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Luminal Ca2+-regulated Mg2+ inhibition of skeletal RyRs reconstituted as isolated channels or coupled clusters.

Laver DR, O'Neill ER, Lamb GD - J. Gen. Physiol. (2004)

Bottom Line: Cytoplasmic Ca(2+) and Cs(+) decreased Mg(2+) affinity by a competitive mechanism.We found that luminal Ca(2+) and cytoplasmic Mg(2+) did not compete at the A-sites of single open RyRs but did compete during multiple channel openings in rafts.Thus it appears that RyRs are effectively "immune" to Ca(2+) emanating from their own pore but sensitive to Ca(2+) from neighboring channels.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences, University of Newcastle and Hunter Medical Research Institute, Callaghan, NSW 2308, Australia. derek.laver@newcastle.edu.au

ABSTRACT
In resting muscle, cytoplasmic Mg(2+) is a potent inhibitor of Ca(2+) release from the sarcoplasmic reticulum (SR). It is thought to inhibit calcium release channels (RyRs) by binding both to low affinity, low specificity sites (I-sites) and to high affinity Ca(2+) sites (A-sites) thus preventing Ca(2+) activation. We investigate the effects of luminal and cytoplasmic Ca(2+) on Mg(2+) inhibition at the A-sites of skeletal RyRs (RyR1) in lipid bilayers, in the presence of ATP or modified by ryanodine or DIDS. Mg(2+) inhibits RyRs at the A-site in the absence of Ca(2+), indicating that Mg(2+) is an antagonist and does not simply prevent Ca(2+) activation. Cytoplasmic Ca(2+) and Cs(+) decreased Mg(2+) affinity by a competitive mechanism. We describe a novel mechanism for luminal Ca(2+) regulation of Ca(2+) release whereby increasing luminal [Ca(2+)] decreases the A-site affinity for cytoplasmic Mg(2+) by a noncompetitive, allosteric mechanism that is independent of Ca(2+) flow. Ryanodine increases the Ca(2+) sensitivity of the A-sites by 10-fold, which is insufficient to explain the level of activation seen in ryanodine-modified RyRs at nM Ca(2+), indicating that ryanodine activates independently of Ca(2+). We describe a model for ion binding at the A-sites that predicts that modulation of Mg(2+) inhibition by luminal Ca(2+) is a significant regulator of Ca(2+) release from the SR. We detected coupled gating of RyRs due to luminal Ca(2+) permeating one channel and activating neighboring channels. This indicated that the RyRs existed in stable close-packed rafts within the bilayer. We found that luminal Ca(2+) and cytoplasmic Mg(2+) did not compete at the A-sites of single open RyRs but did compete during multiple channel openings in rafts. Also, luminal Ca(2+) was a stronger activator of multiple openings than single openings. Thus it appears that RyRs are effectively "immune" to Ca(2+) emanating from their own pore but sensitive to Ca(2+) from neighboring channels.

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The dependence of opening rate on the presence of open channels in the bilayer obtained from individual experiments. For each experiment, the opening rates, k0+, in the absence of open channels (opening to level O1, see Fig. 8) are shown on the abscissa while opening rates in the presence of one open channel, k1+, (opening to level O2) is on the ordinate. The dashed line indicates independent gating of uniform channels. Datum points above this line indicate coupled channel openings while points below the line can arise independent gating of a heterogeneous group of channels. The legend shows the bilayer potential and luminal [Ca2+] in the absence of cytoplasmic Mg2+ (A) or in the presence of 60–230 μM Mg2+ (B). Coupling between channels was promoted by luminal [Ca2+] and cytoplasmic Mg2+ and was abolished by positive bilayer potentials or removing cytoplasmic ATP and using 100 μM Ca2+ as the primary channel activator.
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fig10: The dependence of opening rate on the presence of open channels in the bilayer obtained from individual experiments. For each experiment, the opening rates, k0+, in the absence of open channels (opening to level O1, see Fig. 8) are shown on the abscissa while opening rates in the presence of one open channel, k1+, (opening to level O2) is on the ordinate. The dashed line indicates independent gating of uniform channels. Datum points above this line indicate coupled channel openings while points below the line can arise independent gating of a heterogeneous group of channels. The legend shows the bilayer potential and luminal [Ca2+] in the absence of cytoplasmic Mg2+ (A) or in the presence of 60–230 μM Mg2+ (B). Coupling between channels was promoted by luminal [Ca2+] and cytoplasmic Mg2+ and was abolished by positive bilayer potentials or removing cytoplasmic ATP and using 100 μM Ca2+ as the primary channel activator.

