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Luminal Ca2+ regulation of single cardiac ryanodine receptors: insights provided by calsequestrin and its mutants.

Qin J, Valle G, Nani A, Nori A, Rizzi N, Priori SG, Volpe P, Fill M - J. Gen. Physiol. (2008)

Bottom Line: It does not depend on CSQ2 oligomerization or CSQ2 monomer Ca2+ binding affinity.The R33Q CSQ2 mutant can participate in luminal RyR2 Ca2+ regulation but less effectively than wild-type (WT) CSQ2.CSQ2-L167H does not participate in luminal RyR2 Ca2+ regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, Rush University Medical Center, Chicago, IL 60612, USA.

ABSTRACT
The luminal Ca2+ regulation of cardiac ryanodine receptor (RyR2) was explored at the single channel level. The luminal Ca2+ and Mg2+ sensitivity of single CSQ2-stripped and CSQ2-associated RyR2 channels was defined. Action of wild-type CSQ2 and of two mutant CSQ2s (R33Q and L167H) was also compared. Two luminal Ca2+ regulatory mechanism(s) were identified. One is a RyR2-resident mechanism that is CSQ2 independent and does not distinguish between luminal Ca2+ and Mg2+. This mechanism modulates the maximal efficacy of cytosolic Ca2+ activation. The second luminal Ca2+ regulatory mechanism is CSQ2 dependent and distinguishes between luminal Ca2+ and Mg2+. It does not depend on CSQ2 oligomerization or CSQ2 monomer Ca2+ binding affinity. The key Ca2+-sensitive step in this mechanism may be the Ca2+-dependent CSQ2 interaction with triadin. The CSQ2-dependent mechanism alters the cytosolic Ca2+ sensitivity of the channel. The R33Q CSQ2 mutant can participate in luminal RyR2 Ca2+ regulation but less effectively than wild-type (WT) CSQ2. CSQ2-L167H does not participate in luminal RyR2 Ca2+ regulation. The disparate actions of these two catecholaminergic polymorphic ventricular tachycardia (CPVT)-linked mutants implies that either alteration or elimination of CSQ2-dependent luminal RyR2 regulation can generate the CPVT phenotype. We propose that the RyR2-resident, CSQ2-independent luminal Ca2+ mechanism may assure that all channels respond robustly to large (>5 muM) local cytosolic Ca2+ stimuli, whereas the CSQ2-dependent mechanism may help close RyR2 channels after luminal Ca2+ falls below approximately 0.5 mM.

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Ca2+-dependent CSQ2 oligomerization and CSQ2–triadin interaction. (A) The Ca2+ sensitivity of light (350 nm) scattering of CSQ2-WT (filled circles), CSQ2-R33Q (triangles), and CSQ2-L167H (inverted triangles) proteins in presence of 100 mM CsCl. Samples were stirred for 2 min before measurement. The curve fit to the CSQ2-WT data has an EC50 of 18.1 ± 5.23 mM and a 2.1 Hill coefficient. The curve fit to the CSQ2-R33Q data has an EC50 of 16.4 ± 1.18 mM and a 3.0 Hill coefficient. Both curves were fit with VMAX arbitrarily fixed at 0.6. (B) At left, top panel (i) depicts the Coomassie blue–stained SDS-PAGE of purified, recombinant CSQ2-WT (arrow, MW of ∼52,000). Bottom panel (i) depicts the Western blot with anti-triadin antibodies, revealing two bands having MW of ∼45,000 (glycosylated form) and 40,000 (unglycosylated form), respectively. At right (ii), the Ca2+ sensitivity of the interaction of glycosylated and unglycosylated triadin with CSQ2-WT, CSQ2-R33Q, and CSQ2-L167H was measured with either very low Ca2+ (1 mM EGTA) or 1 mM free Ca2+ present, and data are shown as means ± SEM (n = 5). Filled bars represent glycosylated triadin, open bars represent unglycosylated triadin. Asterisk indicates P < 0.05 using an unpaired Student's t test.
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fig6: Ca2+-dependent CSQ2 oligomerization and CSQ2–triadin interaction. (A) The Ca2+ sensitivity of light (350 nm) scattering of CSQ2-WT (filled circles), CSQ2-R33Q (triangles), and CSQ2-L167H (inverted triangles) proteins in presence of 100 mM CsCl. Samples were stirred for 2 min before measurement. The curve fit to the CSQ2-WT data has an EC50 of 18.1 ± 5.23 mM and a 2.1 Hill coefficient. The curve fit to the CSQ2-R33Q data has an EC50 of 16.4 ± 1.18 mM and a 3.0 Hill coefficient. Both curves were fit with VMAX arbitrarily fixed at 0.6. (B) At left, top panel (i) depicts the Coomassie blue–stained SDS-PAGE of purified, recombinant CSQ2-WT (arrow, MW of ∼52,000). Bottom panel (i) depicts the Western blot with anti-triadin antibodies, revealing two bands having MW of ∼45,000 (glycosylated form) and 40,000 (unglycosylated form), respectively. At right (ii), the Ca2+ sensitivity of the interaction of glycosylated and unglycosylated triadin with CSQ2-WT, CSQ2-R33Q, and CSQ2-L167H was measured with either very low Ca2+ (1 mM EGTA) or 1 mM free Ca2+ present, and data are shown as means ± SEM (n = 5). Filled bars represent glycosylated triadin, open bars represent unglycosylated triadin. Asterisk indicates P < 0.05 using an unpaired Student's t test.

