Luminal Ca²⁺ controls activation of the cardiac ryanodine receptor by ATP

The cardiac RYR2 channels provide the fundamental pathway for release of Ca²⁺ ions from the SR into the cytosol. In cardiac myocytes, cytosolic Ca²⁺ is generally considered to be the main physiological activator of the RYR2 channel (Fabiato, 1985; Ashley and Williams, 1990; Stern, 1992b), whereas luminal Ca²⁺ (Fabiato and Fabiato, 1975, 1978; Sitsapesan and Williams, 1994; Györke and Györke, 1998; Xu and Meissner, 1998; Shannon et al., 2000; Laver, 2007a,b; Györke and Terentyev, 2008; Laver and Honen, 2008; Qin et al., 2008) and cytosolic ATP (Meissner, 1984; Rousseau et al., 1986; Meissner and Henderson, 1987; Copello et al., 2002) together with cytosolic Mg²⁺ (Rousseau et al., 1986; Meissner and Henderson, 1987; Ashley and Williams, 1990; Laver et al., 1997; Copello et al., 2002; Zahradníková et al., 2003; Laver and Honen, 2008; Zahradníková et al., 2010) are considered to play a regulatory role.

At the single-channel level, the regulation of the RYR2 channel by luminal Ca²⁺ has been extensively studied since the late 1990s. However, the precise mechanism underlying this process is still not fully understood because the reported effects of luminal Ca²⁺ are diverse and sometimes even confusing. Currently, there is no consensus about how luminal Ca²⁺ regulates the RYR2 channel. The relative importance of Ca²⁺ binding to a luminal site (Györke and Györke, 1998) and Ca²⁺ binding to cytosolic sites, after having permeated the channel (Xu and Meissner, 1998), is intensively debated (Györke et al., 2002; Laver, 2007b; Liu et al., 2010; Diaz-Sylvester et al., 2011). Likewise, the relative roles of the luminal Ca²⁺ regulatory sites located directly on the channel (Sitsapesan and Williams, 1997; Jiang et al., 2007) and those mediated by the associated luminal protein calsequestrin (CSQ2) (Györke et al., 2004; Qin et al., 2008) are also not clear. Luminal regulation of the RYR2 channel in vivo could involve Ca²⁺-sensing mechanisms on both the luminal (CSQ2-dependent and CSQ2-independent) and cytosolic face of the channel (Sitsapesan and Williams, 1997; Györke et al., 2002; Laver, 2007a,b; Knollmann, 2009), and the prominence of one or the other mechanisms could depend on experimental conditions. Specifically, diastolic activation of RYR2 channels at low cytosolic Ca²⁺ may be regulated differently from activation during Ca²⁺-induced Ca²⁺ release, when local cytosolic Ca²⁺ is elevated to >10 µM (Acsai et al., 2011), as the presence of activating cytosolic Ca²⁺ concentration may compete with Ca²⁺ feed-through (Györke and Györke, 1998; Ching et al., 1999). In addition, when extrapolating from in vitro data on reconstituted purified or recombinant RYR2 channels, it has to be noted that modification or loss of RYR2 sensitivity to luminal Ca²⁺ may occur as a result of dissociation or absence of CSQ2 (Xu and Meissner, 1998; Liu et al., 2010).

Although luminal Ca²⁺ regulates fractional Ca²⁺ release during excitation–contraction coupling (Shannon et al., 2000), its role in modulation of diastolic Ca²⁺ leak (Díaz et al., 1997; Györke et al., 1997; Lukyanenko et al., 2000) and spontaneous Ca²⁺ release (Lukyanenko et al., 1996, 1999) may be considered more important, as it has been shown to be altered in several pathological states such as heart failure (Hobai and O’Rourke, 2001; Piacentino et al., 2003) and Ca²⁺-induced arrhythmias (Jiang et al., 2005; Terentyev et al., 2006). Hence, in this paper, we were interested in diastolic regulation of the native RYR2 channel. We therefore investigated RYR2 activity in planar lipid bilayers at a diastolic level of cytosolic Ca²⁺ (100 nM) at a wide range of ATP and luminal Ca²⁺, and at a diastolic level of luminal Ca²⁺ (1 mM) at a wide range of ATP and cytosolic Ca²⁺. Furthermore, we investigated the inhibition of ATP-activated RYR2 channels by cytosolic Mg²⁺ at a wide range of luminal Ca²⁺. The data were interpreted in the framework of an allosteric model of RYR2 activation by ATP. We show that luminal Ca²⁺ potentiates the ATP-activated RYR2 channel by binding to a luminal site with an apparent affinity in the millimolar range, over which local Ca²⁺ in the lumen of the SR likely varies in cardiac myocytes. In addition, we show that the inhibitory effect of cytosolic Mg²⁺ on ATP-activated RYR2 channels is not affected by luminal Ca²⁺, which is consistent with the action of Mg²⁺ at the cytosolic activation site of the RYR2 channel.

The rats were anesthetized with sodium pentobarbital (100 mg kg⁻¹, intraperitoneally). All anesthetic and surgical procedures were approved by the State veterinary and food administration of the Slovak Republic (Ro-3820/05-221, Ro-2821/09-221, and Ro-2672/08-221). Cardiac SR microsomes were isolated by differential centrifugation from the ventricles of rat hearts, as described previously (Gaburjakova and Gaburjakova, 2006).

RYR2 channels were incorporated into a bilayer lipid membrane (BLM), and single-channel currents were recorded under voltage-clamp conditions. Cardiac SR microsomes were added to the cis chamber near the BLM formed from a 3:1 mixture of phosphatidyl ethanolamine with phosphatidyl serine (Avanti Polar Lipids, Inc.) across a 20–50-µm aperture in the wall of a polystyrene cup. The cis chamber (corresponding to the cytosol) was filled with 1 ml of (in mM): 250 HEPES, 125 Tris, 50 KCl, 1 EGTA, and 0.5–0.754 CaCl₂, pH 7.35. Cytosolic Mg²⁺ and ATP were added as MgSO₄ and ATPNa₂, respectively. The channels responded to the addition of Mg²⁺ and ATP to their cytosolic face within a few seconds. The concentration of EGTA was determined by potentiometric titration with a standard 100-mM CaCl₂ solution (Fluka) at a constant pH of 7.35, using a Ca²⁺-selective electrode (type 25–20+; Elektrochemické Detektory, Ltd.), and free [Ca²⁺]_C and free [Mg²⁺]_C were calculated with WinMaxc32 (version 2.50; Bers et al., 1994).

