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Luminal Mg2+, a key factor controlling RYR2-mediated Ca2+ release: cytoplasmic and luminal regulation modeled in a tetrameric channel.

Laver DR, Honen BN - J. Gen. Physiol. (2008)

Bottom Line: The effects of luminal Ca(2+) and Mg(2+) on sheep RYR2 were measured in lipid bilayers.Ca(2+) and Mg(2+) on opposite sides of the bilayer exhibited competitive effects on RYRs, indicating that they can compete via the pore for common sites.The model predicts that under physiological conditions, SR load-dependent Ca(2+) release (1) is mainly determined by Ca(2+) displacement of Mg(2+) from the L-site as SR loading increases, and (2) depends on the properties of both luminal and cytoplasmic activation mechanisms.

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 cardiac muscle, intracellular Ca(2+) and Mg(2+) are potent regulators of calcium release from the sarcoplasmic reticulum (SR). It is well known that the free [Ca(2+)] in the SR ([Ca(2+)](L)) stimulates the Ca(2+) release channels (ryanodine receptor [RYR]2). However, little is known about the action of luminal Mg(2+), which has not been regarded as an important regulator of Ca(2+) release. The effects of luminal Ca(2+) and Mg(2+) on sheep RYR2 were measured in lipid bilayers. Cytoplasmic and luminal Ca(2+) produced a synergistic increase in the opening rate of RYRs. A novel, high affinity inhibition of RYR2 by luminal Mg(2+) was observed, pointing to an important physiological role for luminal Mg(2+) in cardiac muscle. At diastolic [Ca(2+)](C), luminal Mg(2+) inhibition was voltage independent, with K(i) = 45 microM at luminal [Ca(2+)] ([Ca(2+)](L)) = 100 microM. Luminal and cytoplasmic Mg(2+) inhibition was alleviated by increasing [Ca(2+)](L) or [Ca(2+)](C). Ca(2+) and Mg(2+) on opposite sides of the bilayer exhibited competitive effects on RYRs, indicating that they can compete via the pore for common sites. The data were accurately fitted by a model based on a tetrameric RYR structure with four Ca(2+)-sensing mechanisms on each subunit: activating luminal L-site (40-microM affinity for Mg(2+) and Ca(2+)), cytoplasmic A-site (1.2 microM for Ca(2+) and 60 microM for Mg(2+)), inactivating cytoplasmic I(1)-site (approximately 10 mM for Ca(2+) and Mg(2+)), and I(2)-site (1.2 microM for Ca(2+)). Activation of three or more subunits will cause channel opening. Mg(2+) inhibition occurs primarily by Mg(2+) displacing Ca(2+) from the L- and A-sites, and Mg(2+) fails to open the channel. The model predicts that under physiological conditions, SR load-dependent Ca(2+) release (1) is mainly determined by Ca(2+) displacement of Mg(2+) from the L-site as SR loading increases, and (2) depends on the properties of both luminal and cytoplasmic activation mechanisms.

