4201R, R4496C and V4653F, all exhibit reduced FKBP12.6 binding [28,38]. However, a number of groups, including our own, have shown that many of these CPVT mutations have no effect on the RyR2–FKBP12.6 interaction. Jiang et al. [8,9] have shown that the CPVT mutations Q4201R, I4867M, S2246L, R2474S, R176Q/T2504M and L433P have no significant effect on RyR2–FKBP12.6 association in HEK-293 cells. Similarly, George et al. [39] have shown that S2246L, N4104K and R4497C also have no impact on RyR2–FKBP12.6 binding in HL-1 cells. In the present study, we, likewise, show that V4653F has no effect on RyR2–FKBP12.6 binding. It is unclear why such conflicting results are obtained, but it is likely to be due to a difference in experimental conditions. Nevertheless, this growing body of evidence questions the notion that FKBP12.6 dissociation is the mechanism by which these CPVT RyR2 mutations alter the function of the channel.

Summary

In summary, we have characterized the CPVT mutation V4653F. Consistent with the previously studied CPVT mutations, we found that V4653F results in an increased propensity for SOICR and a corresponding increase in luminal Ca2+ activation. More importantly, by measuring the relative luminal Ca2+ levels, we have directly demonstrated, for the first time, that V4653F and a host of other CPVT RyR2 mutations share a common causal dysfunction – lowering the luminal Ca2+ threshold at which SOICR occurs. These studies reveal new insights into how CPVT-linked mutations of RyR2 cause disease.

D. J. was a recipient of an AHFMR (Alberta Heritage Foundation for Medical Research) Studentship Award. S. R. W. C. is a scientist at the AHFMR. This work was supported by research grants from the CIHR (Canadian Institutes of Health Research) and an NIH (National Institutes of Health) grant to S. R. W. C. We thank Professor Roger Yonchien Tsien (Departments of Pharmacology and Chemistry and Biochemistry, Howard Hughes Medical Institute, University of California at San Diego, San Diego, La Jolla, CA, U.S.A.) for the gift of D1ER pcDNA, and Ms Tina Vo for excellent technical assistance.

