Drugs and chemicals

CaCl₂ standard for calibration was from Word Precision Instruments Inc (Sarasota, FL). Phospholipids were obtained from Avanti (Alabaster, AL), and decane from Aldrich (Milwaukee, WI). BAPTA (1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid), dibromoBAPTA (1,2-bis 2-bis(o-amino-5-bromophenoxy)ethane-N,N,N',N'-tetraacetic acid), Ba(OH)₂, Ca(OH)₂, Mg(OH)₂, Sr(OH)_2, CsOH, CsCl, and HEPES were obtained from Fluka (Boca Raton, Fl). All other drugs and chemicals were from Sigma or were reagent grade.

Sarcoplasmic reticulum microsomes

All procedures with animals were designed to minimize pain and suffering and conformed to the guidelines of the National Institutes of Health. SIUMED animal research procedures have AAALAC accreditation and PHS assurances numbers 000551 and A3209-01 respectively. The committee on the Use and Care of Laboratory Animals of Southern Illinois University School of Medicine reviewed and approved the protocols for animal use in our laboratory (196-05-021 and 196-08-003). Sarcoplasmic reticulum (SR) microsomes were obtained from pig heart ventricle using heart homogenization and ultracentrifugation steps that follow the procedures published by Chamberlain et al. [23]. SR pellets obtained after high speed centrifugation were resuspended in 290 mM sucrose - 5 mM Imidazole buffer (pH = 7), aliquoted in cryovials (300 µl each) and kept in liquid nitrogen (better and safer long-term storage). Every month, a few cryovials were used to generate smaller aliquots of membranes (15 µl each) which were stored at –80°C for easy access. For experiments, aliquots were quickly thawed in water, kept on ice and used within 3–5 hours.

Bilayer technique

Reconstitution of RyR2 in planar lipid bilayers was performed as previously described [10]. Briefly, planar lipid bilayers were formed on 80 to 100 µm-diameter circular holes in teflon septa, separating two 1.3 ml compartments. The trans compartment was filled with HEPES-M²⁺ solution containing HEPES 250 mM and M(OH)₂ 53 mM, pH 7.4 (M²⁺ was either Mg²⁺, Ca²⁺, Sr²⁺ or Ba²⁺). The trans compartment was clamped at 0 mV using an Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA). The cis compartment (ground) was filled with HEPES-Tris solution containing HEPES 250 mM and TrisOH 118 mM, pH 7.4. Bilayers of a 5∶4∶1 mixture of bovine brain phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine (45–50 mg/ml in decane) were painted onto the holes of teflon septa from the cis side. Sarcoplasmic reticulum microsomes (5–15 µg) were then added to the cis solution followed by 500–1000 mM CsCl and 1 mM CaCl₂ to promote vesicle fusion. After RyR currents (or Cl⁻ currents >100 pA at 0 mV) were observed, the cis chamber was perfused with HEPES-TRIS solution for 5 min at 4 ml/min. A mixture of BAPTA and dibromo-BAPTA was used to buffer free [Ca²⁺] on the cytosolic surface of the channel ([Ca²⁺]_cyt) [10]. As previously done [10], RyR channels were identified by current amplitudes (∼3.5 pA at 0 mV), slope conductance (∼100 pS), reversal potential (∼−45 mV, trans - cis) and response to diagnostic ligands (e.g., ryanodine, Ca²⁺, ATP, caffeine and Ruthenium Red). RyR channel currents are depicted as positive (upward deflections of the current) in figures and reflect cation flux from the trans (luminal) to the cis (cytosolic) compartment. Membrane voltages always represent the difference between trans - cis compartments (in mV).

Single channel analysis

Channel currents were first filtered through the Axopatch 200B low-pass Bessel filter at 2 kHz, digitized at 20 kHz with an analog to digital converter (Digidata 1320, Axon Instruments) and stored on DVD. Recordings were analyzed using pClamp9 software (Axon Instruments). Analysis with this program included open times, closed times and open probabilities (P_o), which were determined by half-amplitude threshold analysis of single-channel recordings as done before [10]. In multichannel experiments, the global open probability (nP_o) was estimated. In the figures we show the P_o (for single channels) or nP_o/x (for multiple channels, with x representing the maximal number of current levels observed).

Statistical Analysis

Data are shown as means ± S.E.M. of n measurements. Statistical comparisons between groups were performed with Student's t test for paired samples. Differences were considered statistically significant at P<0.05.

At least two different types of M²⁺ binding sites exist on RyR2 cytosolic surface

Figure 1A shows representative recordings of RyR2 channels activated by Ca²⁺ (left panel) or Sr²⁺ (middle panel) added to the cytosolic compartment. All recordings were made at a holding potential (V_m) of 0 mV with luminal Ca²⁺ (50 mM) as current carrier. As previously reported (reviewed in [1], [2], [3], [4]), the channels activated when cytosolic Ca²⁺ increased to micromolar levels. Figure 1A, right panel summarizes open probability (P_o) data from n = 10 RyR2 experiments (open circles). From these experiments, we estimated that the effective concentration of Ca²⁺ that induces half maximal P_o (EC₅₀) was 2.3±0.1 µM. Channel activation had a Hill coefficient (n_H) of 2.4±0.1. RyR2 were also activated with increasing Sr²⁺ levels, as shown in the recordings (Fig. 1A , middle panel) and in the summary of n = 6 experiments (Fig. 1A, right panel, open triangles). However, EC₅₀ for Sr²⁺ was 20.2±1.0 µM (∼10 times higher than EC₅₀ for Ca²⁺). Similar as with Ca²⁺, n_H with Sr²⁺ was 2.2±0.2. These n_H >1 suggest that multiple interacting M²⁺ binding sites specific for Ca²⁺>Sr²⁺ are involved in Ca²⁺ or Sr²⁺-induced RyR2 activation. As shown in Fig. 1A (right panel, filled circles and triangles), RyR2 did not activate when cytosolic Mg²⁺ or Ba²⁺ levels were increased (from 0.1 to 500 µM). This confirms that cytosolic M²⁺ activating sites are selective for Ca²⁺ and Sr²⁺.

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Figure 1. Effects of M²⁺ added to the cytosolic surface of RyR2.