The trans chamber (corresponding to the lumen) was filled with 1 ml of (in mM): 50 KCl, 250 HEPES, and 0.5–53 Ca(OH)₂. When 0.5 and 1 mM Ca(OH)₂ was present, 7.5 and 7.0 mM Ba(OH)₂, respectively, were added into the solution to increase the current amplitude to a level optimal for recording. In this case, both ions served as charge carriers. Experiments with 8 mM [Ba²⁺]_L as the sole permeant ion mimicked the situation in which no Ca²⁺ is present at the luminal face of the RYR2 channel (Gaburjakova and Gaburjakova, 2006). The level of contaminating Ca²⁺ in the trans solution with 8 mM [Ba²⁺]_L was 5 µM, as determined potentiometrically using a Ca²⁺-selective electrode. To minimize the changes in both ionic strength and osmolality, which have been shown to affect the basal activity of the RYR2 channel (Jiang et al., 2002, 2004, 2007), we kept the concentration of HEPES in the trans chamber constant and Tris (50–140 mM) was used to buffer pH to 7.35. The ionic strength of the trans solutions increased with luminal Ca²⁺ and ranged from 216 mM at 0.005–8 mM [Ca²⁺]_L to 290 mM at 53 mM [Ca²⁺]_L, and their osmolality decreased with luminal Ca²⁺ and ranged from 542 mM at 0.005–8 mM [Ca²⁺]_L to 430 mM at 53 mM [Ca²⁺]_L.

The trans chamber was connected to the head-stage input of an amplifier (MultiClamp 700B; Molecular Devices), and the cis chamber was held at ground. The holding potential was 0 mV. Electrical signals were filtered at 1 kHz, digitized at 4 kHz by an A/D converter (Digidata 1440A; Molecular Devices), and analyzed. Data acquisition and analysis were performed with pCLAMP 10 (Molecular Devices). Channel activity was continuously recorded for >2 min when only a single channel was incorporated into a BLM. To analyze gating behavior, the records were divided into 30-s intervals, and the open probability (P_O) and channel gating parameters were determined for each interval. The average open (t_O) and closed times (t_C) were calculated on these intervals as a standard arithmetic average, and for the calculation of t_O, at least 40 opening events (from several single channels) were collected, as suggested by Laver (2007b).

All chemicals were from Sigma-Aldrich, if not stated otherwise.

The dependence of P_O on ATP concentration was characterized by fitting the values of open probability by the Hill function,

where $POmin$ is the open probability in the absence of ATP, $POmax$ is the maximum achievable open probability induced by ATP, EC₅₀ is the ATP concentration inducing half-maximal activation, and n_H is the Hill coefficient. The values of $POmin$ could not be reliably fitted with Eq. 1; therefore, they were set to the average P_O at the given [Ca²⁺]_L and [Ca²⁺]_C in the absence of ATP.

The dependence of normalized P_O on Mg²⁺ concentration was characterized by fitting the values of relative open probability by the Hill function,

where P_O0 is the open probability in the absence of Mg²⁺, IC₅₀ is the Mg²⁺ concentration leading to half-maximal inactivation, and n_H is the Hill coefficient.

The action of ATP was further quantified within the framework of the allosteric model of RYR2 activation (Monod et al., 1965; Zahradník et al., 2005). The model is depicted in Fig. 1. Because $POmax$ in the presence of luminal Ca²⁺ and ATP reached 1, the low activity and inactivated modes were not accounted for. The model has five closed (Fig. 1, top row) and five open states (Fig. 1, bottom row) in which the channel tetramers contain 0–4 ATP-bound monomers (from left to right). The opening probability in the absence of ATP is determined by the equilibrium constant of the opening transition of the ATP-free tetramer, K_O0 = [C₀]/[O₀], where [C₀] and [O₀] are the probabilities of ATP-free closed and open states, respectively. Binding of ATP to the closed channel is defined by the microscopic ATP dissociation constant of the RYR2 monomer in the closed state, K_ATP. ATP exerts its effect by binding more strongly to RYR2 monomers in the open than to those in the closed states of the channel, and this property is defined by the allosteric factor f_ATP. Because of the principle of detailed balance, the equilibrium constants of the opening transitions of ATP-bound tetramers are affected as well (Monod et al., 1965; Zahradník et al., 2005). As a result, the propensity for opening of the whole channel tetramer is allosterically increased by the binding of ATP to each monomer.

The ATP dependence of RYR2 P_O has the following form:

where [ATP] is ATP concentration, and the remaining parameters have been defined previously.

The effect of Ca²⁺ feed-through on the local cytosolic Ca²⁺ concentrations at a distance r from the RYR2 pore, [Ca²⁺]_C, was calculated according to Stern (1992a) using the equation

where i_Ca is the Ca²⁺ current amplitude, D_Ca = 3 × 10⁻¹⁰ m² s⁻¹ is the Ca²⁺ diffusion coefficient, F = 96,500 C mol⁻¹ is the Faraday constant, and [B] = 1 mM is the concentration of the Ca²⁺ buffer (EGTA or ATP) at the cytosolic side of the channel. The rates of Ca²⁺ binding, k, were 1.5 × 10⁶ M⁻¹ s⁻¹ for EGTA (Stern, 1992a) and 2.25 × 10⁸ M⁻¹ s⁻¹ for ATP (Michailova and McCulloch, 2001).

In all fitting procedures, if a fitted parameter did not change significantly for a set of tested conditions ([Ca²⁺]_L, [Ca²⁺]_C, [ATP], [Mg²⁺]_C), fitting was repeated with a model in which this parameter was independent of these conditions. The quality of the resulting fits was compared using the F-test at a significance level of P = 0.05. When two models did not show a significant difference, the model with fewer parameters was chosen.

Calculations and fitting were performed in Origin (version 8E; OriginLab). Eq. 3 was derived in Mathematica (version 8; Wolfram Research). The results are reported as average ± SEM. The fitted parameters are reported with the standard error of the fit. Statistical comparisons (P < 0.05) of differences were made by either the Student’s t test or ANOVA.

Fig. S1 documents the presence of CSQ2 in the cardiac SR microsomes used for reconstitution of the RYR2 channel into the BLM. Standard Western blotting method was used to detect CSQ2 (see legend to Fig. S1). Fig. S2 displays the theoretical dependence of local [Ca²⁺]_C at a distance r from the channel pore for 1, 8, 26, and 53 mM [Ca²⁺]_L, calculated using Eq. 4. Parameters for calculation were taken from Stern (1992a). Figs. S1 and S2 are available.

Rat cardiac SR microsomes were fused into a BLM, and single-channel currents were recorded under asymmetric conditions with luminal Ca²⁺ and/or Ba²⁺ as charge carriers at a membrane potential of 0 mV.