Abbreviations

     
  • ARVD2

    arrhythmogenic right ventricular cardiomyopathy type 2

  •  
  • CPVT

    catecholaminergic polymorphic ventricular tachycardia

  •  
  • DAD

    delayed-after-depolarization

  •  
  • ER

    endoplasmic reticulum

  •  
  • FKBP12.6

    FK506-binding protein 12.6 kDa

  •  
  • FRET

    fluorescence resonance energy transfer

  •  
  • HEK-293 cells

    human embryonic kidney cells

  •  
  • KRH

    Krebs–Ringer Hepes

  •  
  • RyR2

    cardiac ryanodine receptor

  •  
  • SOICR

    store-overload-induced Ca2+ release

  •  
  • SR

    sarcoplasmic reticulum

  •  
  • TM

    transmembrane

  •  
  • VT

    ventricular tachycardia

  •  
  • wt

    wild-type

References

References
1
Marban
E.
Cardiac channelopathies
Nature
2002
, vol. 
415
 (pg. 
213
-
218
)
2
Pogwizd
S.
Bers
D.
Calcium cycling in heart failure: the arrhythmia connection
J. Cardiovasc. Electrophysiol.
2002
, vol. 
13
 (pg. 
88
-
91
)
3
Kass
R. S.
Tsien
R. W.
Fluctuations in membrane current driven by intracellular calcium in cardiac Purkinje fibers
Biophys. J.
1982
, vol. 
38
 (pg. 
259
-
269
)
4
Orchard
C.
Eisner
D.
Allen
D.
Oscillations of intracellular Ca2+ in mammalian cardiac muscle
Nature
1983
, vol. 
304
 (pg. 
735
-
738
)
5
Stern
M.
Kort
A.
Bhatnagar
G.
Lakatta
E.
Scattered-light intensity fluctuations in diastolic rat cardiac muscle caused by spontaneous Ca++-dependent cellular mechanical oscillations
J. Gen. Physiol.
1983
, vol. 
82
 (pg. 
119
-
153
)
6
Wier
W.
Kort
A.
Stern
M.
Lakatta
E.
Marban
E.
Cellular calcium fluctuations in mammalian heart: direct evidence from noise analysis of aequorin signals in Purkinje fibers
Proc. Natl. Acad. Sci. U.S.A.
1983
, vol. 
80
 (pg. 
7367
-
7371
)
7
Marban
E.
Robinson
S. W.
Wier
W. G.
Mechanisms of arrhythmogenic delayed and early after depolarizations in ferret ventricular muscle
J. Clin. Invest.
1986
, vol. 
78
 (pg. 
1185
-
1192
)
8
Jiang
D.
Xiao
B.
Yang
D.
Wang
R.
Choi
P.
Zhang
L.
Cheng
H.
Chen
S. R. W.
RyR2 mutations linked to ventricular tachycardia and sudden death reduce the threshold for store-overload-induced Ca2+ release (SOICR)
Proc. Natl. Acad. Sci. U.S.A.
2004
, vol. 
101
 (pg. 
13062
-
13067
)
9
Jiang
D.
Wang
R.
Xiao
B.
Kong
H.
Hunt
D. J.
Choi
P.
Zhang
L.
Chen
S. R. W.
Enhanced store overload-induced Ca2+ release and channel sensitivity to luminal Ca2+ activation are common defects of RyR2 mutations linked to ventricular tachycardia and sudden death
Circ. Res.
2005
, vol. 
97
 (pg. 
1173
-
1181
)
10
Ter Keurs
H. E.
Boyden
P. A.
Calcium and arrhythmogenesis
Physiol. Rev.
2007
, vol. 
87
 (pg. 
457
-
506
)
11
George
C. H.
Jundi
H.
Thomas
N. L.
Fry
D. L.
Lai
F. A.
Ryanodine receptors and ventricular arrhythmias: emerging trends in mutations, mechanisms and therapies
J. Mol. Cell. Cardiol.
2007
, vol. 
42
 (pg. 
34
-
50
)
12
Mohamed
U.
Napolitano
C.
Priori
S. G.
Molecular and electrophysiological bases of catecholaminergic polymorphic ventricular tachycardia
J. Cardiovasc. Electrophysiol.
2007
, vol. 
18
 (pg. 
791
-
797
)
13
Priori
S. G.
Napolitano
C.
Tiso
N.
Memmi
M.
Vignati
G.
Bloise
R.
Sorrentino
V. V.
Danieli
G. A.
Mutations in the cardiac ryanodine receptor gene (hRyR2) underlie catecholaminergic polymorphic ventricular tachycardia
Circulation
2001
, vol. 
103
 (pg. 
196
-
200
)
14
Laitinen
P. J.
Brown
K. M.
Piippo
K.
Swan
H.
Devaney
J. M.
Brahmbhatt
B.
Donarum
E. A.
Marino
M.
Tiso
N.
Viitasalo
M.
, et al. 
Mutations of the cardiac ryanodine receptor (RyR2) gene in familial polymorphic ventricular tachycardia
Circulation
2001
, vol. 
103
 (pg. 
485
-
490
)
15
Tiso
N.
Stephan
D. A.
Nava
A.
Bagattin
A.
Devaney
J. M.
Stanchi
F.
Larderet
G.
Brahmbhatt
B.
Brown
K.
Bauce
B.
, et al. 
Identification of mutations in the cardiac ryanodine receptor gene in families affected with arrhythmogenic right ventricular cardiomyopathy type 2 (ARVD2)
Hum. Mol. Genet.
2001
, vol. 
10
 (pg. 
189
-
194
)
16
Bauce
B.
Rampazzo
A.
Basso
C.
Bagattin
A.
Daliento
L.
Tiso
N.
Turrini
P.
Thiene
G.
Danieli
G. A.
Nava
A.
Screening for ryanodine receptor type 2 mutations in families with effort-induced polymorphic ventricular arrhythmias and sudden death: early diagnosis of asymptomatic carriers
J. Am. Coll. Cardiol.
2002
, vol. 
40
 (pg. 
341
-
349
)
17
Priori
S. G.
Napolitano
C.
Memmi
M.
Colombi
B.
Drago
F.
Gasparini
M.
DeSimone
L.
Coltorti
F.
Bloise
R.
Keegan
R.
, et al. 
Clinical and molecular characterization of patients with catecholaminergic polymorphic ventricular tachycardia
Circulation
2002
, vol. 
106
 (pg. 
69
-
74
)
18
Laitinen
P.
Swan
H.
Piippo
K.
Viitasalo
M.
Toivonen
L.
Kontula
K.
Genes, exercise and sudden death: molecular basis of familial catecholaminergic polymorphic ventricular tachycardia
Ann. Med.
2004
, vol. 
36
 