(A) RyR2 are activated by micromolar levels of cytosolic Ca²⁺/Sr²⁺ but not Mg²⁺/Ba²⁺. Representative RyR2 recordings for channels activated by Ca²⁺ (left panel) or Sr²⁺ (middle panel) added to the cytosolic compartment. All recordings were made at a holding potential (V_m) of 0 mV with luminal Ca²⁺ (50 mM) as the current carrier. The right panel shows mean open probability (P_o) of RyR2 channels as a function of free earth alkaline divalent cation concentration [M²⁺] varying from 100 nM to 500 µM. P_o's are mean values from n = 10 (Ca²⁺, open circles), 6 (Sr²⁺, open triangles), and 5 experiments (Mg²⁺ and Ba²⁺, filled circles and triangles respectively). Values are shown as mean ± SEM. Ca²⁺ activated the channels with an EC₅₀ = 2.3±0.1 µM and n_H = 2.4±0.1. With Sr²⁺ EC₅₀ = 20.2±1.0 µM and n_H = 2.2±0.2. (B) RyR2 are inhibited by high (mM) concentrations of M²⁺. Left panels show representative recordings of RyR2 fully activated by cytosolic Ca²⁺ (100 µM) which were exposed to cumulative doses of Ca²⁺ added to the cytosolic side of channel. The Mean P_o of RyR2 as a function of [M²⁺] varying from 0.25 to 10 mM is shown in the right panel. Again, luminal Ca²⁺ (50 mM) was current carrier and V_m = 0 mV. All M²⁺ tested had similar inhibitory action when applied at millimolar concentrations. IC₅₀'s were: 3.5±1.3; 4.5±0.4; 5.6±0.7 and 5.5±0.3 mM (Ba²⁺, Mg²⁺, Sr²⁺ and Ca²⁺ respectively). (C) Cytosolic Mg²⁺ and Ba²⁺ interfered Ca²⁺activation of RyR2. Mean P_o of RyR2 at 5 µM free cytosolic Ca²⁺ was inhibited by addition of 1 mM Mg²⁺ (circles) or 1 mM Ba²⁺ (Triangles) (n = 5 each). Subsequent increase of cytosolic Ca²⁺ to 100 µM counteracted the inhibition by Mg²⁺ or Ba²⁺. (D) Ca²⁺ and Sr²⁺ counteract inhibition by Mg²⁺ of caffeine-activated RyR. RyR2 studies were performed at V_m = 0 mV with luminal Ca²⁺ (50 mM) as current carrier. In the presence of 0.1 µM free cytosolic Ca²⁺, RyR2 are fully activated by 10 mM caffeine (top traces). 1 mM cytosolic Mg²⁺ inhibited RyR2 (middle traces). Increasing cytosolic Ca²⁺ 1 µM (bottom left trace) or Sr²⁺ to ∼20 µM (bottom right trace) recovered RyR2 activity. The right panel shows that mean P_o of RyR2 at 0.1 µM cytosolic Ca²⁺ was greatly enhanced by adding caffeine (10 mM). Addition of 1 mM Mg²⁺ inhibited the channels. Subsequent increase in cytosolic Ca²⁺ levels to >1 µM or Sr²⁺ to 5–50 µM recovered channel activity (n = 5).

https://doi.org/10.1371/journal.pone.0026693.g001

It is well known that RyR2 response to cytosolic Ca²⁺ is biphasic. Micromolar Ca²⁺ levels activate RyR2 while millimolar Ca²⁺ levels inhibit the channel ([10] reviewed in [2], [5]). Yet, the RyR2 response to millimolar Ca²⁺ is heterogeneous, as only a fraction of the channels would be significantly inhibited by 5 mM Ca²⁺ [10]. This population of sensitive RyR2 was used to examine the M²⁺ selectivity of the cytosolic low affinity inhibitory sites. For that, we first exposed RyR2 to 200 µM cytosolic Ca²⁺ (to elevate P_o to a peak level) and subsequently, added cumulative doses of either Ca²⁺, Sr²⁺, Ba²⁺ or Mg²⁺. As shown in Fig. 1B, all the M²⁺ tested had a similar inhibitory action on RyR2 P_o. The concentrations for half maximal inhibition (IC₅₀) were also similar, ranging from 3.5 to 5.7 mM (see Fig. 1B, legend).

We found that Mg²⁺ and Ba²⁺ interfere with Ca²⁺/Sr²⁺-induced activation of RyR2. Figure 1C shows that in the presence of 5 µM cytosolic Ca²⁺ RyR2 activated, reaching a high P_o value. Subsequent addition of either 1 mM Ba²⁺ or 1 mM Mg²⁺. reduced P_o by 90.5±2.8% and 92.7±4.2% respectively. The inhibition induced by cytosolic Mg²⁺ or Ba²⁺ was counteracted by further increasing cytosolic Ca²⁺ to 100 µM (Fig. 1 C).

It is known that caffeine increases RyR2 sensitivity to cytosolic Ca²⁺ activation [2], [4], [24]. In Fig. 1D we show that in the presence of 10 mM caffeine, only 100 nM cytosolic Ca²⁺ is required to reach a high P_o level (comparable to that in Fig. 1C, with 5 µM cytosolic Ca²⁺). Still, the Mg²⁺ concentrations required to induce equivalent degrees of inhibition are the same, regardless of the presence/absence of caffeine. However, much less Ca²⁺ was required to counteract the effect of Mg²⁺ (1 µM versus 50–100 µM Ca²⁺ in the absence of caffeine; compare Fig. 1C and 1D). Our current and previous results [10], [11], [24] indicate that caffeine increases RyR2 sensitivity to cytosolic Sr²⁺ and Ca²⁺ activation by ∼20–50 times but it does not significantly increase RyR2 sensitivity to Mg²⁺ and Ba²⁺ inhibition.

Luminal M²⁺ affect the response of RyR2 to Ca²⁺ and caffeine

It has been reported that RyR2 channel function can also be regulated by luminal Ca²⁺ [13], [14], [20], [25], [26]. Fewer studies have explored the sensitivity of these luminal sites to different divalent cations [13], [15]. Figure 2A shows RyR2 sensitivity to cytosolic Ca²⁺ with different luminal cations (Ca²⁺, Sr²⁺, Mg²⁺, Ba²⁺ and Cs⁺). In these experiments, the luminal cations were also the charge carriers (the current flows in lumen-to-cytosol direction). With luminal Cs⁺, Ca²⁺ or Sr²⁺, the channels were activated by cytosolic Ca²⁺ with an EC₅₀ ∼ 3 µM (Fig. 2A). In contrast, significantly higher cytosolic Ca²⁺ levels were required to activate RyR2 in the presence of luminal Ba²⁺ or Mg²⁺ (EC₅₀ ∼ 6.7 µM and 10.4 µM, respectively). At high cytosolic Ca²⁺ levels (>100 µM), the maximal P_o (plateau) value was similar in the presence of luminal Ca²⁺, Sr²⁺, Mg²⁺ or Ba²⁺. However, this value was significantly reduced (from P_o ∼0.8 to P_o ∼0.4) when we used Cs⁺ as charge carrier (luminal solution contained 100 mM Cs⁺ plus ∼500 µM luminal Ca²⁺). Open probabilities were much lower with luminal Cs⁺ solutions (where only micromolar contaminant Ca²⁺ was present) , but P_o at 0 mV cannot be accurately determined using this approach because the channels are prone to inactivation (results not shown). In Supporting Information (Fig. S1) we show that cytosolic Ca²⁺ increases P_o of RyR2 bathed with luminal Ca²⁺ by increasing the number of openings and by increasing event duration. A similar pattern (albeit with overal briefer events) is observed when RyR2 are bathed with luminal Sr²⁺, Mg²⁺ and Ba²⁺.