Mentions: The differences in CSQ2-dependent RyR2 function (Fig. 5 B) could conceivably arise due to mutant vs. WT-dependent differences in CSQ2 oligomerization and/or the CSQ2–RyR2 interaction. Light scattering was used to measure the Ca2+ dependence of CSQ2 oligomerization in conditions (100 mM CsCl) similar to those used for bilayer experiments (Fig. 6 A). Increased light scattering here reflects more CSQ2 oligomerization. The CSQ2-WT and CSQ2-R33Q proteins oligomerized at Ca2+ levels >3 mM and this oligomerization had similar Ca2+ dependency. Virtually no oligomerization of the CSQ2-L167H protein was observed over the Ca2+ concentration range tested.


Luminal Ca2+ regulation of single cardiac ryanodine receptors: insights provided by calsequestrin and its mutants.

Qin J, Valle G, Nani A, Nori A, Rizzi N, Priori SG, Volpe P, Fill M - J. Gen. Physiol. (2008)

Ca2+-dependent CSQ2 oligomerization and CSQ2–triadin interaction. (A) The Ca2+ sensitivity of light (350 nm) scattering of CSQ2-WT (filled circles), CSQ2-R33Q (triangles), and CSQ2-L167H (inverted triangles) proteins in presence of 100 mM CsCl. Samples were stirred for 2 min before measurement. The curve fit to the CSQ2-WT data has an EC50 of 18.1 ± 5.23 mM and a 2.1 Hill coefficient. The curve fit to the CSQ2-R33Q data has an EC50 of 16.4 ± 1.18 mM and a 3.0 Hill coefficient. Both curves were fit with VMAX arbitrarily fixed at 0.6. (B) At left, top panel (i) depicts the Coomassie blue–stained SDS-PAGE of purified, recombinant CSQ2-WT (arrow, MW of ∼52,000). Bottom panel (i) depicts the Western blot with anti-triadin antibodies, revealing two bands having MW of ∼45,000 (glycosylated form) and 40,000 (unglycosylated form), respectively. At right (ii), the Ca2+ sensitivity of the interaction of glycosylated and unglycosylated triadin with CSQ2-WT, CSQ2-R33Q, and CSQ2-L167H was measured with either very low Ca2+ (1 mM EGTA) or 1 mM free Ca2+ present, and data are shown as means ± SEM (n = 5). Filled bars represent glycosylated triadin, open bars represent unglycosylated triadin. Asterisk indicates P < 0.05 using an unpaired Student's t test.
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fig6: Ca2+-dependent CSQ2 oligomerization and CSQ2–triadin interaction. (A) The Ca2+ sensitivity of light (350 nm) scattering of CSQ2-WT (filled circles), CSQ2-R33Q (triangles), and CSQ2-L167H (inverted triangles) proteins in presence of 100 mM CsCl. Samples were stirred for 2 min before measurement. The curve fit to the CSQ2-WT data has an EC50 of 18.1 ± 5.23 mM and a 2.1 Hill coefficient. The curve fit to the CSQ2-R33Q data has an EC50 of 16.4 ± 1.18 mM and a 3.0 Hill coefficient. Both curves were fit with VMAX arbitrarily fixed at 0.6. (B) At left, top panel (i) depicts the Coomassie blue–stained SDS-PAGE of purified, recombinant CSQ2-WT (arrow, MW of ∼52,000). Bottom panel (i) depicts the Western blot with anti-triadin antibodies, revealing two bands having MW of ∼45,000 (glycosylated form) and 40,000 (unglycosylated form), respectively. At right (ii), the Ca2+ sensitivity of the interaction of glycosylated and unglycosylated triadin with CSQ2-WT, CSQ2-R33Q, and CSQ2-L167H was measured with either very low Ca2+ (1 mM EGTA) or 1 mM free Ca2+ present, and data are shown as means ± SEM (n = 5). Filled bars represent glycosylated triadin, open bars represent unglycosylated triadin. Asterisk indicates P < 0.05 using an unpaired Student's t test.
