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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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Recordings from one experiment on a single skeletal RyR (RyR1) showing the effects of luminal [Ca2+] and cytoplasmic [Mg2+] on channel gating. Increasing luminal [Ca2+] markedly increased the channel open probability and decreased the channel sensitivity to Mg2+ inhibition. The cytoplasmic solution contained (in mM) 250 CsCH3O3S, 10 TES (pH 7.4), 4.5 BAPTA (∼1 nM free Ca2+), 2 ATP plus the various [Mg2+]. The free Mg2+ is indicated at the left and right of each row. The luminal solution contained (in mM) 30 CsCH3O3S, 20 CsCl, 10 TES, and the indicated [Ca2+]. Under these conditions, Mg2+ is thought to inhibit primarily by binding at the high affinity Ca2+ activation site. Membrane potential was held at +40 mV. The current baselines are shown by dashed lines.
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fig2: Recordings from one experiment on a single skeletal RyR (RyR1) showing the effects of luminal [Ca2+] and cytoplasmic [Mg2+] on channel gating. Increasing luminal [Ca2+] markedly increased the channel open probability and decreased the channel sensitivity to Mg2+ inhibition. The cytoplasmic solution contained (in mM) 250 CsCH3O3S, 10 TES (pH 7.4), 4.5 BAPTA (∼1 nM free Ca2+), 2 ATP plus the various [Mg2+]. The free Mg2+ is indicated at the left and right of each row. The luminal solution contained (in mM) 30 CsCH3O3S, 20 CsCl, 10 TES, and the indicated [Ca2+]. Under these conditions, Mg2+ is thought to inhibit primarily by binding at the high affinity Ca2+ activation site. Membrane potential was held at +40 mV. The current baselines are shown by dashed lines.

Mentions: Consistent with previous findings (Smith et al., 1986), cytoplasmic ATP (2 mM) amplified the bell-shaped Ca2+ dependence without greatly altering the half-activating [Ca2+]c, Ka(Ca2+)c (Fig. 1 B and Table I), and activated RyRs in the absence of cytoplasmic Ca2+. We also found that ATP increased the half-inhibitory [Ca2+], Ki(Ca2+)c, by approximately threefold (Table I). Luminal Ca2+ ([Ca2+]l) increased channel activity in 2 mM ATP and 1 nM [Ca2+]c (Pi; Fig. 2, top). The mean Pi increased from 0.25 ± 0.07 in 0.1 mM luminal Ca2+ (number of bilayers, N = 7, and number of channels, n = 21) to 0.50 ± 0.06 in 1 mM (N = 15, n = 37) and 0.63 ± 0.08 in 3 mM (N = 12, n = 43). However, luminal Ca2+ had no effect on the maximum of bell-shaped Ca2+ dependence, Pmax (Table I).


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)

Recordings from one experiment on a single skeletal RyR (RyR1) showing the effects of luminal [Ca2+] and cytoplasmic [Mg2+] on channel gating. Increasing luminal [Ca2+] markedly increased the channel open probability and decreased the channel sensitivity to Mg2+ inhibition. The cytoplasmic solution contained (in mM) 250 CsCH3O3S, 10 TES (pH 7.4), 4.5 BAPTA (∼1 nM free Ca2+), 2 ATP plus the various [Mg2+]. The free Mg2+ is indicated at the left and right of each row. The luminal solution contained (in mM) 30 CsCH3O3S, 20 CsCl, 10 TES, and the indicated [Ca2+]. Under these conditions, Mg2+ is thought to inhibit primarily by binding at the high affinity Ca2+ activation site. Membrane potential was held at +40 mV. The current baselines are shown by dashed lines.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2234024&req=5

fig2: Recordings from one experiment on a single skeletal RyR (RyR1) showing the effects of luminal [Ca2+] and cytoplasmic [Mg2+] on channel gating. Increasing luminal [Ca2+] markedly increased the channel open probability and decreased the channel sensitivity to Mg2+ inhibition. The cytoplasmic solution contained (in mM) 250 CsCH3O3S, 10 TES (pH 7.4), 4.5 BAPTA (∼1 nM free Ca2+), 2 ATP plus the various [Mg2+]. The free Mg2+ is indicated at the left and right of each row. The luminal solution contained (in mM) 30 CsCH3O3S, 20 CsCl, 10 TES, and the indicated [Ca2+]. Under these conditions, Mg2+ is thought to inhibit primarily by binding at the high affinity Ca2+ activation site. Membrane potential was held at +40 mV. The current baselines are shown by dashed lines.
Mentions: Consistent with previous findings (Smith et al., 1986), cytoplasmic ATP (2 mM) amplified the bell-shaped Ca2+ dependence without greatly altering the half-activating [Ca2+]c, Ka(Ca2+)c (Fig. 1 B and Table I), and activated RyRs in the absence of cytoplasmic Ca2+. We also found that ATP increased the half-inhibitory [Ca2+], Ki(Ca2+)c, by approximately threefold (Table I). Luminal Ca2+ ([Ca2+]l) increased channel activity in 2 mM ATP and 1 nM [Ca2+]c (Pi; Fig. 2, top). The mean Pi increased from 0.25 ± 0.07 in 0.1 mM luminal Ca2+ (number of bilayers, N = 7, and number of channels, n = 21) to 0.50 ± 0.06 in 1 mM (N = 15, n = 37) and 0.63 ± 0.08 in 3 mM (N = 12, n = 43). However, luminal Ca2+ had no effect on the maximum of bell-shaped Ca2+ dependence, Pmax (Table I).

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