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Figure 2. Effects of M²⁺ added to the luminal surface of RyR2.

(A) Luminal M²⁺ alters open probability of Ca²⁺-activated RyR2. Representative recordings of RyR exposed to increasing concentration of cytosolic Ca²⁺, in the presence of luminal Ba²⁺ (left panel) or Mg²⁺ (middle panel). Mean P_o as a function of cytosolic Ca²⁺ observed in RyR2 bathed with different luminal cations (right panel). Maximal open probability values of channels exposed to 50 mM luminal M²⁺ were higher than those observed with 100 mM luminal Cs⁺ (p<0.05). The EC₅₀ for cytosolic Ca²⁺ activation in the presence of luminal Mg²⁺ was 10.4±0.6 µM (n = 4). This is substantially higher than the EC₅₀ with luminal Cs⁺, Ca²⁺ or Sr²⁺ (3.2±0.3 µM, n = 5; 2.9±0.3, n = 7; and 3.4±0.4 µM, n = 5 respectively). EC₅₀ with Ba²⁺ was intermediate (6.7±0.3 µM, n = 7). (B) Luminal M²⁺ effect is more evident in the presence of caffeine. Representative recordings of RyR exposed to increasing concentration of cytosolic caffeine, in the presence of luminal Ca²⁺ (left panel) or Ba²⁺ (middle panel). The left panel shows mean P_o as a function of cytosolic caffeine observed in RyR2 bathed with different luminal M²⁺ (n = 5 in each condition). Recordings were made at 100 nM cytosolic free Ca²⁺. In the presence of luminal Ca²⁺ or Sr²⁺, caffeine activated RyR2 to high P_o (EC₅₀'s were 5.8±0.5 and 6.4±0.4 mM respectively). With luminal Ba²⁺ or Mg²⁺, RyR2 poorly activated or remained closed. (C) Luminal M²⁺ affects the gating of caffeine-activated RyR2. Left panels: representative single channel recordings. Right panels: Open and closed dwell-time distribution histograms of caffeine-activated RyR2 bathed with different luminal M²⁺. All recordings were made at a holding potential of 0 mV. The cytosolic solutions contained 100 nM cytosolic Ca²⁺ and 20 mM caffeine. Dwell open (τ^o) and closed (τ^c) times with different luminal M²⁺ were obtained by fitting the logarithmic dwell time distributions (open or close events distributions) with two components. Openings with luminal Ca²⁺ distributed with τ^o = 154 ± 1 ms (100% events) while closures distributed with τ^c₁ = 1.00 ± 0.21 ms (17% of the events) and τ^c₂ = 143 ± 9 ms (83%). Values with Sr²⁺ were τ^o₁ = 1.32 ± 0.14 ms (18%), τ^o₂ = 114 ± 5 ms (82%), τ^c₁ = 2.2 ± 0.3 ms (28%) and τ^c₂ = 23.0 ± 2.5 ms (72%). Values with Ba²⁺ were τ^o = 0.6 ± 0.1 ms (100%), τ^c₁ = 1.3 ± 0.1 ms (37%) and τ^c₂ = 23.0 ± 1.1 (63%). Values with Mg²⁺ were τ^o = 0.8 ± 0.1 ms (100%), τ^c₁ = 0.5 ± 0.1 ms (43%) and τ^c₂ = 11.2 ± 1.2 (57%).

https://doi.org/10.1371/journal.pone.0026693.g002

As shown in Fig. 2B, we then tested the effect of different luminal M²⁺ on the sensitivity of single RyR2 to caffeine. These experiments were conducted in the presence of 100 nM cytosolic Ca²⁺. As shown in the examples with luminal Ca²⁺ or Ba²⁺ (Fig. 2B, left and center panels), in the absence of caffeine (control conditions), the RyR2 had low P_o. With luminal Ca²⁺ or Sr²⁺, caffeine activated RyR2 with similar EC₅₀ values (Fig. 2B, right panel, open circles and open triangles). These caffeine-activated channels displayed long events (openings and closures), as shown in recordings (Fig. 2C, left top panels) and in dwell-time distribution histograms (Fig. 2C, right top panels). In contrast, when the current carrier was luminal Ba²⁺ or Mg²⁺, caffeine had little effect on RyR2, which were poorly activated or remained closed (Fig. 2B, right panel filled circles and triangles). In these conditions, most opening events were brief, as shown in recordings and dwell-time distributions (Fig. 2C bottom panels). A subsequent increase in cytosolic Ca²⁺ to 1 µM (Supporting Information, Fig. S2), activated these channels to high P_o, suggesting they were still sensitive to caffeine. However, the length of openings did not reach values observed with caffeine and luminal Ca²⁺. In summary, caffeine enhanced the differential effects of luminal M²⁺ on RyR2 behavior. It is also apparent that the stabilization of RyR2 long openings, which has been associated with a conformational state denominated “high P_o gating mode” [27], [28], [29], requires luminal Ca²⁺. Indeed, in the absence of luminal Ca²⁺, RyR2 display a bursting behavior with alternating periods of low (flickering) and high P_o, which is denominated modal gating [2], [28].

Luminal M²⁺ affect RyR2 voltage-dependence

As shown in Fig. 2, there was a shift in the EC₅₀ for cytosolic Ca²⁺ activation and the changes in RyR2 gating kinetics observed with different luminal M²⁺ (Fig. 2). This suggests either the presence of two different luminal sites (one for all M²⁺ and one selective for Ca²⁺ and Sr²⁺) or that luminal M²⁺ can affect cytosolic sites when flowing through the channel (feed-through) [4], [15], [17], [18], [19], [20]. If feed-through regulation exists, it could positively or negatively modulate RyR2 function. On one hand, luminal Ca²⁺or Sr²⁺ flowing through the channels could bind to the high affinity activating cytosolic M²⁺ binding sites and increase RyR2 activity. On the other hand, any M²⁺ coming from the lumen could interact with low affinity inhibitory cytosolic M²⁺ binding sites and decrease RyR2 activity. If feed-through produces further activation of RyR2, increasing SR membrane voltage (which increases the magnitude of lumen-to cytosol M²⁺ flux) should induce an increase in the P_o of RyR2 exposed to luminal Ca²⁺ or Sr²⁺ (i.e., voltage-dependence with a positive slope would be observed only with luminal Ca²⁺ or Sr²⁺). In contrast, if feed-through negatively modulates RyR2 function, the voltage-dependence curve for any M²⁺ should have a negative slope.