Suppl. 1
(pg. 
81
-
86
)
19
Bagattin
A.
Veronese
C.
Bauce
B.
Wuyts
W.
Settimo
L.
Nava
A.
Rampazzo
A.
Danieli
G. A.
Denaturing HPLC-based approach for detecting RYR2 mutations involved in malignant arrhythmias
Clin. Chem.
2004
, vol. 
50
 (pg. 
1148
-
1155
)
20
Choi
G.
Kopplin
L. J.
Tester
D. J.
Will
M. L.
Haglund
C. M.
Ackerman
M. J.
Spectrum and frequency of cardiac channel defects in swimming-triggered arrhythmia syndromes
Circulation
2004
, vol. 
110
 (pg. 
2119
-
2124
)
21
Tester
D. J.
Spoon
D. B.
Valdivia
H. H.
Makielski
J. C.
Ackerman
M. J.
Targeted mutational analysis of the RyR2-encoded cardiac ryanodine receptor in sudden unexplained death: a molecular autopsy of 49 medical examiner/coroner's cases
Mayo Clin. Proc.
2004
, vol. 
79
 (pg. 
1380
-
1384
)
22
Priori
S. G.
Napolitano
C.
Cardiac and skeletal muscle disorders caused by mutations in the intracellular Ca2+ release channels
J. Clin. Invest.
2005
, vol. 
115
 (pg. 
2033
-
2038
)
23
Wagenknecht
T.
Radermacher
M.
Grassucci
R.
Berkowitz
J.
Xin
H. B.
Fleischer
S.
Locations of calmodulin and FK506-binding protein on the three-dimensional architecture of the skeletal muscle ryanodine receptor
J. Biol. Chem.
1997
, vol. 
272
 (pg. 
32463
-
32471
)
24
Sharma
M. R.
Jeyakumar
L. H.
Fleischer
S.
Wagenknecht
T.
Three-dimensional visualization of FKBP12.6 binding to cardiac ryanodine receptor (RyR2) in open buffer condition
Biophys. J.
2002
, vol. 
82
 pg. 
644a
 