The activity of three to seven channels was recorded at 0.005, 1, 8, 15, 26, and 53 mM [Ca²⁺]_L; 100 nM [Ca²⁺]_C; and 0–30 mM ATP (27 single channels). Typical current traces of a single RYR2 channel in the presence of 100 nM [Ca²⁺]_C at 0.005, 8, and 53 mM [Ca²⁺]_L are illustrated in Fig. 2 A, and the effect of luminal Ca²⁺ on the dose–response of the RYR2 channel to ATP is illustrated in Fig. 2 B (top). Solid lines are the best fits by the Hill equation (Eq. 1). The fitted parameters are summarized in Table 1. Substantial activation of the RYR2 channel could be achieved only at high levels of luminal Ca²⁺ (P_O = 0.267 ± 0.047 at 2.5 mM ATP for 8 mM [Ca²⁺]_L and 0.955 ± 0.015 at 2 mM ATP for [Ca²⁺]_L = 53 mM), whereas at 5 µM [Ca²⁺]_L, the increase of activity, although over 10-fold, led to only a marginal open probability (0.0041 ± 0.0009 at 30 mM ATP).

The luminal Ca²⁺ dependence of RYR2 open probability in the absence of ATP, $POmin$⁠, and of the fitted values of $POmax$ and EC₅₀ is depicted in Fig. 2 B (middle and bottom). Elevation of luminal Ca²⁺ from 5 µM to 1 mM led to an increase in $POmax$ from 0.0048 ± 0.0007 to 0.051 ± 0.017 (Fig. 2 B, middle, and Table 1). Despite the 10-fold increase in $POmax$⁠, the resulting open probability was still in the low range. Further increases of [Ca²⁺]_L progressively increased maximal activation of the channel with EC₅₀ for luminal Ca²⁺ of 14.3 ± 2.0 mM (Fig. 2 B, middle, solid line), and full activation occurred at [Ca²⁺]_L = 53 mM. Notably, luminal Ca²⁺ also modulated RYR2 activity in the absence of ATP, albeit to a lesser extent. $POmin$ changed only negligibly for [Ca²⁺]_L < 26 mM, but it increased significantly at 26 and 53 mM [Ca²⁺]_L (P < 0.05; Fig. 2 B, middle). Although high luminal Ca²⁺ was necessary to achieve substantial activation of the RYR2 channel by ATP, 1 mM [Ca²⁺]_L was sufficient to decrease the value of EC₅₀ for ATP from 9.9 ± 1.8 mM to 0.549 ± 0.035 mM. EC₅₀ did not further decrease at higher [Ca²⁺]_L (Fig. 2 B, bottom).

The mechanism by which luminal Ca²⁺ modulated RYR2 activation by ATP was investigated by analyzing the average open (t_O) and closed times (t_C). Channel openings were much shorter at micromolar (Fig. 2 A, top) than at millimolar levels of luminal Ca²⁺ (Fig. 2 A, middle and bottom). In Fig. 2 C, the values of t_O and t_C of RYR2 channels activated by different concentrations of ATP (0–30 mM) are plotted against P_O. Notably, at 5 µM and 1 mM [Ca²⁺]_L, the data are available only for very low P_O because under these conditions, ATP induced only slight activation of the channel (Fig. 2 C, insets). Nevertheless, in the P_O interval of 0–0.1, a steep increase of t_O was apparent even at 1 mM [Ca²⁺]_L, and a steep decrease of t_C was prominent under all tested conditions. Importantly, both t_O and t_C were larger at millimolar than at micromolar levels of luminal Ca²⁺ for any P_O value (Fig. 2 C, insets). In addition, in the [Ca²⁺]_L range of 8 to 26 mM, the values of t_O and t_C at a given P_O were independent of [Ca²⁺]_L, although the ATP concentrations at these P_O values were substantially different for different [Ca²⁺]_L. Furthermore, Fig. 2 C demonstrates that at all [Ca²⁺]_L, the values of t_O did not change appreciably with channel open probability in the P_O interval of 0.2–0.9, whereas the values of t_C decreased substantially in this P_O interval. Full activation of the RYR2 channel in the presence of 53 mM [Ca²⁺]_L was manifested by a further increase in t_O and a decrease in t_C for P_O > 0.9. It is possible that these changes of average dwell times were caused by incompletely resolved channel closures under these conditions (see Fig. 2 A, bottom trace in the bottom panel).

Although t_C decreased with increasing [ATP] regardless of the presence of luminal Ca²⁺ (Fig. 2 D, bottom), a different situation was found for t_O. At 5 µM of luminal Ca²⁺, t_O was not significantly prolonged in the presence of up to 30 mM ATP. In the presence of millimolar levels of luminal Ca²⁺, the increase in open time saturated at [ATP] < 1 mM. Importantly, under conditions when ATP led to substantial activation of the channel ([Ca²⁺]_L ≥ 8 mM and [ATP] ≥ 1 mM), the open time was dependent neither on luminal Ca²⁺ concentration nor on ATP concentration (Fig. 2 D, top), despite the large differences in P_O (0.2–1; Fig. 2 B, top). Although ATP modulated both gating parameters, clearly the rise in P_O was predominantly associated with a decrease in the closed time, which spanned over two orders of magnitude, was graded with both luminal Ca²⁺ and ATP concentration, and was the steepest and the largest for 53 mM [Ca²⁺]_L (Fig. 2 D, bottom). Collectively, luminal Ca²⁺ markedly amplified most effects of ATP on the activity and gating of RYR2 channels at 100 nM [Ca²⁺]_C by decreasing the closed times at all ATP concentrations.

It was quite surprising that under quasi-physiological conditions, when 1 mM [Ca²⁺]_L was present, the maximal activity of the RYR2 channel induced by ATP was very low at 100 nM [Ca²⁺]_C. This led us to the question of whether cytosolic Ca²⁺, which is the main physiological activator, could modulate the effect of luminal Ca²⁺ on the ATP-activated RYR2 channel. To address this question, we performed a series of experiments in which we fixed [Ca²⁺]_L at the physiological level of 1 mM and examined the whole dose–response of the RYR2 channel to ATP (0–10 mM) at five different sub-activating cytosolic Ca²⁺ concentrations. Activity of three to six channels was recorded at 100, 180, 250, 330, and 410 nM [Ca²⁺]_C (22 single channels). In the absence of ATP, the RYR2 channel had a P_O of <1% when [Ca²⁺]_C was varied from 100 to 410 nM. This indicates that cytosolic Ca²⁺ in this range did not activate the RYR2 channel substantially. Fig. 3 A illustrates the activating effect of ATP on representative RYR2 channels in the presence of 100, 180, and 410 nM [Ca²⁺]_C. At 410 nM [Ca²⁺]_C, submillimolar levels of ATP activated RYR2 channels to P_O > 0.9 (Fig. 3 A, bottom). Fig. 3 B (top panel) describes the relationship between P_O and ATP concentration at various [Ca²⁺]_C. Solid lines are fits by Eq. 1, and their parameters are given in Table 2. In Fig. 3 B (middle and bottom), the values of $POmin$ calculated from the experimental data and the fitted values of $POmax$ and EC₅₀ are plotted as a function of [Ca²⁺]_C. In contrast to the effect of luminal Ca²⁺, all parameters (⁠$POmin$⁠, $POmax$ , and EC₅₀) were considerably changed by cytosolic Ca²⁺. $POmin$ increased by about an order of magnitude, $POmax$ increased 20 times, and EC₅₀ decreased 5 times when [Ca²⁺]_C was changed from 100 to 410 nM. Our results indicate that a relatively small change in [Ca²⁺]_C, which does not induce substantial activation of the RYR2 channel in the absence of ATP, leads to dramatic changes in the $POmax$ and EC₅₀ of ATP in the presence of 1 mM [Ca²⁺]_L.

