Limits...
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.

Show MeSH

Related in: MedlinePlus

Recordings from an experiment with ∼4 RyRs in the bilayer showing coupled gating at −40 mV. At +40 mV, the channels appeared to gate independently (see text). The baths contained symmetric 250 mM Cs+ solutions with [Ca2+]c = 1 nM and [Ca2+]l = 1 mM. The current baselines are shown by dashed lines and the dashed lines (O1–O4) indicate current levels associated with increasing numbers of open channels.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2234024&req=5

fig8: Recordings from an experiment with ∼4 RyRs in the bilayer showing coupled gating at −40 mV. At +40 mV, the channels appeared to gate independently (see text). The baths contained symmetric 250 mM Cs+ solutions with [Ca2+]c = 1 nM and [Ca2+]l = 1 mM. The current baselines are shown by dashed lines and the dashed lines (O1–O4) indicate current levels associated with increasing numbers of open channels.

Mentions: On average, 20% of fusion events incorporated groups of four to eight RyRs into the bilayer. Under the right experimental conditions (e.g., −40 mV and the presence of ATP), the opening of one RyR in a group tended to promote the opening of other RyRs. Fig. 8 shows the activity of four, ATP-activated RyRs at positive and negative bilayer potentials. The current trace shows transitions between the current baseline (labeled C) and four equally spaced levels (O1–O4) corresponding to one to four open channels. At positive potentials, the weighting of each current level followed a binomial distribution expected from the gating in independent channels in the bilayer (unpublished data). At negative potentials, downward current steps frequently “bypassed” some of the current levels, indicating that several channels were opening in near synchrony. The weighting of current levels in these records markedly deviated from a binomial distribution. HMM analysis of these records (see materials and methods) shows that the mean RyR opening rate associated with transitions between the current baseline and level O1, k0+, was significantly slower than opening rates associated with transitions between levels O1–O4, k1+–k4+ (Fig. 9 A, □; P = 0.003, paired t test), while there was no significant difference in the rates associated with transitions between levels O1–O4. Channel closing rates, k-, did not depend on the number of open RyRs (Fig. 9 B). Thus, the coupling between RyRs was primarily due to the opening rate, which for each channel depended on whether or not one other channel was open in the bilayer.


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 an experiment with ∼4 RyRs in the bilayer showing coupled gating at −40 mV. At +40 mV, the channels appeared to gate independently (see text). The baths contained symmetric 250 mM Cs+ solutions with [Ca2+]c = 1 nM and [Ca2+]l = 1 mM. The current baselines are shown by dashed lines and the dashed lines (O1–O4) indicate current levels associated with increasing numbers of open channels.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Recordings from an experiment with ∼4 RyRs in the bilayer showing coupled gating at −40 mV. At +40 mV, the channels appeared to gate independently (see text). The baths contained symmetric 250 mM Cs+ solutions with [Ca2+]c = 1 nM and [Ca2+]l = 1 mM. The current baselines are shown by dashed lines and the dashed lines (O1–O4) indicate current levels associated with increasing numbers of open channels.
Mentions: On average, 20% of fusion events incorporated groups of four to eight RyRs into the bilayer. Under the right experimental conditions (e.g., −40 mV and the presence of ATP), the opening of one RyR in a group tended to promote the opening of other RyRs. Fig. 8 shows the activity of four, ATP-activated RyRs at positive and negative bilayer potentials. The current trace shows transitions between the current baseline (labeled C) and four equally spaced levels (O1–O4) corresponding to one to four open channels. At positive potentials, the weighting of each current level followed a binomial distribution expected from the gating in independent channels in the bilayer (unpublished data). At negative potentials, downward current steps frequently “bypassed” some of the current levels, indicating that several channels were opening in near synchrony. The weighting of current levels in these records markedly deviated from a binomial distribution. HMM analysis of these records (see materials and methods) shows that the mean RyR opening rate associated with transitions between the current baseline and level O1, k0+, was significantly slower than opening rates associated with transitions between levels O1–O4, k1+–k4+ (Fig. 9 A, □; P = 0.003, paired t test), while there was no significant difference in the rates associated with transitions between levels O1–O4. Channel closing rates, k-, did not depend on the number of open RyRs (Fig. 9 B). Thus, the coupling between RyRs was primarily due to the opening rate, which for each channel depended on whether or not one other channel was open in the bilayer.

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