Mentions: One measure for the degree of RyR coupling is the difference between k0+ and k1+. The data from individual experiments are summarized in Fig. 10 by comparing k0+ (abscissa) with k1+ (ordinate). Identical RyRs that gate independently would produce data points that lie on the diagonal dashed line, while data from coupled RyRs would lie above the line. Points that lie below the diagonal line can result from channel-to-channel differences in activity, which tends to bias the gating of the low activity RyRs to transitions between the upper current levels. In a large proportion of experiments where [Ca2+]l = 1 mM and bilayer potentials were negative, RyR groups showed coupled gating. The scatter in the data reflects genuine differences between gating kinetics in different experiments rather than statistical uncertainties in the HMM analysis (relative error <25%, see materials and methods). In the absence of Mg2+ (Fig. 10 A, ▪) three out of eight experiments exhibited significant coupling effects. This increased to seven out of seven in the same experiments performed in the presence of Mg2+ (Fig. 10 B, ▪). Fig. 10 shows that RyR coupling is decreased by reducing [Ca2+]l from 1 to 0.1 mM and abolished by positive bilayer potentials that oppose Ca2+ flow from the luminal to cytoplasmic baths (Fig. 10, A and B, empty symbols). Coupling is reversibly abolished by the absence of cytoplasmic ATP when the channels are activated by cytoplasmic Ca2+ (100 μM; Fig. 10 B, crosses).


Luminal Ca2+-regulated Mg2+ inhibition of skeletal RyRs reconstituted as isolated channels or coupled clusters.

Laver DR, O'Neill ER, Lamb GD - J. Gen. Physiol. (2004)

The dependence of opening rate on the presence of open channels in the bilayer obtained from individual experiments. For each experiment, the opening rates, k0+, in the absence of open channels (opening to level O1, see Fig. 8) are shown on the abscissa while opening rates in the presence of one open channel, k1+, (opening to level O2) is on the ordinate. The dashed line indicates independent gating of uniform channels. Datum points above this line indicate coupled channel openings while points below the line can arise independent gating of a heterogeneous group of channels. The legend shows the bilayer potential and luminal [Ca2+] in the absence of cytoplasmic Mg2+ (A) or in the presence of 60–230 μM Mg2+ (B). Coupling between channels was promoted by luminal [Ca2+] and cytoplasmic Mg2+ and was abolished by positive bilayer potentials or removing cytoplasmic ATP and using 100 μM Ca2+ as the primary channel activator.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2234024&req=5