Luminal Ca²⁺ considerably affected the gating profile of ATP-activated RYR2 channels (Fig. 2, C and D). In contrast, no noticeable changes in the gating parameters were visible in the current traces that could be generated by the changes in cytosolic Ca²⁺ from 100 to 330 nM (Fig. 3 A). Fig. 3 C shows that the dependence of t_O and t_C on the value of P_O at 100–330 nM [Ca²⁺]_C is virtually identical. However, at 410 nM [Ca²⁺]_C, both t_O and t_C were slightly shorter at any value of P_O, which resulted in a higher frequency of channel openings (Fig. 3 A, the third trace in the bottom panel). The changes of t_O and t_C at the lower and upper extremes of P_O were similar to those obtained by varying luminal Ca²⁺. The main factor causing the increase in channel P_O in the range of 0.2 to 0.9 was the decrease in t_C.

The values of t_O gradually increased and those of t_C decreased with increasing [ATP] regardless of [Ca²⁺]_C (Fig. 3 D). In contrast to luminal Ca²⁺, cytosolic Ca²⁺ strongly affected the ATP dependence of t_O. The ATP dependence of t_C changed with [Ca²⁺]_C in a similar manner as it did with [Ca²⁺]_L: the data for intermediate values of [Ca²⁺]_C (180–330 nM) superimposed, whereas t_C at 100 nM [Ca²⁺]_C was larger and at 410 nM [Ca²⁺]_C was smaller. In all cases, the main factor causing the increase in channel P_O was the decrease in t_C accompanied by less extensive changes in t_O. Collectively, our results reveal that Ca²⁺ ions at low cytosolic concentrations, at which they by themselves do not activate the RYR2 channel substantially, do synergistically increase the activation effect of ATP on RYR2 channels in the presence of 1 mM [Ca²⁺]_L, predominantly by decreasing the closed times at all ATP concentrations.

To understand the impact of cytosolic Ca²⁺ on the luminal Ca²⁺ activation of the RYR2 channel, we characterized the dependence of RYR2 open probability on luminal Ca²⁺ in the presence of 10 mM ATP at 180 and 410 nM [Ca²⁺]_C. Activity of three to five channels was recorded at 0.5, 1, 8, and 53 mM [Ca²⁺]_L (16 single channels).

The ATP concentration was sufficient to achieve the maximal level of RYR2 activation at all tested [Ca²⁺]_L. Fig. 4 A summarizes this series of experiments where P_O is plotted against [Ca²⁺]_L for 180 and 410 nM [Ca²⁺]_C. The dashed line is the fit of the data at 100 nM [Ca²⁺]_C replotted from Fig. 2 B (middle). It is obvious that increasing [Ca²⁺]_C from 100 to 180 nM effectively shifted EC₅₀ for luminal Ca²⁺ from 14.3 ± 2.0 mM to 0.897 ± 0.085 mM, which is already a physiologically relevant value (Fig. 4 B). Further, elevation of [Ca²⁺]_C to 410 nM led to a much smaller decrease in EC₅₀ to 0.631 ± 0.049 mM (Fig. 4 B).

The open times in the absence of ATP dose-dependently increased with [Ca²⁺]_L (Fig. 4 C). Although the extent of t_O prolongation was larger at 410 than at 100 nM [Ca²⁺]_C, the EC₅₀ for t_O prolongation by luminal Ca²⁺ did not change with [Ca²⁺]_C and was 1.68 ± 0.55 mM. At 10 mM ATP, the mean open times did not significantly depend on cytosolic Ca²⁺ (Fig. 4 D). Again, they dose-dependently increased with [Ca²⁺]_L, with EC₅₀ = 2.7 ± 1.0 mM. The values at 53 mM [Ca²⁺]_L were omitted from the fit, as the proportion of unresolved short closures, leading to overestimation of the open time, was very high under these conditions (see Fig. 2 A, bottom trace in the bottom panel). Interestingly, the values of EC₅₀ for the prolongation of open times by luminal Ca²⁺ in the absence and presence of ATP were not significantly different.

Although the exact nature of ATP-binding sites is not certain, it is assumed that these sites reside on individual monomers (Nakai et al., 1990; Otsu et al., 1990). Therefore, we attempted to describe the effect of luminal and cytosolic Ca²⁺ on ATP activation of the RYR2 channel as modulation of ATP binding to individual monomers. The mechanism of channel activation by ATP in the model (Fig. 1) was analogous to that of allosteric activation of the RYR2 channel by cytosolic Ca²⁺ (Zahradník et al., 2005); i.e., the binding of ATP to each monomer was assumed to allosterically increase the propensity for opening of the whole channel tetramer. The open probability in the absence of ATP is determined by the equilibrium constant of the opening transition of the ATP-free tetramer, K_O0, which reflects the level of channel activation by luminal and cytosolic Ca²⁺. Binding of ATP to the closed channel is defined by the microscopic ATP dissociation constant of the RYR2 monomer in the closed state, K_ATP. ATP exerts its effect by binding more strongly to RYR2 monomers in the open than to those in the closed states of the channel, and this property is defined by the allosteric factor f_ATP. The results of fitting the ATP dependence of RYR2 open probabilities at different concentrations of luminal and cytosolic Ca²⁺ (data from Figs. 2 and 3) by the allosteric model of RYR activation (Eq. 3) are shown in Fig. 5. Optimal fit required the sharing of K_ATP for all conditions except at 5 µM of luminal Ca²⁺. The theoretical prediction of this model was in a very good agreement with the experimental data. The parameters of the best fit are shown in Table 3.