We tested the effect of changing membrane voltage on RyR2 activity. Tested voltages ranged from −20 to +40 mV, which changed the magnitude of Ca²⁺ flux from ∼1 to 8 pA. As reported before for various aspects of channel function [10], [11], we found here that RyR2 are heterogeneous. Kinetic analysis of individual RyR2 was used to sort them into two groups: one displaying low-mid P_o mode with abundance of short lived gating events lasting from 1 to a few ms and the other with higher P_o and slower kinetics (long lasting events usually ranging from 10 to 100 ms). In Fig. 3, we show paired recordings performed on the same mid-low P_o mode RyR2 with either luminal Ca²⁺ (Fig. 3A) or luminal Ba²⁺ (Fig. 3B) as current carriers. We observed that increasing lumen-to-cytosol M²⁺ flux (by making V_m more positive) decreases P_o regardless of the identity of the luminal M²⁺. Notice, however, that P_o values at comparable cytosolic Ca²⁺ levels are higher with luminal Ca²⁺ than with luminal Ba²⁺. The differences in P_o correlate with more abundant and longer openings and shorter closed times in luminal Ca²⁺ versus Ba²⁺ (See Supporting Information, Fig. S3 and Table S1). For these voltage-dependent RyR2, we found that the probability to transition from closed to open (P_C→O), estimated as number of openings divided by (1 – P_o) recording time, also decreases with voltage but it is more marked with luminal Ba²⁺ (not shown).

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Figure 3. Effects of luminal M²⁺ on RyR2 voltage-dependence.

(A-B) Effect of luminal M²⁺ on cytosolic Ca²⁺ sensitivity and voltage dependence of a RyR2 that displays modal gating. Single-channel recordings of a RyR2 exposed to different [Ca²⁺]_cyt with 50 mM luminal Ca²⁺ as current carrier (V_m = 0 mV). (B) Traces of the same channel shown in (A) after replacement of the luminal solution with 50 mM Ba²⁺. In (A) and (B), the bottom panels summarize the open probabilities as a function of holding voltage of the channel exposed to the indicated [Ca²⁺]_cyt. Notice that increasing lumen → cytosol M²⁺ flux (by making V_m more positive) decreased P_o regardless of the identity of the luminal M²⁺. (C–D) Effect of luminal M²⁺ on cytosolic Ca²⁺ sensitivity and voltage dependence of a RyR2 that displays high P_o mode. Single-channel recordings of a RyR2 exposed to different [Ca²⁺]_cyt with 50 mM luminal Ca²⁺ (C) or 50 mM luminal Ba²⁺ (D) as current carrier (V_m = 0 mV). Bottom panels show open probabilities as a function of voltage. In contrast to the RyR2 shown in (A) and (B), this channel is virtually voltage insensitive when luminal Ca²⁺ is the current carrier. However, in the presence of [Ca²⁺]_cyt≤4 µM and luminal Ba²⁺, increasing V_m decreased P_o. This voltage sensitivity was abolished by further activating the channel upon addition of Ca²⁺ to the cytosol ([Ca²⁺]_cyt ≥9 µM).

https://doi.org/10.1371/journal.pone.0026693.g003

Paired recordings showed in Fig 3C and 3D were taken from a single RyR2 displaying high P_o gating mode (slow kinetics). For this type of channel we detected little (if any) voltage-induced change in P_o when Ca²⁺ was the current carrier (Fig. 3C). However, when Ca²⁺ was replaced with Ba²⁺ (Fig. 3D) the channel displayed more frequent shorter events and definite voltage-dependence. Interestingly, voltage-dependence is not observed when the channels are fully activated by cytosolic Ca²⁺ (Fig. 3C and Fig. 3D). Indeed, full or partial suppression of voltage-dependence by increasing cytosolic Ca²⁺ levels was observed in most RyR2 (both populations). Increasing cytosolic Ca²⁺ levels from 2 µM to 4 µM in the presence of luminal Ba²⁺ increased P_o to values similar to those observed in the presence of luminal Ca²⁺ with 2 µM cytosolic Ca²⁺. However, openings are still shorter with luminal Ba²⁺ compared to luminal Ca²⁺ and the increase in P_o is mainly due to a shortening of closed events. Additionally, the decrease in P_o at more positive SR voltage observed with luminal Ba²⁺ results from a decrease in open times and an increase in closed times (See Supporting Information, Fig. S4 and Table S2). For this type of RyR2, the probability of transition from closed to open is voltage-independent with luminal Ca²⁺ but decreased with voltage with luminal Ba²⁺ (not shown).

Replacement of 10% of luminal Ba²⁺ with Ca²⁺ suffices to match RyR2 behavior in 100% luminal Ca²⁺

As mentioned above, RyR2 activity, gating kinetics and voltage-dependence varied according to the identity of the luminal M²⁺. Specifically, RyR2 display slower kinetics, higher P_o and less voltage-dependence with 50 mM luminal Ca²⁺ than with 50 mM luminal Ba²⁺. To test how much luminal Ca²⁺ is required to observe this behavior, we recorded partially activated (by 4 µM cytosolic Ca²⁺) RyR2 bathed with luminal 50 mM Ba²⁺ before and after adding increasing concentrations of Ca²⁺ to the luminal chamber. Subsequently, the luminal Ba²⁺ was completely replaced by 50 mM Ca²⁺. As shown in Fig. 4A, 5 mM Ca²⁺ suffices to increase P_o to values observed with 50 mM luminal Ca²⁺. As show in Fig. 4B, similar results were obtained when testing RyR2 partially activated by caffeine ([Caffeine] = 20 mM; [Ca²⁺ ]_cyt = 100 nM). Indeed, addition of 5 mM Ca²⁺ to the luminal chamber, increased the P_o to the same levels observed with 50 mM luminal Ca²⁺. Notice that 0.5 mM luminal Ca²⁺ induced a significant increase in the P_o of caffeine-activated channels while it did not affect Ca²⁺-activated channels. This would suggest that the interplay caffeine - luminal Ca²⁺ may produce a more robust change in RyR2 activity than luminal Ca²⁺ alone [24]. The main point to be taken from these experiments is that although the Ca²⁺ fluxes through the open RyR2 would be substantially different with luminal 5 mM Ca²⁺/45 mM Ba²⁺ versus 50 mM Ca²⁺ (as RyR2 Ca²⁺/Ba²⁺ permeability ratio is ∼1; [30]) there is no significant difference in RyR2 behavior between these conditions.