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Kinetic schemes for luminal-triggered Ca2+ feedthrough. Ca2+ activation (scheme 1, A- and L- sites) and Ca2+ inactivation (scheme 3, I1 and I2-sites). Each pair of open and closed states is associated with different functional states of individual RYR2 subunits, and the numbers refer to the entries in Table IV describing these stoichiometries. Asterisks indicate changes in subunit stoichiometry, and these depend on binding of Ca2+ or Mg2+. Also shown are their corresponding simplified schemes (schemes 2 and 4) and the overall scheme (scheme 5) resulting from the combined action of activation and inactivation. The reaction rates in scheme 5 have complex dependencies on [Ca2+] and [Mg2+], and these are derived in Eqs. 2–13. Reaction rates that depend on the Ca2+ and Mg2+ concentrations at the cytoplasmic pore mouth will depend on the permeability of these ions in the channel (Eqs. 1–4). Hence, they are given subscripts “o” and “c” to indicate the different rates associated with open and closed channels, respectively. In scheme 5, the open and closed status of the channel associated with each kinetic state is indicated by the subscripts “O” and “C,” respectively.
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fig10: Kinetic schemes for luminal-triggered Ca2+ feedthrough. Ca2+ activation (scheme 1, A- and L- sites) and Ca2+ inactivation (scheme 3, I1 and I2-sites). Each pair of open and closed states is associated with different functional states of individual RYR2 subunits, and the numbers refer to the entries in Table IV describing these stoichiometries. Asterisks indicate changes in subunit stoichiometry, and these depend on binding of Ca2+ or Mg2+. Also shown are their corresponding simplified schemes (schemes 2 and 4) and the overall scheme (scheme 5) resulting from the combined action of activation and inactivation. The reaction rates in scheme 5 have complex dependencies on [Ca2+] and [Mg2+], and these are derived in Eqs. 2–13. Reaction rates that depend on the Ca2+ and Mg2+ concentrations at the cytoplasmic pore mouth will depend on the permeability of these ions in the channel (Eqs. 1–4). Hence, they are given subscripts “o” and “c” to indicate the different rates associated with open and closed channels, respectively. In scheme 5, the open and closed status of the channel associated with each kinetic state is indicated by the subscripts “O” and “C,” respectively.

Mentions: Previous work by Zahradnik et al. (2005) established that each subunit has independent Ca2+ sites that cause channel opening by an allosteric mechanism. We have incorporated these findings into the gating model for luminal and cytoplasmic Ca2+ and Mg2+ (Fig. 10, scheme 1). In this model, the pairs of open and closed states (C↔O) are associated with particular subunit stoichiometries of the RYR (i.e., particular combinations of subunits with bound sites). The Ca2+ and Mg2+ dependencies in gating arise from the transition rates between different subunit stoichiometries (Fig. 10, scheme 1, asterisks), whereas the rates within each C↔O pair are independent of Ca2+ and Mg2+.


Luminal Mg2+, a key factor controlling RYR2-mediated Ca2+ release: cytoplasmic and luminal regulation modeled in a tetrameric channel.

Laver DR, Honen BN - J. Gen. Physiol. (2008)

Kinetic schemes for luminal-triggered Ca2+ feedthrough. Ca2+ activation (scheme 1, A- and L- sites) and Ca2+ inactivation (scheme 3, I1 and I2-sites). Each pair of open and closed states is associated with different functional states of individual RYR2 subunits, and the numbers refer to the entries in Table IV describing these stoichiometries. Asterisks indicate changes in subunit stoichiometry, and these depend on binding of Ca2+ or Mg2+. Also shown are their corresponding simplified schemes (schemes 2 and 4) and the overall scheme (scheme 5) resulting from the combined action of activation and inactivation. The reaction rates in scheme 5 have complex dependencies on [Ca2+] and [Mg2+], and these are derived in Eqs. 2–13. Reaction rates that depend on the Ca2+ and Mg2+ concentrations at the cytoplasmic pore mouth will depend on the permeability of these ions in the channel (Eqs. 1–4). Hence, they are given subscripts “o” and “c” to indicate the different rates associated with open and closed channels, respectively. In scheme 5, the open and closed status of the channel associated with each kinetic state is indicated by the subscripts “O” and “C,” respectively.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2553390&req=5