At a constant cytosolic Ca²⁺ of 100 nM, the concentration dependence of allosteric activation of the RYR2 channel by ATP at different [Ca²⁺]_L is shown in Fig. 5 A. The increase of luminal Ca²⁺ from 5 µM to 1 mM led to a decrease of K_ATP by over an order of magnitude and was accompanied by an increase of the allosteric effect of ATP (decrease of f_ATP). The decrease of K_O0 was not significant. Further increase of [Ca²⁺]_L up to 53 mM did not affect ATP binding to the closed RYR2 channel. At lower [Ca²⁺]_L (≤8 mM), K_O0 and f_ATP decreased in parallel, but for [Ca²⁺]_L ≥ 8 mM, K_O0 changed by more than one order of magnitude, whereas f_ATP did not change significantly (Fig. 5 C). As a result, there was a marginally significant correlation (R = 0.99 and P = 0.066) between log(K_O0), proportional to the energy difference between the open and the closed state in the absence of ATP, and f_ATP for [Ca²⁺]_L ≤ 8 mM, and no correlation (R = 0.62 and P = 0.37) between these quantities for [Ca²⁺]_L ≥ 8 mM. These results explain why EC₅₀ for ATP did not change significantly at [Ca²⁺]_L ≥ 8 mM (Fig. 2 B, bottom). At the same time, there was a significant linear correlation (Fig. 5 D; R = 0.99 and P < 0.005) between t_O0 and f_ATP for all [Ca²⁺]_L, indicating that the prolongation of the open states of the ATP-free RYR2 channel by luminal Ca²⁺ occurred in parallel with the decrease of their microscopic ATP dissociation constant. Of note, t_O0 and f_ATP changed significantly only at [Ca²⁺]_L ≤ 8 mM, showing that the increase in $POmax$ at elevated [Ca²⁺]_L (≥8 mM; Fig. 2 B, middle) was caused solely by the decrease in K_O0, i.e., by the increased open probability in the absence of ATP.

At a constant luminal Ca²⁺ of 1 mM, the concentration dependence of allosteric activation of the RYR2 channel by ATP at different [Ca²⁺]_C is shown in Fig. 5 B. The value of K_ATP was not affected by [Ca²⁺]_C (Table 3). This result means that the substantial changes of EC₅₀ for ATP that are evoked by changing [Ca²⁺]_C (Fig. 3 B, bottom) occur in the absence of changes in the microscopic ATP dissociation constant of the closed RYR2 channel. The variation in EC₅₀ was fully accounted for by the changes of f_ATP, i.e., by the decrease of the microscopic ATP dissociation constant of the RYR2 open state with increasing [Ca²⁺]_C. Under these conditions, the increase in $POmax$ at elevated [Ca²⁺]_C (Fig. 3 B, middle) was brought about by changes of both K_O0 and f_ATP. There was a significant linear correlation (Fig. 5 C; R = 0.97 and P < 0.01) between log(K_O0) and f_ATP. On the other hand, no significant correlation was observed between t_O0 and f_ATP for variation of [Ca²⁺]_C (R = 0.5 and P = 0.39). The changes of f_ATP upon variation of cytoplasmic Ca²⁺ suggest that although ATP binds more strongly to the long open states of the RYR2 channel, induced by low (<8 mM) luminal Ca²⁺, the stability of the ATP-bound open states is further increased by cytoplasmic Ca²⁺ in the absence of an effect on channel open times.

To this point, the functional profile of the ATP-activated RYR2 channels was examined in the absence of Mg²⁺. However, in cardiac myocytes, cytoplasmic Mg²⁺ is a potent inhibitor of Ca²⁺ release from the SR; therefore, we assessed how Mg²⁺ would antagonize the potentiating effects of luminal Ca²⁺ on ATP-activated RYR2 channels. Specifically, to determine whether luminal Ca²⁺ affects the inhibitory potency of cytosolic Mg²⁺, we have examined the effect of cytosolic Mg²⁺ on the activity of RYR2 channels fully activated by 10 mM ATP at 410 nM [Ca²⁺]_C. Under the conditions of these experiments, the concentration of free [ATP²⁻] did not decrease below 2.5 mM. Thus, both [ATP²⁻] and total [ATP] were sufficient to saturate the ATP-binding sites. Activity of three to five channels was recorded at 0.5, 1, 8, and 53 mM [Ca²⁺]_L (17 single channels).

Typical current traces of a single RYR2 channel at 0.5, 1, and 8 mM [Ca²⁺]_L are illustrated in Fig. 6 A. In all cases, 0.4–0.5 mM of free [Mg²⁺]_C caused almost full inhibition of the channel activity. The effect of luminal Ca²⁺ on the dose–response of the RYR2 channel to Mg²⁺ is summarized in Fig. 6 B. Normalized P_O is plotted against free [Mg²⁺]_C for channels recorded at 0.5, 1, 8, and 53 mM [Ca²⁺]_L. The solid line in Fig. 6 B is the best fit by the Hill equation (Eq. 2). The value of IC₅₀ did not depend on [Ca²⁺]_L and was 61.5 ± 4.2 µM.

CSQ2 is important in regulation of the RYR2 channel by luminal Ca²⁺. To ascertain the presence of CSQ2, standard Western blotting was performed, confirming that our preparation contained a detectable amount of CSQ2 (Fig. S1). The stability of CSQ2 binding to the RYR2 channel was further tested by monitoring RYR2 activity after exposure to 8 mM [Ca²⁺]_L. When the channels were negligibly activated by cytosolic Ca²⁺ (100–391 nM) at 8 mM [Ca²⁺]_L, their open probability remained stationary (0.0105 ± 0.0081) over a time period of at least 15 min (not depicted). Furthermore, when the channels were activated by cytosolic Ca²⁺ at concentrations (950 nM to 2 µM) that have been shown to activate CSQ2-bound RYR2 channel considerably but to induce only partial activation of the CSQ2-stripped channel (Qin et al., 2008), the open probability of 50% of RYR2 channels was stable during a 20-min exposure to 8 mM [Ca²⁺]_L, and the activity of the remaining 50% of RYR2 channels significantly and abruptly declined to 37.1 ± 9.2% after 10–20 min. This reduction of RYR2 open probability may be attributed to CSQ2 dissociation (Qin et al., 2008; Liu et al., 2010; Wei et al., 2009).

In this study, the effects of luminal Ca²⁺ on ATP-activated RYR2 channels were investigated. Our main aim was to characterize the regulation of the RYR2 channel relevant to diastolic Ca²⁺ release. To determine the principles by which diastolic RYR2 activation by luminal Ca²⁺ and ATP is governed, we have used a wide range of luminal Ca²⁺ and cytosolic ATP to resolve the shape of the concentration dependence of RYR2 open probability. The experiments revealed strong nonlinearities in the behavior of the RYR2 channel, namely, a strong dependence of activation by ATP on both luminal and cytosolic Ca²⁺ and a strong dependence of activation by luminal Ca²⁺ on cytosolic Ca²⁺.

