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Modulation of cardiac ryanodine receptor channels by alkaline earth cations.

Diaz-Sylvester PL, Porta M, Copello JA - PLoS ONE (2011)

Bottom Line: To better characterize Ca(2+) and Mg(2+) binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M(2+): Mg(2+), Ca(2+), Sr(2+), Ba(2+)) were studied on RyR2 from pig ventricle reconstituted in bilayers.However, with luminal Ba(2+)or Mg(2+), RyR2 were less sensitive to cytosolic Ca(2+) and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca(2+)or Sr(2+)).In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M(2+) binding sites (specific for Ca(2+) and unspecific for Ca(2+)/Mg(2+)) that dynamically modulate channel activity and gating status, depending on SR voltage.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Southern Illinois School of Medicine, Springfield, Illinois, United States of America.

ABSTRACT
Cardiac ryanodine receptor (RyR2) function is modulated by Ca(2+) and Mg(2+). To better characterize Ca(2+) and Mg(2+) binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M(2+): Mg(2+), Ca(2+), Sr(2+), Ba(2+)) were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M(2+) binding to high affinity activating sites at the cytosolic channel surface, specific for Ca(2+) or Sr(2+). This activation was interfered by Mg(2+) and Ba(2+) acting at low affinity M(2+)-unspecific binding sites. When testing the effects of luminal M(2+) as current carriers, all M(2+) increased maximal RyR2 open probability (compared to Cs(+)), suggesting the existence of low affinity activating M(2+)-unspecific sites at the luminal surface. Responses to M(2+) vary from channel to channel (heterogeneity). However, with luminal Ba(2+)or Mg(2+), RyR2 were less sensitive to cytosolic Ca(2+) and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca(2+)or Sr(2+)). Kinetics of RyR2 with mixtures of luminal Ba(2+)/Ca(2+) and additive action of luminal plus cytosolic Ba(2+) or Mg(2+) suggest luminal M(2+) differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca(2+)/Sr(2+)-specific sites, which stabilize high P(o) mode (less voltage-dependent) and increase RyR2 sensitivity to cytosolic Ca(2+) activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M(2+) binding sites (specific for Ca(2+) and unspecific for Ca(2+)/Mg(2+)) that dynamically modulate channel activity and gating status, depending on SR voltage.

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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 (Vm) of 0 mV with luminal Ca2+ (50 mM) as the current carrier. The right panel shows mean open probability (Po) of RyR2 channels as a function of free earth alkaline divalent cation concentration [M2+] varying from 100 nM to 500 µM. Po'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 nH = 2.4±0.1. With Sr2+ EC50 = 20.2±1.0 µM and nH = 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 Po 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 Vm = 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 Po 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 Vm = 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 Po 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).
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pone-0026693-g001: 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 (Vm) of 0 mV with luminal Ca2+ (50 mM) as the current carrier. The right panel shows mean open probability (Po) of RyR2 channels as a function of free earth alkaline divalent cation concentration [M2+] varying from 100 nM to 500 µM. Po'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 nH = 2.4±0.1. With Sr2+ EC50 = 20.2±1.0 µM and nH = 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 Po 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 Vm = 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 Po 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 Vm = 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 Po 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).

Mentions: 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 (Vm) 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 (Po) data from n = 10 RyR2 experiments (open circles). From these experiments, we estimated that the effective concentration of Ca2+ that induces half maximal Po (EC50) was 2.3±0.1 µM. Channel activation had a Hill coefficient (nH) 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+, nH with Sr2+ was 2.2±0.2. These nH >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+.


Modulation of cardiac ryanodine receptor channels by alkaline earth cations.

Diaz-Sylvester PL, Porta M, Copello JA - PLoS ONE (2011)

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 (Vm) of 0 mV with luminal Ca2+ (50 mM) as the current carrier. The right panel shows mean open probability (Po) of RyR2 channels as a function of free earth alkaline divalent cation concentration [M2+] varying from 100 nM to 500 µM. Po'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 nH = 2.4±0.1. With Sr2+ EC50 = 20.2±1.0 µM and nH = 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 Po 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 Vm = 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 Po 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 Vm = 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 Po 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).
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pone-0026693-g001: 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 (Vm) of 0 mV with luminal Ca2+ (50 mM) as the current carrier. The right panel shows mean open probability (Po) of RyR2 channels as a function of free earth alkaline divalent cation concentration [M2+] varying from 100 nM to 500 µM. Po'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 nH = 2.4±0.1. With Sr2+ EC50 = 20.2±1.0 µM and nH = 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 Po 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 Vm = 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 Po 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 Vm = 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 Po 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).
Mentions: 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 (Vm) 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 (Po) data from n = 10 RyR2 experiments (open circles). From these experiments, we estimated that the effective concentration of Ca2+ that induces half maximal Po (EC50) was 2.3±0.1 µM. Channel activation had a Hill coefficient (nH) 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+, nH with Sr2+ was 2.2±0.2. These nH >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+.

Bottom Line: To better characterize Ca(2+) and Mg(2+) binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M(2+): Mg(2+), Ca(2+), Sr(2+), Ba(2+)) were studied on RyR2 from pig ventricle reconstituted in bilayers.However, with luminal Ba(2+)or Mg(2+), RyR2 were less sensitive to cytosolic Ca(2+) and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca(2+)or Sr(2+)).In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M(2+) binding sites (specific for Ca(2+) and unspecific for Ca(2+)/Mg(2+)) that dynamically modulate channel activity and gating status, depending on SR voltage.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Southern Illinois School of Medicine, Springfield, Illinois, United States of America.

ABSTRACT
Cardiac ryanodine receptor (RyR2) function is modulated by Ca(2+) and Mg(2+). To better characterize Ca(2+) and Mg(2+) binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M(2+): Mg(2+), Ca(2+), Sr(2+), Ba(2+)) were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M(2+) binding to high affinity activating sites at the cytosolic channel surface, specific for Ca(2+) or Sr(2+). This activation was interfered by Mg(2+) and Ba(2+) acting at low affinity M(2+)-unspecific binding sites. When testing the effects of luminal M(2+) as current carriers, all M(2+) increased maximal RyR2 open probability (compared to Cs(+)), suggesting the existence of low affinity activating M(2+)-unspecific sites at the luminal surface. Responses to M(2+) vary from channel to channel (heterogeneity). However, with luminal Ba(2+)or Mg(2+), RyR2 were less sensitive to cytosolic Ca(2+) and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca(2+)or Sr(2+)). Kinetics of RyR2 with mixtures of luminal Ba(2+)/Ca(2+) and additive action of luminal plus cytosolic Ba(2+) or Mg(2+) suggest luminal M(2+) differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca(2+)/Sr(2+)-specific sites, which stabilize high P(o) mode (less voltage-dependent) and increase RyR2 sensitivity to cytosolic Ca(2+) activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M(2+) binding sites (specific for Ca(2+) and unspecific for Ca(2+)/Mg(2+)) that dynamically modulate channel activity and gating status, depending on SR voltage.

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Related in: MedlinePlus