Mentions: The differences in CSQ2-dependent RyR2 function (Fig. 5 B) could conceivably arise due to mutant vs. WT-dependent differences in CSQ2 oligomerization and/or the CSQ2–RyR2 interaction. Light scattering was used to measure the Ca2+ dependence of CSQ2 oligomerization in conditions (100 mM CsCl) similar to those used for bilayer experiments (Fig. 6 A). Increased light scattering here reflects more CSQ2 oligomerization. The CSQ2-WT and CSQ2-R33Q proteins oligomerized at Ca2+ levels >3 mM and this oligomerization had similar Ca2+ dependency. Virtually no oligomerization of the CSQ2-L167H protein was observed over the Ca2+ concentration range tested.

Bottom Line: It does not depend on CSQ2 oligomerization or CSQ2 monomer Ca2+ binding affinity.The R33Q CSQ2 mutant can participate in luminal RyR2 Ca2+ regulation but less effectively than wild-type (WT) CSQ2.CSQ2-L167H does not participate in luminal RyR2 Ca2+ regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, Rush University Medical Center, Chicago, IL 60612, USA.

ABSTRACT
The luminal Ca2+ regulation of cardiac ryanodine receptor (RyR2) was explored at the single channel level. The luminal Ca2+ and Mg2+ sensitivity of single CSQ2-stripped and CSQ2-associated RyR2 channels was defined. Action of wild-type CSQ2 and of two mutant CSQ2s (R33Q and L167H) was also compared. Two luminal Ca2+ regulatory mechanism(s) were identified. One is a RyR2-resident mechanism that is CSQ2 independent and does not distinguish between luminal Ca2+ and Mg2+. This mechanism modulates the maximal efficacy of cytosolic Ca2+ activation. The second luminal Ca2+ regulatory mechanism is CSQ2 dependent and distinguishes between luminal Ca2+ and Mg2+. It does not depend on CSQ2 oligomerization or CSQ2 monomer Ca2+ binding affinity. The key Ca2+-sensitive step in this mechanism may be the Ca2+-dependent CSQ2 interaction with triadin. The CSQ2-dependent mechanism alters the cytosolic Ca2+ sensitivity of the channel. The R33Q CSQ2 mutant can participate in luminal RyR2 Ca2+ regulation but less effectively than wild-type (WT) CSQ2. CSQ2-L167H does not participate in luminal RyR2 Ca2+ regulation. The disparate actions of these two catecholaminergic polymorphic ventricular tachycardia (CPVT)-linked mutants implies that either alteration or elimination of CSQ2-dependent luminal RyR2 regulation can generate the CPVT phenotype. We propose that the RyR2-resident, CSQ2-independent luminal Ca2+ mechanism may assure that all channels respond robustly to large (>5 muM) local cytosolic Ca2+ stimuli, whereas the CSQ2-dependent mechanism may help close RyR2 channels after luminal Ca2+ falls below approximately 0.5 mM.

Show MeSH
Related in: MedlinePlus