Our results clearly show that luminal Ca²⁺ is a potent regulator of ATP-activated RYR2 channels, as documented by a dramatic increase in $POmax$ from almost zero to almost one when [Ca²⁺]_L was changed from 1 to 53 mM at 100 nM [Ca²⁺]_C (Fig. 2, A and B). Three separate effects of luminal Ca²⁺ on the activity of the RYR2 channel could be discerned (Fig. 7). The first effect (Fig. 7, left) is fully developed at 1 mM [Ca²⁺]_L and 100 nM [Ca²⁺]_C, but it affects neither RYR2 open probability in the absence of ATP nor the maximum attainable open probability in the presence of ATP (⁠$POmax$⁠). It transpires as an increase in the sensitivity of the RYR2 channel to ATP (decrease of EC₅₀ for ATP) as a result of a decrease of the microscopic ATP dissociation constant of the closed RYR2 channel (K_ATP). The EC₅₀ values of the cytosolic A-site and the luminal L-site are 1 and 40–60 µM, respectively (Laver, 2007b, 2009; Laver and Honen, 2008). Calculations of local [Ca²⁺]_C in the vicinity of the channel pore (Fig. S2) predict local [Ca²⁺]_C of 27–45 µM at [Ca²⁺]_L of 1 mM, so that both the A-site and L-site should be saturated under these conditions. This effect is therefore consistent with action at previously observed cytosolic or luminal Ca²⁺-binding sites.

The second effect (Fig. 7, middle) has millimolar affinity (1.7 ± 0.6 mM), is independent of cytosolic Ca²⁺, and transpires as prolongation of the mean open time in the absence of ATP (t_O0) and as an increase in the relative stability of the ATP-bound open state (decrease of f_ATP) without a change of EC₅₀ for ATP. Because even small changes in the cytosolic Ca²⁺ increased $POmax$ in parallel with decreasing EC₅₀ for ATP, and because local [Ca²⁺]_C during RYR2 openings was sufficient to fully saturate the cytosolic A-site (Fig. S2), neither an increase of t_O0 nor a decrease of f_ATP can be attributed to a feed-through effect of luminal Ca²⁺. We propose that these effects are mediated by a previously uncharacterized true luminal Ca²⁺ site (L_A-site), different from the L-site described by Laver (2009).

The third effect (Fig. 7, right) is apparent at ≥8 mM [Ca²⁺]_L, transpires as an increase of RYR2 open probability in the absence of ATP (decrease of K_O0), and thus indirectly increases activation of the channel in the presence of ATP without affecting ATP binding to either closed (K_ATP) or open states (f_ATP). The low diastolic open probabilities in the absence of ATP precluded reliable determination of the concentration dependence of this process, and therefore it is not clear whether it is caused by Ca²⁺ binding to the L_A-site or by another mechanism.

It has been observed previously that activation of RYR2 channel by luminal Ca²⁺ is most prominent in the presence of agonists (Sitsapesan and Williams, 1994, 1997; Lukyanenko et al., 1996; Györke and Györke, 1998; Laver, 2007b). Two competing hypotheses were proposed to explain this phenomenon: that the luminal Ca²⁺ sites are inaccessible in the absence of the agonist (Sitsapesan and Williams, 1997), or that luminal-triggered Ca²⁺ feed-through is necessary to sufficiently amplify the effect of ATP on the relative stability of the open and closed RYR2 conformation (Laver, 2007b). Our experiments showed that the effect of luminal Ca²⁺ is operative in the absence of ATP but that it is strongly amplified by ATP binding to the channel. The amplification was not caused by Ca²⁺ feed-through but rather by allosteric interaction between the cytosolic and luminal Ca²⁺-binding sites and the ATP-binding site (Fig. 7).

The increase of t_O by ATP in the presence of luminal Ca²⁺ was proposed previously to be caused by Ca²⁺ binding to the A-site (Laver, 2007b). In contrast with this supposition, we have observed a significant and prominent increase of the open time in the absence of ATP (t_O0) by luminal Ca²⁺ (Fig. 4 C). Moreover, the increase of open time by luminal Ca²⁺ was half-maximal at 1.7–2.7 mM [Ca²⁺]_L independently of [ATP] and of [Ca²⁺]_C. The steady-state gradient around the open RYR2 channel leads to local [Ca²⁺]_C >10 µM under these conditions (Fig. S2); thus, binding of the permeating Ca²⁺ ions to the A-site via feed-through could not be responsible for this effect. Luminal Ca²⁺ therefore affects RYR2 open time without acting at the cytosolic site; i.e., occupation of the L_A site allosterically increases the kinetic stability of the open RYR2 conformation regardless of the presence of ATP. This result is important, as prolongation of open times by luminal Ca²⁺ has been considered previously the hallmark of “feed-through” regulation (Xu and Meissner, 1998; Laver, 2007b).

When expressed as the effect on t_O0 (EC₅₀ = 1.7 ± 0.6 mM), the EC₅₀ of the L_A site is comparable to that described by Györke and Györke (1998): EC₅₀ = 2–3 mM. When expressed as the effect on $POmax$⁠, the EC₅₀ of the L_A site, although substantially higher than that described by Györke and Györke (1998) at 100 nM [Ca²⁺]_C (EC₅₀ = 14.3 ± 2.0 mM), decreases to a comparable value (0.5–1 mM) at 180–410 nM [Ca²⁺]_C. Because the data of Györke and Györke (1998) were obtained at a cytosolic Ca²⁺ of 1 µM, the concentration dependence of the effects observed by Györke and Györke (1998) is in a good accordance with the luminal Ca²⁺ sensitivity of the L_A site for both prolongation of RYR2 open time and increasing RYR2 open probability.