fig10: Kinetic schemes for luminal-triggered Ca2+ feedthrough. Ca2+ activation (scheme 1, A- and L- sites) and Ca2+ inactivation (scheme 3, I1 and I2-sites). Each pair of open and closed states is associated with different functional states of individual RYR2 subunits, and the numbers refer to the entries in Table IV describing these stoichiometries. Asterisks indicate changes in subunit stoichiometry, and these depend on binding of Ca2+ or Mg2+. Also shown are their corresponding simplified schemes (schemes 2 and 4) and the overall scheme (scheme 5) resulting from the combined action of activation and inactivation. The reaction rates in scheme 5 have complex dependencies on [Ca2+] and [Mg2+], and these are derived in Eqs. 2–13. Reaction rates that depend on the Ca2+ and Mg2+ concentrations at the cytoplasmic pore mouth will depend on the permeability of these ions in the channel (Eqs. 1–4). Hence, they are given subscripts “o” and “c” to indicate the different rates associated with open and closed channels, respectively. In scheme 5, the open and closed status of the channel associated with each kinetic state is indicated by the subscripts “O” and “C,” respectively.
Mentions: Previous work by Zahradnik et al. (2005) established that each subunit has independent Ca2+ sites that cause channel opening by an allosteric mechanism. We have incorporated these findings into the gating model for luminal and cytoplasmic Ca2+ and Mg2+ (Fig. 10, scheme 1). In this model, the pairs of open and closed states (C↔O) are associated with particular subunit stoichiometries of the RYR (i.e., particular combinations of subunits with bound sites). The Ca2+ and Mg2+ dependencies in gating arise from the transition rates between different subunit stoichiometries (Fig. 10, scheme 1, asterisks), whereas the rates within each C↔O pair are independent of Ca2+ and Mg2+.

Bottom Line: The effects of luminal Ca(2+) and Mg(2+) on sheep RYR2 were measured in lipid bilayers.Ca(2+) and Mg(2+) on opposite sides of the bilayer exhibited competitive effects on RYRs, indicating that they can compete via the pore for common sites.The model predicts that under physiological conditions, SR load-dependent Ca(2+) release (1) is mainly determined by Ca(2+) displacement of Mg(2+) from the L-site as SR loading increases, and (2) depends on the properties of both luminal and cytoplasmic activation mechanisms.

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 cardiac muscle, intracellular Ca(2+) and Mg(2+) are potent regulators of calcium release from the sarcoplasmic reticulum (SR). It is well known that the free [Ca(2+)] in the SR ([Ca(2+)](L)) stimulates the Ca(2+) release channels (ryanodine receptor [RYR]2). However, little is known about the action of luminal Mg(2+), which has not been regarded as an important regulator of Ca(2+) release. The effects of luminal Ca(2+) and Mg(2+) on sheep RYR2 were measured in lipid bilayers. Cytoplasmic and luminal Ca(2+) produced a synergistic increase in the opening rate of RYRs. A novel, high affinity inhibition of RYR2 by luminal Mg(2+) was observed, pointing to an important physiological role for luminal Mg(2+) in cardiac muscle. At diastolic [Ca(2+)](C), luminal Mg(2+) inhibition was voltage independent, with K(i) = 45 microM at luminal [Ca(2+)] ([Ca(2+)](L)) = 100 microM. Luminal and cytoplasmic Mg(2+) inhibition was alleviated by increasing [Ca(2+)](L) or [Ca(2+)](C). Ca(2+) and Mg(2+) on opposite sides of the bilayer exhibited competitive effects on RYRs, indicating that they can compete via the pore for common sites. The data were accurately fitted by a model based on a tetrameric RYR structure with four Ca(2+)-sensing mechanisms on each subunit: activating luminal L-site (40-microM affinity for Mg(2+) and Ca(2+)), cytoplasmic A-site (1.2 microM for Ca(2+) and 60 microM for Mg(2+)), inactivating cytoplasmic I(1)-site (approximately 10 mM for Ca(2+) and Mg(2+)), and I(2)-site (1.2 microM for Ca(2+)). Activation of three or more subunits will cause channel opening. Mg(2+) inhibition occurs primarily by Mg(2+) displacing Ca(2+) from the L- and A-sites, and Mg(2+) fails to open the channel. The model predicts that under physiological conditions, SR load-dependent Ca(2+) release (1) is mainly determined by Ca(2+) displacement of Mg(2+) from the L-site as SR loading increases, and (2) depends on the properties of both luminal and cytoplasmic activation mechanisms.

Show MeSH