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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 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 (Vm = 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 Vm more positive) decreased Po 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 Po 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 (Vm = 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 Vm decreased Po. This voltage sensitivity was abolished by further activating the channel upon addition of Ca2+ to the cytosol ([Ca2+]cyt ≥9 µM).
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pone-0026693-g003: 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 (Vm = 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 Vm more positive) decreased Po 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 Po 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 (Vm = 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 Vm decreased Po. This voltage sensitivity was abolished by further activating the channel upon addition of Ca2+ to the cytosol ([Ca2+]cyt ≥9 µM).

Mentions: 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 Po mode with abundance of short lived gating events lasting from 1 to a few ms and the other with higher Po 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 Po 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 Vm more positive) decreases Po regardless of the identity of the luminal M2+. Notice, however, that Po values at comparable cytosolic Ca2+ levels are higher with luminal Ca2+ than with luminal Ba2+. The differences in Po 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 (PC→O), estimated as number of openings divided by (1 – Po) recording time, also decreases with voltage but it is more marked with luminal Ba2+ (not shown).


Modulation of cardiac ryanodine receptor channels by alkaline earth cations.

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

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 (Vm = 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 Vm more positive) decreased Po 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 Po 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 (Vm = 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 Vm decreased Po. This voltage sensitivity was abolished by further activating the channel upon addition of Ca2+ to the cytosol ([Ca2+]cyt ≥9 µM).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198824&req=5

pone-0026693-g003: 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 (Vm = 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 Vm more positive) decreased Po 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 Po 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 (Vm = 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 Vm decreased Po. This voltage sensitivity was abolished by further activating the channel upon addition of Ca2+ to the cytosol ([Ca2+]cyt ≥9 µM).
Mentions: 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 Po mode with abundance of short lived gating events lasting from 1 to a few ms and the other with higher Po 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 Po 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 Vm more positive) decreases Po regardless of the identity of the luminal M2+. Notice, however, that Po values at comparable cytosolic Ca2+ levels are higher with luminal Ca2+ than with luminal Ba2+. The differences in Po 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 (PC→O), estimated as number of openings divided by (1 – Po) recording time, also decreases with voltage but it is more marked with luminal Ba2+ (not shown).

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