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Figure 4. Effect of increasing luminal Ca²⁺ on the activity RyR2 bathed with luminal Ba²⁺

. (A) Single-channel recordings of a Ca²⁺-activated RyR2 ([Ca²⁺]_cyt = 4 µM) exposed to the indicated luminal M²⁺ mixtures (V_m = 0 mV). The bottom panel summarizes the open probabilities obtained at three different holding voltages. Notice that the channels display maximal activation and no voltage-sensitivity when luminal Ca²⁺ is higher than 5 mM. (B) Single-channel recordings of a caffeine-activated RyR2 ([Caffeine] = 20 mM; Ca²⁺ = 100 nM) exposed to the indicated luminal M²⁺ mixtures (V_m = 0 mV). As shown in the bottom panel, a small increase in luminal Ca²⁺ (0.5 mM) was enough to induce a significant increase in P_o. Further increase in luminal Ca²⁺ (5 mM) caused maximal activation and removed voltage-sensitivity.

https://doi.org/10.1371/journal.pone.0026693.g004

As shown in Fig. 4 caffeine-activated and Ca²⁺-activated RyR2 channels reached high P_o after luminal addition of 5 mM Ca²⁺, which could mask voltage-dependence. This lack of voltage-dependence was also observed in caffeine-activated RyR2 displaying partial activation (10 nM cytosolic Ca²⁺) with 50 mM luminal Ca²⁺ (Supporting Information, Fig. S5). This could reflect the fact that caffeine locks the channels in high P_o mode and RyR2 displaying this kind of gating are voltage-insensitive.

Voltage-dependence does not seem to be related to luminal → cytosol M²⁺ flux

The decreased sensitivity to cytosolic Ca²⁺ observed with luminal Ba²⁺/Mg²⁺ and the negative slope of RyR2 voltage-dependence could reflect the action of the M²⁺ feeding through the channel and producing: i) interference of flowing Ba²⁺/Mg²⁺ with cytosolic Ca²⁺-mediated activation; ii) interaction of flowing M²⁺ with cytosolic inhibitory low affinity M²⁺ binding sites or iii) a combination of both effects (only for luminal Ba²⁺/Mg²⁺). In any case, very high levels of the M²⁺ flowing through the channels should be reached at the cytosolic surface to produce inhibition. In previous experiments, we found that 1 mM cytosolic Mg²⁺ produces an increase in RyR2 EC₅₀ for cytosolic Ca²⁺ from ∼2 µM to ∼10-20 µM [10], [11]. A similar EC₅₀ for cytosolic Ca²⁺ was found when using luminal Mg²⁺ as charge carrier with no cytosolic Mg²⁺ added (EC₅₀ ∼ 10 µM; Fig. 2A). If this effect results from luminal Mg²⁺ feeding through the channels we would expect the levels of Mg²⁺ reaching the cytosolic RyR2 surface to be around 1 mM. However, adding 1 mM Mg²⁺ to the cytosolic surface of channels bathed with 50 mM luminal Mg²⁺ produces a very large inhibition (Fig. 5A and B). This suggests that, if any, the levels of luminal Mg²⁺ reaching RyR2 cytosolic sites would be much less than 1 mM. Likewise, adding 250 µM cytosolic Ba²⁺ to RyR2 bathed with luminal Ba²⁺ induced a significant decrease in P_o, without affecting voltage dependence (Fig. 5C and D). This again suggests that the levels of M²⁺ reaching cytosolic sites are lower than 250 µM.

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Figure 5. Effect of cytosolic Mg²⁺/Ba²⁺ on the behavior of RyR2 exposed to luminal Mg²⁺/Ba²⁺. (A)

Multiple-channel recording of nine RyR2 exposed to 2 µM [Ca²⁺]_cyt with 50 mM luminal Mg²⁺ as current carrier (V_m = 0 mV) before (top) and after (bottom) addition of 1 mM cytosolic Mg²⁺. (B) Chart summarizing mean open probabilities (± S.E.M.) as a function of holding voltage of channels bathed with luminal Mg²⁺ under the indicated cytosolic conditions (n = 9 experiments). (C) Single-channel recording of a RyR2 exposed to 2 µM [Ca²⁺]_cyt with 50 mM luminal Ba²⁺ as current carrier (V_m = 0 mV) before (top) and after (bottom) addition of 250 µM cytosolic Ba²⁺. (D) Mean open probabilities (± S.E.M.) as a function of holding voltage of channels bathed with luminal Ba²⁺ before (filled circles) and after (open circles) addition of 250 µM cytosolic Ba²⁺ (n = 8 experiments). (E) Dwell open time distribution histograms of a single RyR2 exposed to luminal Ba²⁺ in the presence (grey outline) or absence (black outline) of 250 µM cytosolic Ba²⁺. Openings in the absence of cytosolic Ba²⁺ distributed with τ^o₁ = 1.61 ± 0.17 ms (85% events) and τ^o₂ = 6.18 ± 0.83 ms (15%). In the presence of cytosolic Ba²⁺, values were τ^o₁ = 1.09 ± 0.21 ms (70%) and τ^o₂ = 4.25 ± 0.36 ms (30%). (F) Dwell close time distribution histograms of RyR2 bathed with luminal Ba²⁺ before (black outline) and after (grey outline) addition of 250 µM cytosolic Ba²⁺. Closures in the absence of cytosolic Ba²⁺ distributed with τ^c₁ = 0.79 ± 0.24 ms (80% events) and τ^c₂ = 2.95 ± 0.57 ms (20%). In the presence of cytosolic Ba²⁺, values were τ^c₁ = 2.54 ± 0.12 ms (72%) and τ^c₂ = 13.37 ± 0.29 ms (28%)

https://doi.org/10.1371/journal.pone.0026693.g005

An important observation in this paper is that luminal Ba²⁺ (or Mg²⁺) produce high frequency of short lived events, even when the channels are exposed to fully activating cytosolic Ca²⁺ levels (Fig. 2C). This could be attributed to Ba²⁺ (or Mg²⁺) feeding through the channel and transiently inhibiting RyR2 by binding to cytosolic sites [15], [17], [19]. This would be in agreement with Fig. 3 and Fig. 4 where we found large effects of voltage (or luminal to cytosol Ba²⁺ flux) on channel kinetics. However, it has been reported that in the absence of luminal Mg²⁺ (i.e., with luminal Ca²⁺ or Cs⁺), cytosolic Mg²⁺ increases the length of closures but does not significantly affect open times [12]. We have found similar results for the effects of both cytosolic Mg²⁺ and Ba²⁺ on channels bathed with luminal Ca²⁺ (results not shown). Indeed, in our paired experiments, the same RyR2 now bathed with luminal Ba²⁺, still displayed longer closures when exposed to 250 µM cytosolic Ba²⁺, but open event distribution did not change (Fig. 5E and 5F). Thus, previous studies and our current results suggest that the flickering observed in the presence of luminal Ba²⁺ (or Mg²⁺) would not be due to Ba²⁺ feed-through acting on a cytosolic site but it would be a luminal phenomenon.