25
Jiang
D.
Chen
W.
Wang
R.
Zhang
L.
Chen
S. R. W.
Loss of luminal Ca2+ activation in the cardiac ryanodine receptor is associated with ventricular fibrillation and sudden death
Proc. Natl. Acad. Sci. U.S.A.
2007
, vol. 
104
 (pg. 
18309
-
18314
)
26
Welch
W.
Rheault
S.
West
D. J.
Williams
A. J.
A model of the putative pore region of the cardiac ryanodine receptor channel
Biophys. J.
2004
, vol. 
87
 (pg. 
2335
-
2351
)
27
Du
G. G.
Sandhu
B.
Khanna
V. K.
Guo
X. H.
MacLennan
D. H.
Topology of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (RyR1)
Proc. Natl. Acad. Sci. U.S.A.
2002
, vol. 
99
 (pg. 
16725
-
16730
)
28
Lehnart
S. E.
Wehrens
X. H. T.
Laitinen
P. J.
Reiken
S. R.
Deng
S.
Cheng
Z.
Landry
D. W.
Kontula
K.
Swan
H.
Marks
A. R.
Sudden death in familial polymorphic ventricular tachycardia associated with calcium release channel (ryanodine receptor) leak
Circulation
2004
, vol. 
109
 (pg. 
3208
-
3214
)
29
Masumiya
H.
Wang
R.
Zhang
J.
Xiao
B.
Chen
S. R. W.
Localization of the 12.6-kDa FK506-binding protein (FKBP12.6) binding site to the NH2-terminal domain of the cardiac Ca2+ release channel (ryanodine receptor)
J. Biol. Chem.
2003
, vol. 
278
 (pg. 
3786
-
3792
)
30
Ikemoto
N.
Yamamoto
T.
Regulation of calcium release by interdomain interaction within ryanodine receptors
Front. Biosci.
2002
, vol. 
7
 (pg. 
671
-
683
)
31
George
C. H.
Jundi
H.
Thomas
N. L.
Scoote
M.
Walters
N.
Williams
A. J.
Lai
F. A.
Ryanodine receptor regulation by intramolecular interaction between cytoplasmic and transmembrane domains
Mol. Biol. Cell
2004
, vol. 
15
 (pg. 
2627
-
2638
)
32
Palmer
A. E.
Jin
C.
Reed
J. C.
Tsien
R. Y.
Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor
Proc. Natl. Acad. Sci. U.S.A.
2004
, vol. 
101
 (pg. 
17404
-
17409
)
32a
Fabiato
A.
Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands
Methods Enzymol.
1988
, vol. 
157
 (pg. 
378
-
417
)
33
Laemmli
U. K.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4
Nature
1970
, vol. 
227
 (pg. 
680
-
685
)
34
Towbin
H.
Staehelin
T.
Gordon
J.
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications
Proc. Natl. Acad. Sci. U.S.A.
1979
, vol. 
76
 (pg. 
4350
-
4354
)
35
Jiang
D.
Xiao
B.
Zhang
L.
Chen
S. R.
Enhanced basal activity of a cardiac Ca2+ release channel (ryanodine receptor) mutant associated with ventricular tachycardia and sudden death
Circ. Res.
2002
, vol. 
91
 (pg. 
218
-
225
)
36
Shannon
T. R.
Pogwizd
S. M.
Bers
D. M.
Elevated sarcoplasmic reticulum Ca2+ leak in intact ventricular myocytes from rabbits in heart failure
Circ. Res.
2003
, vol. 
93
 (pg. 
592
-
594
)
37
Kubalova
Z.
Gyorke
I.
Terentyeva
R.
Viatchenko-Karpinski
S.
Terentyev
D.
Williams
S. C.
Gyorke
S.
Modulation of cytosolic and intra-sarcoplasmic reticulum calcium waves by calsequestrin in rat cardiac myocytes
J. Physiol.
2004
, vol. 
561
 (pg. 
515
-
524
)
38
Wehrens
X.
Lehnart
S.
Huang
F.
Vest
J.
Reiken
S.
Mohler
P.
Sun
J.
Guatimosim
S.
Song
L.
Rosemblit
N.
, et al. 
FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death
Cell
2003
, vol. 
113
 (pg. 
829
-
840
)
39
George
C. H.
Higgs
G. V.
Lai
F. A.
Ryanodine receptor mutations associated with stress-induced ventricular tachycardia mediate increased calcium release in stimulated cardiomyocytes
Circ. Res.
2003
, vol. 
93
 (pg. 
531
-
540
)