Analysis of the concentration dependence of ATP activation using an allosteric model of RYR2 activation by ATP has revealed two important differences between the action of luminal and cytosolic Ca²⁺ on the ATP-activated RYR2 channel. First, luminal Ca²⁺ affected f_ATP inasmuch as it prolonged the mean open time of the ATP-free RYR2 channel (t_O0), whereas cytosolic Ca²⁺, despite having a stronger effect on f_ATP, did not affect t_O0 significantly. Second, the effect of high luminal Ca²⁺ (≥8 mM) on the open probability of the ATP-free RYR2 channel (a decrease of K_O0) did not affect the allosteric action of ATP (f_ATP), whereas cytosolic Ca²⁺ affected both parameters in parallel (Fig. 5 C). This implies that ATP-free open states with different properties were induced by variation of [Ca²⁺]_L and [Ca²⁺]_C. The combined effect of cytosolic Ca²⁺ on K_O0 and f_ATP at [Ca²⁺]_L = 1 mM indicates that increased occupancy of cytosolic Ca²⁺ sites increases the relative stability of ATP binding to the open states of the RYR2 channel. These results suggest that the effect of ATP might be mediated by the free energy of the RYR2 open states in the absence of ATP, i.e., indirectly by the allosteric effect of the occupancy of the channel by Ca²⁺ at the cytosolic A-site and at the luminal L_A site.

CSQ2 is an important dynamic regulator of SR Ca²⁺ release not only because of its Ca²⁺-buffering function but also as a putative luminal Ca²⁺ sensor for the RYR2 channel (Györke et al., 2004; Liu et al., 2010). CSQ2 presence was confirmed by Western blotting (Fig. S1), indicating that this protein was likely tightly bound to RYR2 channels, as it has been reported that the final crude SR preparation contains only ∼5% of the CSQ2 that had been originally present in the ventricular tissue (Murphy et al., 2011). The presence of CSQ2 in our membrane preparation also implies that triadin and junctin were present as well, as these membrane proteins are localized in the close proximity to RYR2 channels and anchor CSQ2 to these channels (Zhang et al., 1997).

Most of our experiments were performed at high [Ca²⁺]_L (≥5 mM) that has been used by several laboratories to dissociate CSQ2 from the RYR2 channel complex after a prolonged exposure (Zhang et al., 1997; Beard et al., 2002; Qin et al., 2008, 2009; Wei et al., 2009). Each laboratory used specific conditions defined by [Ca²⁺]_L, luminal ionic strength, duration of high [Ca²⁺]_L exposure, and the extent of RYR2 activation by cytosolic agonists, the significance of which in the CSQ2 dissociation process is not known. Our data demonstrate that under the conditions of our experiments, 8 mM [Ca²⁺]_L is not strong enough to release CSQ2 from RYR2 channels within 10 min. Thus, it is most likely that CSQ2 remained bound to the RYR2 channel at 8 mM [Ca²⁺]_L in the experiments described in our study. At 0.005 and 1 mM [Ca²⁺]_L, CSQ2 can be assumed to be present in the RYR2 channel complex (Györke et al., 2004; Qin et al., 2008, Wei et al., 2009; Liu et al., 2010). For [Ca²⁺]_L of 15, 26, and 53 mM, CSQ2 may be presumed to have been dissociated from the RYR2 channel, considering the fact that the rise in [Ca²⁺]_L gradually reduces the ability of CSQ2 to bind to other proteins (Mitchell et al., 1988; Park et al., 2004).

The only effect of high [Ca²⁺]_L (26–53 mM) was an increase of RYR2 open probability in the absence of ATP. This P_O increase was fully responsible for the increased ability of ATP to activate the channel. If CSQ2 dissociation causes an increase of RYR2 P_O (Györke et al., 2004), the increased RYR2 open probability at high [Ca²⁺]_L may be attributed to the absence of CSQ2 from the channel. However, many laboratories report a decrease of RYR2 P_O after dissociation of CSQ2 (Qin et al., 2008, 2009; Wei et al., 2008; Liu et al., 2010). It is possible, as suggested by Qin et al. (2009), that CSQ2 inhibits the channel under conditions where the luminal Ca²⁺-dependent RYR2 deactivation (Györke et al., 2004) is operative, whereas it activates the channel otherwise (Qin et al., 2008, 2009). If this is the case, both inhibition of the RYR2 channels after prolonged incubation with high luminal Ca²⁺ under activating conditions and increased RYR2 open probability at high luminal Ca²⁺ under diastolic conditions can be explained by CSQ2 dissociation. On the whole, the observed allosteric interactions between the L_A site and the two cytosolic binding sites (for Ca²⁺ and ATP) suggest that the L_A site resides directly on the RYR2 channel. Nevertheless, if CSQ2 acts by modulating the properties of RYR2 Ca²⁺-binding sites, as suggested by Qin et al. (2008), it may be involved in the observed effects of luminal Ca²⁺. However, the role of CSQ2 in the observed effects cannot be decided with certainty.

Currently, it is not clear whether only free ATP²⁻ or also divalent ion-bound ATP activates the RYR2 channel, although the importance of the negative charges on the phosphates of ATP for its effect on RYR2 open probability (Chan et al., 2003) would suggest the first to be the case. In our experiments, a significant decrease of [ATP²⁻] may have occurred during RYR2 openings caused by the binding of the permeating Ca²⁺ to ATP in the pore vicinity. Using Eq. 4, an up to 60% decrease was predicted at 53 mM [Ca²⁺]_L and 100 µM of total ATP for a distance of the ATP-binding site from the pore, r = 12 nM. However, [ATP²⁻] changes were <10% for ATP concentrations that evoked significant RYR2 activation; therefore, our data cannot help to resolve this question.

Our results have significant physiological and pathophysiological implications for joint regulation of Ca²⁺ release by ATP, cytosolic and luminal Ca²⁺, as well as by cytosolic Mg²⁺.

Variation of luminal Ca²⁺ in the range of 0.005 to 1 mM at physiological ATP levels (400–600 µM of free [Mak et al., 1999] and ∼3 mM of total ATP [Bers, 2001]) does not bring about RYR2 activation to high levels under diastolic conditions (100 nM [Ca²⁺]_C; Fig. 2). However, the relative increase of RYR2 P_O upon increasing [Ca²⁺]_L is very prominent (48- or 57-fold increase of P_O if, respectively, free or total ATP is considered the activating species; see Fig. 5 A). Because RYR2 inhibition by cytosolic Mg²⁺ is not affected by luminal Ca²⁺ (Fig. 6), the same relative increase of P_O would be observed in its presence. The increase in spark frequency brought about by a 50-fold increase in RYR2 open probability under diastolic conditions can be estimated to be ∼20-fold (Fig. 4 of Zahradníková et al., 2010). Using Eq. 3 and Table 3, it can be calculated that the change of EC₅₀ for ATP, evoked by the previously described high affinity processes involving the A-site and the L-site (Laver, 2007b), is responsible for only 18–23% of the increase in RYR2 P_O over the physiological [Ca²⁺]_L range. Thus, the L_A site, despite having a relatively low affinity, can be considered to significantly contribute to the physiological regulation of diastolic Ca²⁺ leak.