fig10: The dependence of opening rate on the presence of open channels in the bilayer obtained from individual experiments. For each experiment, the opening rates, k0+, in the absence of open channels (opening to level O1, see Fig. 8) are shown on the abscissa while opening rates in the presence of one open channel, k1+, (opening to level O2) is on the ordinate. The dashed line indicates independent gating of uniform channels. Datum points above this line indicate coupled channel openings while points below the line can arise independent gating of a heterogeneous group of channels. The legend shows the bilayer potential and luminal [Ca2+] in the absence of cytoplasmic Mg2+ (A) or in the presence of 60–230 μM Mg2+ (B). Coupling between channels was promoted by luminal [Ca2+] and cytoplasmic Mg2+ and was abolished by positive bilayer potentials or removing cytoplasmic ATP and using 100 μM Ca2+ as the primary channel activator.
Mentions: One measure for the degree of RyR coupling is the difference between k0+ and k1+. The data from individual experiments are summarized in Fig. 10 by comparing k0+ (abscissa) with k1+ (ordinate). Identical RyRs that gate independently would produce data points that lie on the diagonal dashed line, while data from coupled RyRs would lie above the line. Points that lie below the diagonal line can result from channel-to-channel differences in activity, which tends to bias the gating of the low activity RyRs to transitions between the upper current levels. In a large proportion of experiments where [Ca2+]l = 1 mM and bilayer potentials were negative, RyR groups showed coupled gating. The scatter in the data reflects genuine differences between gating kinetics in different experiments rather than statistical uncertainties in the HMM analysis (relative error <25%, see materials and methods). In the absence of Mg2+ (Fig. 10 A, ▪) three out of eight experiments exhibited significant coupling effects. This increased to seven out of seven in the same experiments performed in the presence of Mg2+ (Fig. 10 B, ▪). Fig. 10 shows that RyR coupling is decreased by reducing [Ca2+]l from 1 to 0.1 mM and abolished by positive bilayer potentials that oppose Ca2+ flow from the luminal to cytoplasmic baths (Fig. 10, A and B, empty symbols). Coupling is reversibly abolished by the absence of cytoplasmic ATP when the channels are activated by cytoplasmic Ca2+ (100 μM; Fig. 10 B, crosses).

Bottom Line: Cytoplasmic Ca(2+) and Cs(+) decreased Mg(2+) affinity by a competitive mechanism.We found that luminal Ca(2+) and cytoplasmic Mg(2+) did not compete at the A-sites of single open RyRs but did compete during multiple channel openings in rafts.Thus it appears that RyRs are effectively "immune" to Ca(2+) emanating from their own pore but sensitive to Ca(2+) from neighboring channels.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences, University of Newcastle and Hunter Medical Research Institute, Callaghan, NSW 2308, Australia. derek.laver@newcastle.edu.au

ABSTRACT
In resting muscle, cytoplasmic Mg(2+) is a potent inhibitor of Ca(2+) release from the sarcoplasmic reticulum (SR). It is thought to inhibit calcium release channels (RyRs) by binding both to low affinity, low specificity sites (I-sites) and to high affinity Ca(2+) sites (A-sites) thus preventing Ca(2+) activation. We investigate the effects of luminal and cytoplasmic Ca(2+) on Mg(2+) inhibition at the A-sites of skeletal RyRs (RyR1) in lipid bilayers, in the presence of ATP or modified by ryanodine or DIDS. Mg(2+) inhibits RyRs at the A-site in the absence of Ca(2+), indicating that Mg(2+) is an antagonist and does not simply prevent Ca(2+) activation. Cytoplasmic Ca(2+) and Cs(+) decreased Mg(2+) affinity by a competitive mechanism. We describe a novel mechanism for luminal Ca(2+) regulation of Ca(2+) release whereby increasing luminal [Ca(2+)] decreases the A-site affinity for cytoplasmic Mg(2+) by a noncompetitive, allosteric mechanism that is independent of Ca(2+) flow. Ryanodine increases the Ca(2+) sensitivity of the A-sites by 10-fold, which is insufficient to explain the level of activation seen in ryanodine-modified RyRs at nM Ca(2+), indicating that ryanodine activates independently of Ca(2+). We describe a model for ion binding at the A-sites that predicts that modulation of Mg(2+) inhibition by luminal Ca(2+) is a significant regulator of Ca(2+) release from the SR. We detected coupled gating of RyRs due to luminal Ca(2+) permeating one channel and activating neighboring channels. This indicated that the RyRs existed in stable close-packed rafts within the bilayer. We found that luminal Ca(2+) and cytoplasmic Mg(2+) did not compete at the A-sites of single open RyRs but did compete during multiple channel openings in rafts. Also, luminal Ca(2+) was a stronger activator of multiple openings than single openings. Thus it appears that RyRs are effectively "immune" to Ca(2+) emanating from their own pore but sensitive to Ca(2+) from neighboring channels.

Show MeSH
Related in: MedlinePlus