Additional experiments were carried out with the purpose of preventing the putative effects of luminal M²⁺ reaching cytosolic binding sites. For that, we increased the buffering power of the cytosolic solution keeping [Ca²⁺]_cyt constant by adding mixtures containing Ca²⁺ and fast buffers (BAPTA/di-Bromo-BAPTA). If feed-through positively modulates RyR2 function, when using Ca²⁺ as charge carrier, higher levels of buffer would be expected to chelate the Ca²⁺ flowing from the lumen and decrease P_o by preventing Ca²⁺ interaction with cytosolic activating sites. Conversely, if inhibitory feed-through occurs, increasing buffering should only increase the activity of RyR2 exposed to luminal Ca²⁺ because BAPTA and di-Bromo-BAPTA are less effective as Ba²⁺ chelators and they do not significantly bind to Mg²⁺ [31]. Unexpectedly, increasing cytosolic buffer concentration induced a concentration-dependent increase in P_o, regardless of the current carrier identity (Mg²⁺, Ca²⁺or Ba²⁺) (Results not shown). Some RyR2 activation is also found with EGTA or EDTA, which bind much more slowly to RyR2, suggesting that the buffering effect does not depend on the on-rate or affinity of the chelator for binding to the flowing M²⁺. This may suggests that BAPTA at high concentrations may have some Ca²⁺ buffering independent effects [32]. Another possibility is that contaminating transition metals, which may inhibit RyR2 activity, could be removed by high levels of BAPTA. Consequently, we used anions known to precipitate M²⁺, such as 50 mM sulfate (for Ba²⁺), or 10 mM fluoride (for Ca²⁺), as alternative chelators to sequester luminal M²⁺ flowing into the cytosol and prevent feed-through. However, we did not find significant changes in activity (negative results, not shown).

We tested the possibility that physiological monovalent ions (K⁺ and Na⁺), which are more electronegative than Tris⁺ or Cs⁺, could compete with M²⁺ flux effects in the RyR2 cytosolic surface or the vestibule. In this regard, it has been reported that K⁺ and Na⁺ may affect Ca²⁺ sensitivity of [³H]ryanodine binding [3], [8], [10], [33]. However, we and others found similar EC₅₀ for Ca²⁺ using variable levels (100–250 mM) of different cytosolic cations such as Cs⁺ or Tris⁺ [1], [2], [4], [8], [10], [12]. Supporting these observations, we found that the P_o of RyR2 channels bathed with partially activating cytosolic Ca²⁺ (2 µM, near EC₅₀ levels) or maximally activating cytosolic Ca²⁺ (10–100 µM) was not affected by Na⁺ or K⁺ (from 0 to 150 mM; n = 5 experiments each; negative results, not shown).

Cytosolic M²⁺ binding sites for RyR2 activation and inhibition

In the presence of 50 mM luminal Ca²⁺, both Ca²⁺ and Sr²⁺ bind to M²⁺ activating sites in the cytosolic surface of RyR and activate the channels in a cooperative fashion (n_H >1) to high P_o levels (>0.80) with different relative affinities (EC₅₀ = 1.5 and 15 µM respectively). Selectivity of skeletal and cardiac RyRs for Ca²⁺ and Sr²⁺ as activators has been previously reported [4], [34], [35], however, there are discrepancies among these reports regarding the relative affinities for Ca²⁺ versus Sr²⁺. This could reflect differences between isoforms (RyR1 versus RyR2) and/or experimental conditions (e.g. presence/absence of Mg²⁺/ATP).

Mg²⁺ and Ba²⁺ do not induce channel openings, instead, they interfered with Ca²⁺ with Sr²⁺ activation. Many studies have previously defined the modulation of RyRs by Mg²⁺ [2], [4], [11], [12]. The novelty here is that Mg²⁺, the alkaline earth cation with the smallest radius (0.078 nm) and Ba²⁺ (the biggest one with 0.143 nm) bind to inhibitory cytosolic sites with equivalent affinity. The characteristics of selectivity usually described for M²⁺ binding sites [36] suggest that Ca²⁺ and Sr²⁺ (which have intermediate sizes, 0.105 and 0.127 nm respectively) would be able to bind to the same inhibitory sites. Mg²⁺ competition with Ca²⁺ binding to the activating cytosolic M²⁺ binding site has been proposed [1], [2], [3], [4], [11], [12]. In principle, the results presented in Fig. 1B and 1C would be in agreement with the hypothesis of competition for the same site, as Mg²⁺ and Ba²⁺ were more effective inhibitors at lower cytosolic Ca²⁺ concentrations. However, in the presence of caffeine, Ca²⁺ and Sr²⁺ activated RyRs more efficiently, while Mg²⁺ inhibitory efficiency was unchanged (Fig. 1D). This suggests that micromolar Ca²⁺ and Sr²⁺ bind to different cytosolic sites (referred as “Site A” [15]) than millimolar Mg²⁺ and Ba²⁺ (which bind to “Inhibitory M²⁺ Site 1”).

Our data also suggests that the low affinity inhibitory M²⁺ binding sites responsible for the bell-shaped response to cytosolic Ca²⁺ are non-selective (Fig. 1B). These sites have been proposed to be a different entity (putatively, “Inhibitory M²⁺ Site 2”) than the sites interfering with Ca²⁺ activation (Site 1) [15], [19]. As found here, these low affinity inhibitory M²⁺ Sites 1 and 2 cannot be distinguished based on M²⁺ selectivity. In our opinion, functional studies of RyR2 sensitivity to M²⁺ are also inconclusive. As shown here and as previously reported, occupation of Site 1 or 2 results in different kinetics [11], [12], [13]. Indeed, cytosolic Mg²⁺ and Ba²⁺ increase closed times of partially activated RyRs whereas addition of millimolar cytosolic M²⁺ correlates with a decrease in open times. In principle, these differences in response to M²⁺ may suggest two different cytosolic inhibitory M²⁺ sites. However, RyR2 gating status, which is heavily influenced by Ca²⁺ may also play a role [28], [29]. As we previously reported, low P_o mode is more sensitive to Mg²⁺ inhibition [10]. Thus, predominant inhibition of short events, which are more abundant at 5 µM cytosolic Ca²⁺, may mask flickery block during long openings. In contrast, at >100 µM cytosolic Ca²⁺, long openings are much more frequent [28] and the weight of the flickering block is much more manifest.

All partially activated RyR2 (cytosolic Ca²⁺<5 µM) are sensitive to inhibition by 1 mM Mg²⁺ [10], [12], while a significant fraction of fully activated RyR2 (cytosolic Ca²⁺ ∼ 200 µM) are not inhibited by increasing cytosolic Ca²⁺ to 5 mM [10], [37]. This could be indicative of different entities, with M²⁺ Site 1 present in all RyR2 and Site 2 present in ∼50% of the channels. Still, the observed differences may also reflect variability in RyR2 sensitivity to M²⁺. Indeed, we found that IC₅₀ for RyR2 inhibition by Mg²⁺ (Site 1) range from 0.2 to 1 mM [11]. Thus, for Site 2, inhibition ranges may also be variable and much higher than 5–10 mM. Increasing cytosolic M²⁺ from 1 to 10 mM could generate additional unspecific electrostatic effects on the cytosolic RyR2 surface. Still, to confirm if the cytosolic inhibitory M²⁺ binding sites encompass one or more domains (Site 1 and Site 2) in the RyR2 molecule further molecular/structural data are required.

