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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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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 (Vm = 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 (Vm = 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 τo1 = 1.61 ± 0.17 ms (85% events) and τo2 = 6.18 ± 0.83 ms (15%). In the presence of cytosolic Ba2+, values were τo1 = 1.09 ± 0.21 ms (70%) and τo2 = 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 τc1 = 0.79 ± 0.24 ms (80% events) and τc2 = 2.95 ± 0.57 ms (20%). In the presence of cytosolic Ba2+, values were τc1 = 2.54 ± 0.12 ms (72%) and τc2 = 13.37 ± 0.29 ms (28%)
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pone-0026693-g005: 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 (Vm = 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 (Vm = 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 τo1 = 1.61 ± 0.17 ms (85% events) and τo2 = 6.18 ± 0.83 ms (15%). In the presence of cytosolic Ba2+, values were τo1 = 1.09 ± 0.21 ms (70%) and τo2 = 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 τc1 = 0.79 ± 0.24 ms (80% events) and τc2 = 2.95 ± 0.57 ms (20%). In the presence of cytosolic Ba2+, values were τc1 = 2.54 ± 0.12 ms (72%) and τc2 = 13.37 ± 0.29 ms (28%)

Mentions: 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 Po 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 Po 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 Po to the same levels observed with 50 mM luminal Ca2+. Notice that 0.5 mM luminal Ca2+ induced a significant increase in the Po 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.


Modulation of cardiac ryanodine receptor channels by alkaline earth cations.

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

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 (Vm = 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 (Vm = 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 τo1 = 1.61 ± 0.17 ms (85% events) and τo2 = 6.18 ± 0.83 ms (15%). In the presence of cytosolic Ba2+, values were τo1 = 1.09 ± 0.21 ms (70%) and τo2 = 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 τc1 = 0.79 ± 0.24 ms (80% events) and τc2 = 2.95 ± 0.57 ms (20%). In the presence of cytosolic Ba2+, values were τc1 = 2.54 ± 0.12 ms (72%) and τc2 = 13.37 ± 0.29 ms (28%)
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198824&req=5

pone-0026693-g005: 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 (Vm = 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 (Vm = 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 τo1 = 1.61 ± 0.17 ms (85% events) and τo2 = 6.18 ± 0.83 ms (15%). In the presence of cytosolic Ba2+, values were τo1 = 1.09 ± 0.21 ms (70%) and τo2 = 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 τc1 = 0.79 ± 0.24 ms (80% events) and τc2 = 2.95 ± 0.57 ms (20%). In the presence of cytosolic Ba2+, values were τc1 = 2.54 ± 0.12 ms (72%) and τc2 = 13.37 ± 0.29 ms (28%)
Mentions: 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 Po 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 Po 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 Po to the same levels observed with 50 mM luminal Ca2+. Notice that 0.5 mM luminal Ca2+ induced a significant increase in the Po 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.

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.

Show MeSH
Related in: MedlinePlus