Under physiological conditions, [ATP²⁻] is close to the EC₅₀ for ATP observed under diastolic conditions (100 nM [Ca²⁺]_C and 1 mM [Ca²⁺]_L), and the total [ATP] is about six times larger. However, in failing human myocardium and in hearts of animal models of severe heart failure, [ATP] is decreased (Ingwall and Weiss, 2004); therefore, diastolic [ATP²⁻] may drop below the EC₅₀. In the absence of changes in RYR2 gating properties, the resulting decrease of diastolic leak would tend to counteract the parallel decrease of Ca²⁺ ATPase (SERCA2) function. Considering the net reduction of SR Ca²⁺ content observed in heart failure (Belevych et al., 2007), the decrease of SERCA2 function likely exceeds the decrease of diastolic RYR2 activity in the failing heart.

Furthermore, the dramatic increase of the maximal P_O induced by ATP at diastolic [Ca²⁺]_C by elevated [Ca²⁺]_L may play a significant role in the Ca²⁺ overload-induced Ca²⁺ release during the diastole (Berlin et al., 1987; Bassani et al., 1995; Lukyanenko et al., 1996, 1999). Increased SR Ca²⁺ content (Ca²⁺ overload) is a characteristic feature of various pathological states such as metabolic inhibition, ischemia/reperfusion, digitalis poisoning, and the early stage of heart failure (Kass et al., 1978; Levi et al., 1993; Pogwizd et al., 2001), and arises because of imbalance between Ca²⁺ entry and efflux (Trafford et al., 1997, 2001). It is well established that Ca²⁺ overload causes certain forms of arrhythmia that are triggered by Ca²⁺ waves propagating along the myocyte (Cheng et al., 1996; Pogwizd and Bers, 2004). Such arrhythmias account for ∼70% of the cases of ventricular tachycardia in patients without structural heart disease (Lerman et al., 1986). Ca²⁺ waves occur only under certain conditions, and one of them is enhanced activation of the RYR2 channel in the diastole (Lukyanenko et al., 1999). Collectively, potentiation of the RYR2 channel by [Ca²⁺]_L that is elevated beyond physiological levels might play a significant role in the generation of Ca²⁺ overload-induced cardiac arrhythmias.

Finally, the increase of $POmax$ and decrease of EC₅₀ for ATP at physiological [Ca²⁺]_L by small elevations of [Ca²⁺]_C may increase diastolic Ca²⁺ release, even in the absence of changes of RYR2 gating properties under conditions in which the Ca²⁺ uptake into the SR is compromised, such as during heart failure (Piacentino et al., 2003; Armoundas et al., 2007), or when Ca²⁺ signaling via IP₃R channels, located in the close proximity to the RYR2 channels, is enhanced, such as in hypertrophied hearts (Harzheim et al., 2009).

The elevated Ca²⁺ leak is becoming an increasingly recognized feature of the diseased heart. It is generally accepted that changes in RYR2 gating significantly contribute to the increased diastolic Ca²⁺ leak (Lehnart et al., 2004; Belevych et al., 2007; Terentyev et al., 2008; Tateishi et al., 2009). Relatively small changes in the parameters of RYR2 interaction with ATP (Fig. 5 and Table 3) may result in large changes in the open probability under diastolic conditions. Specifically, the ligand-independent opening transition (K_O0) seems to be essential for the joint RYR2 activation by ATP and luminal Ca²⁺. Our data show that any non-ATP–related open probability increase of the RYR2 channel will be greatly amplified by ATP and luminal Ca²⁺. In this way it can be explained why mutant RYR2 channels that exhibit increased ryanodine binding in the absence of agonists (Jiang et al., 2002, 2004) show increased luminal Ca²⁺ sensitivity (Jiang et al., 2005) and enhanced store overload–induced Ca²⁺ release (Jiang et al., 2004), and conversely, why RYR2 mutants that show decreased ryanodine binding under these conditions show decreased luminal Ca²⁺ sensitivity and reduced store overload–induced Ca²⁺ release (Jiang et al., 2007). Additionally, allosteric interactions between the occupation of binding sites for cytosolic and luminal Ca²⁺ and for ATP on one hand and the opening transition of the RYR2 channel tetramer on the other hand result in potentiation of the effects of each of these ligands by the remaining ones (Figs. 2, 3, and 5). This property of RYR2 channels might explain some of the apparent discrepancies when different laboratories attributed the causes of activity changes in RYR2 channels containing the same mutation to different mechanisms (V4653F: Lehnart et al., 2004 and Jones et al., 2008; S2246L: George et al., 2003, 2006, Jones et al., 2008, and Suetomi et al., 2011; R4496C: Jiang et al., 2002, 2004, George et al., 2003, 2006, and Fernández-Velasco et al., 2009; R2474S: Laver et al., 2008, Uchinoumi et al., 2010, and Xu et al., 2010).

Our results show that the potentiating effect of ATP on the activity of the RYR2 channel is antagonized by cytosolic Mg²⁺. This inhibitory effect was not dependent on luminal Ca²⁺ (Fig. 6), and the IC₅₀ for Mg²⁺ inhibition was in a very good accordance with the previously reported values for action of cytosolic Mg²⁺ at the A-site (Laver et al., 1997; Zahradníková et al., 2003, 2010; Gusev and Niggli, 2008). In view of our results, the diastolic Ca²⁺ leak is effectively controlled by cytosolic Mg²⁺ in the healthy heart. A different situation may occur under diseased conditions in which either lowered [Mg²⁺]_C (Haigney et al., 1995; Griffiths, 2000; Tong and Rude, 2005) or the decreased sensitivity of RYR2 channels to cytosolic Mg²⁺ has been reported (Lehnart et al., 2004). In both scenarios, the RYR2 channel is less inhibited by cytosolic Mg²⁺; thus, the synergetic activation effect of ATP and of cytosolic and luminal Ca²⁺ described in our study can become more prominent and thus may further contribute to cardiac dysfunction.

We thank Ivan Zahradník for his instrumental advice in experiment design, single-channel data analysis, and data interpretation, as well as for his critical and constructive comments that greatly improved the manuscript. We also thank Gizela Gajdošíková for technical assistance with animal sacrifice and Adela Kniesová and Jitka Žurmanová for help with Western blotting.

This work was supported by European Union Contracts (LSHM-CT-2005–018802/CONTICA and LSHM-CT-2005-018833/EUGeneHeart), by the Slovak Scientific Grant Agency (VEGA 2/0033/11 and 2/0190/10), and by the Slovak Research and Development Agency (APVV-0139-06, APVV-0721-10, and APVV-SK-CZ-0172-09). This publication is also a result of implementation of the project TRANSMED (ITMS: 2624012000), supported by the Research and Development Operational Program funded by the European Regional Development Fund.

Richard L. Moss served as editor.