RyR2 activation by luminal M²⁺

We found that the maximal Po reached by RyR2 in the presence of luminal M²⁺ is higher than that with luminal Cs⁺. Accordingly, it has been reported that there are Ca²⁺-sensing sites, accessible from the SR lumen, which participate in the regulation of RyRs [14], [15], [20], [25], [26]. Our data suggest that one type of luminal M²⁺ binding site, selective for Ca²⁺ and Sr²⁺, would affect RyR2 behavior. We also found that there are non-selective luminal M²⁺ binding sites where all M²⁺ (including Mg²⁺) may produce an increase in maximal Po. Calsequestrin (CSQ), a regulatory protein closely associated to RyR2, has been proposed to have a role in sensing luminal Ca²⁺ [1], [2], [3], [4], [38]. However, it has been suggested that luminal Ca²⁺ levels greater than 5 mM dissociate CSQ from RyR [38]. Since all of our channel reconstitutions were performed in the presence of 50 mM luminal Ca²⁺, RyR2-CSQ association may have been disrupted in our experiments. Thus, the luminal M²⁺ activation observed here would more likely involve M²⁺ binding sites located on the RyR2 protein itself.

In our hands, Sr²⁺ is the closest substitute for luminal Ca²⁺ (regarding the responses to cytosolic Ca²⁺ and caffeine). Notice, however, that saturating Sr²⁺ levels did not produce the same stabilization of high Po mode in caffeine-activated RyR2 as observed with luminal Ca²⁺ (Fig. 2C). Furthermore, the EC50 for cytosolic Sr²⁺ activation of RyR2 in the presence of luminal Ca²⁺ was less (∼ 20 µM) than that reported in previous studies for cytosolic Sr²⁺ activation in presence of luminal Sr²⁺ (EC50 ∼50 µM) [39]. Studies in cells also indicate that Ca²⁺ and Sr²⁺ differ in their ability to generate sparks [39].

Large differences in RyR2 behavior were found with luminal Ca²⁺ versus luminal Ba²⁺ or Mg²⁺. We estimate (paired comparisons) the probability of a RyR2 to open from the closed state (PC→O). At equivalent cytosolic Ca²⁺, PC→O was 2–20 times higher in RyR2 bathed with luminal Ca²⁺ versus luminal Ba²⁺ (the differences were more marked at positive SR holding voltages). This suggests that luminal Ba²⁺ decreases the on-rate and increases the off-rate of Ca²⁺ binding to activating cytosolic sites. A previous report found that at 0 mV, the effects of luminal Ba²⁺ on Po are only noticeable in caffeine-activated RyR2 [13]. However, in Ca²⁺-activated RyR2, equivalent Po (in the presence of luminal Ba²⁺ versus luminal Ca²⁺) was maintained by increasing the frequency of openings, which compensated the decrease in open times [13]. In our paired experiments, we only reached equivalent Po values by increasing cytosolic Ca²⁺ levels of RyR2 bathed with luminal Ba²⁺ (Supporting Information, Fig. S4 and Table S2). At equivalent Po values, the number of events with luminal Ba²⁺ was ∼5 times the number of events observed with luminal Ca²⁺. Three studies carried out in the presence of cytosolic ATP (1–2 mM) found that RyR function is largely affected by changes in luminal Ca²⁺ levels, but there were discrepancies regarding the sensitivity of luminal Ca²⁺ binding sites, with reported values ranging from micromolar to millimolar [14], [15], [16]. In the absence of ATP, increasing luminal Ca²⁺ to millimolar levels was reported to have no effect on maximal Po [14]. This is in disagreement with our results and a recent report [18] where high levels of luminal M²⁺ significantly increased maximal Po compared to that in the presence of luminal Cs⁺.

Using ATP-activated channels Laver et al. (2008) found that 50 µM Mg²⁺ decreases the activity of RyR2 both at negative (no significant Mg²⁺ flux) and positive SR voltages. The effects of luminal Mg²⁺ were attributed to competition with luminal Ca²⁺ sites and with cytosolic Ca²⁺ sites (feed-through modulation) [15]. However, other reports indicated that in absence of ATP, Mg²⁺ fluxes larger than 1 pA are required to observe voltage-dependence [17]. Different to our studies with saturating luminal Mg²⁺ and absence of ATP, these previous reports did not describe an increase in maximal Po compared with luminal Cs⁺ alone.

Is RyR2 voltage-dependence a consequence of feed-through regulation?

Voltage dependence, observed with high flux of luminal divalent ions has been attributed to the interaction of the M²⁺ moving through the pore with activating and inhibitory sites in the cytosolic side of the channel [15], [17], [18], [19].

In our experiments we used saturated luminal M²⁺ which should maximize feed-through effects. All RyR2 bathed with luminal Ba²⁺ or Mg²⁺ display modal gating and exhibit voltage-dependence, even in the presence of caffeine. In contrast, when RyR2 are bathed with luminal Ca²⁺ a population displaying long openings (high P_o – slow kinetics gating mode) is not sensitive to voltage. Our results differ from a previous study where inhibitory effects of flux were found to be nearly equivalent with both Mg²⁺ and Ca²⁺ [18]. Indeed, in the presence of luminal Ca²⁺ only the subset displaying modal gating was voltage-dependent but addition of caffeine switched them to high P_o mode and abolished their voltage-dependence. These observations suggest that modal gating (ability to switch between high and low P_o modes) is required to observe voltage-dependence.

In the population of RyR2 where voltage dependence is observed, P_o decreased with increased M²⁺ flux (i.e., with increased SR positive voltage). It has been suggested that activating and/or inhibitory cytosolic Ca²⁺ binding sites of cardiac RyR2 and skeletal RyR1 can sense the Ca²⁺ flux through the open channel pore (feed-through M²⁺ regulation) [18], [19], [25]. Here we found that the EC₅₀ for cytosolic Ca²⁺ is higher with luminal Mg²⁺/Ba²⁺ versus Ca²⁺/Sr²⁺ flowing through the channel (Fig. 2A). Previous studies suggested that if luminal Ba²⁺ or Mg²⁺ flowing through the pore reaches a concentration of ∼ 1 mM at the channel cytosolic surface, we should expect a change in the EC₅₀ for cytosolic Ca²⁺ from 2 to ∼10–20 µM [11], [12], [13]. However, the data in Fig. 5 indicate that the amount of Ba²⁺ (or Mg²⁺) feeding through the channel would be much less than 0.25 mM, which could not explain the magnitude of the effect of luminal Ba²⁺ (or Mg²⁺) versus luminal Ca²⁺ (Fig. 2). Moreover, voltage-dependence of RyR2 bathed with luminal Ba²⁺ was not affected by the addition of cytosolic 0.25 mM Ba²⁺ (Fig. 5D), which is unexpected as cytosolic levels would be higher than those reached by the luminal Ba²⁺ feeding through the pore. In this regard, the percentage of inhibition of P_o by cytosolic Ba²⁺ at positive voltages is higher than that at negative voltages. However, higher fluxes of lumenal Ba²⁺ feeding through the pore at positive voltages should have better outcompeted cytosolic Ba²⁺ inhibitory effects. The additive effects of luminal Ba²⁺ with cytosolic Ba²⁺ may be a consequence of luminal Ba²⁺ promoting flickering (i.e. fast kinetics with short events and low P_o mode) and of cytosolic Ba²⁺ being more effective to block RyR displaying “low P_o” mode [11]. Notice that “low P_o” mode is promoted by positive voltages; i.e., by increased flux of luminal M²⁺ (See Figs. S3 and S4 and Tables S1 and S2).

Replacement of 5 mM of the luminal Mg²⁺ or Ba²⁺ with Ca²⁺ makes RyR2 behavior (activity, kinetics and voltage dependence) indistinguishable from that obtained with 100% (50 mM) luminal Ca²⁺. As RyR2 are equally permeable to all M²⁺ [2], [4], 5 mM Ca²⁺ would only account for 10% of the M²⁺ feeding through the pore, which may not explain the magnitude of the effect on channel properties. A possible explanation would be that 5 mM Ca²⁺ saturates the Ca²⁺-selective luminal M²⁺ binding sites, as suggested by previous studies [2], [14], [19].

Our experiments also support the idea of open RyR2 being insensitive to activation by Ca²⁺ feeding-through the channel as proposed by others in the absence of ATP and stimulating cofactors [19]. Although in this article we used fast chelating buffers (BAPTA and Bromo BAPTA), in a series of early experiments we compared RyR2 activity measured at 200 nM cytosolic Ca²⁺ using EGTA (n = 16) versus BAPTA/diBromoBAPTA (n = 37) and found no significant differences in P_o (0.014±0.004, n = 16 and 0.012±.026, n = 25). Analogous conclusions were obtained from similar experiments performed using even larger skeletal RyR1 populations (data not shown). If there were any activating effects of Ca²⁺ flux, we would expect large differences in P_o among experiments with different buffers, as it is estimated that Ca²⁺ levels at the cytosolic surface would reach much higher levels with EGTA (thousand times slower k_on for binding Ca²⁺) than with BAPTA [40]. Even with BAPTA, Ca²⁺ fluxes at positive voltages would have increased cytosolic [Ca²⁺] to micromolar levels [40]. This increase in cytosolic Ca²⁺ should have activated RyR2 (i.e., the slope of the voltage-dependence curve should be positive) and this effect should have been more evident for partially activated RyR2 (bathed with 100 nM Ca²⁺) in the presence of caffeine, where RyR2 sensitivity to cytosolic Ca²⁺ increases ∼20 times and full activation only requires cytosolic [Ca²⁺] ∼ 500 nM. As indicated, much higher levels of Ca²⁺ accumulate at the cytosolic surface of an open RyR2 with Ca²⁺ currents of up to 10 pA [40]. Our results suggest that activating effects of Ca²⁺ feeding through the pore may not be observed even in the presence of the stimulating cofactor caffeine.

As stated above, our results suggest that voltage-dependence requires modal gating. It is apparent that SR voltage depolarization destabilizes long openings and then short openings and long closures become more and more abundant. As shown, the effect of voltage can be counteracted in part by increasing cytosolic Ca²⁺ levels. This RyR2 behavior mirrors that of BK channels, which are also voltage- and Ca²⁺-gated channels [41]. Modal gating in K⁺ channels seems to result from dynamic interactions between various channel structures, including the pore helix, selectivity filter and external vestibule [42]. Similar mechanism could be in play for RyR2, which apparently have some structural homology with K⁺ channels [43], [44].

Speculations on the role of Ca²⁺/Mg²⁺-mediated regulation of RyR2 function in cells

Our current and previously published data [10], [11], indicate that in cells, triggering of RyR2-mediated SR Ca²⁺ release by Ca²⁺ will depend on luminal and cytosolic resting levels of Ca²⁺ and Mg²⁺ as well as on the SR – cytosol membrane voltage. During the Ca²⁺ release event (usually generated by an array of RyR2 activating/deactivating in synchrony), luminal Ca²⁺ levels will decrease and cytosolic Ca²⁺ levels will increase. In contrast, cytosolic and luminal Mg²⁺ levels are expected to remain relatively constant (due to the Donnan effect of the polyanion CSQ, the SR luminal Mg²⁺ concentration would be expected to be similar or higher than that in the cytosol, which is ∼1 mM). Consequently, when intra-SR Ca²⁺ levels fall, Mg²⁺ would maintain occupancy of luminal non-selective M²⁺ binding sites and keep the channels active (at least partially) while cytosolic Ca²⁺ remain higher than 10 µM, which are the estimated Ca²⁺ levels on the RyR2 cytosolic surface [40], [45]. RyR2 activity could be reduced by a decrease in luminal Ca²⁺ levels, which in concomitance with a decrease in the driving force for Ca²⁺ flux, could greatly decrease SR Ca²⁺ release. On the other hand, the increase in cytosolic Ca²⁺ levels would tend to maintain RyR2 channels active even with lower levels of luminal Ca²⁺. How RyR2 close in cells for the termination of Ca²⁺ release is a process of still unknown nature [46].

RyR2 are known to alternate between two gating modes: low P_o mode and high P_o mode [27], [28]. According to our data, Ca²⁺-selective luminal sites may be important to stabilize RyR2 in high P_o mode. This suggests that a switch from high to low P_o mode may have physiological significance for RyR2 deactivation following SR Ca²⁺ depletion while the cytosolic Ca²⁺ levels remain elevated. Additionally, our results and previous reports suggest that open RyR2 do not sense the local increase in cytosolic Ca²⁺ they produce via luminal-to-cytosol flux. This may imply that if multiple RyR2 open simultaneously [47], [48] as during a Ca²⁺ spark [5] and they do not sense the released Ca²⁺ after activation, they might still be able to close in some synchrony, even when cytosolic Ca²⁺ levels remain high. For this, the RyR2 should remain refractory to activation by cytosolic Ca²⁺ for some time after they close (few milliseconds), as to allow for the dissipation of the local Ca²⁺ levels.

In conclusion, our studies indicate that the regulation of single RyR2 by M²⁺ (including physiological relevant ions such as Mg²⁺ and Ca²⁺) is a complex process which involves several interacting M²⁺ binding sites (luminal and cytosolic) that may dynamically modulate the function of heterogeneous RyR2 during local and global Ca²⁺ release events.