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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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CSQ2 shifted the cytosolic Ca2+ sensitivity of single RyR2 channels. Holding potential was 0 mV and the luminal solution contained 100 mM Cs+. (A) Summary Po results from CSQ2-stripped (open circles; n = 8) and CSQ2-replaced (filled circles; n = 6) channels. The CSQ2-replaced channels were associated with CSQ2-WT (0.5 μg/ml in luminal chamber). Luminal free Ca2+ concentration was 1 mM and cytosolic Ca2+ was titrated from 0.1 to 100 μM. The curve fit to the filled circles has an EC50 of 1.04 ± 0.17 μM and a 3.4 Hill coefficient. The curve fit to the CSQ2-stripped data has an EC50 of 2.01 ± 0.34 μM and a 2.6 Hill coefficient. An unpaired t test was used to determine if the Po between CSQ2-replaced and stripped channels at each Ca2+ concentration was statistically different (**, P < 0.01; *, P < 0.05). Dotted curve represents the cytosolic Ca2+ sensitivity of CSQ2-stripped channels when 10 mM luminal Ca2+ was present. (B) Luminal Ca2+ and Mg2+ sensitivity of CSQ2-stripped channels. These stripped channels were maximally activated by high cytosolic Ca2+ (100 μM) and then luminal Ca2+ (open diamond; n = 13) or Mg2+ (open square; n = 16) was varied. The curve fit to the Ca2+ data has an EC50 of 379 ± 247 μM and a 0.70 Hill coefficient. The curve fit to the Mg2+ data has an EC50 of 972 ± 208 μM and a 0.77 Hill coefficient.
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fig4: CSQ2 shifted the cytosolic Ca2+ sensitivity of single RyR2 channels. Holding potential was 0 mV and the luminal solution contained 100 mM Cs+. (A) Summary Po results from CSQ2-stripped (open circles; n = 8) and CSQ2-replaced (filled circles; n = 6) channels. The CSQ2-replaced channels were associated with CSQ2-WT (0.5 μg/ml in luminal chamber). Luminal free Ca2+ concentration was 1 mM and cytosolic Ca2+ was titrated from 0.1 to 100 μM. The curve fit to the filled circles has an EC50 of 1.04 ± 0.17 μM and a 3.4 Hill coefficient. The curve fit to the CSQ2-stripped data has an EC50 of 2.01 ± 0.34 μM and a 2.6 Hill coefficient. An unpaired t test was used to determine if the Po between CSQ2-replaced and stripped channels at each Ca2+ concentration was statistically different (**, P < 0.01; *, P < 0.05). Dotted curve represents the cytosolic Ca2+ sensitivity of CSQ2-stripped channels when 10 mM luminal Ca2+ was present. (B) Luminal Ca2+ and Mg2+ sensitivity of CSQ2-stripped channels. These stripped channels were maximally activated by high cytosolic Ca2+ (100 μM) and then luminal Ca2+ (open diamond; n = 13) or Mg2+ (open square; n = 16) was varied. The curve fit to the Ca2+ data has an EC50 of 379 ± 247 μM and a 0.70 Hill coefficient. The curve fit to the Mg2+ data has an EC50 of 972 ± 208 μM and a 0.77 Hill coefficient.

Mentions: To this point, RyR2 luminal Ca2+ sensitivity has been defined in the presence of a constant 1 μM cytosolic bath Ca2+ concentration. Fig. 4 A shows how CSQ2 affects RyR2 cytosolic Ca2+ sensitivity at a constant luminal Ca2+ concentration (1 mM). The cytosolic Ca2+ sensitivity of CSQ2-stripped (Fig. 4 A, open diamonds) and CSQ2-replaced channels (Fig. 4 A, filled circles) is compared. The EC50 of cytosolic Ca2+ activation was 2.01 ± 0.34 μM (maximum Po ∼0.6) when no CSQ2 was associated with the channel. It was 1.04 ± 0.17 μM (maximum Po ∼0.8) when CSQ2 was present. An unpaired T test was used to determine if mean Po's of the CSQ2-replaced and stripped channels at each of the different cytosolic Ca2+ levels were statistical different. The Po was significantly different at all cytosolic Ca2+ levels >0.5 μM. Note that the lumen-to-cytosolic Ca2+ flux was essentially constant in these experiments because luminal Ca2+ was always 1 mM. Thus, this CSQ2-dependent change in cytosolic RyR2 Ca2+ sensitivity was not the result of feed-through Ca2+ modulation.


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)

CSQ2 shifted the cytosolic Ca2+ sensitivity of single RyR2 channels. Holding potential was 0 mV and the luminal solution contained 100 mM Cs+. (A) Summary Po results from CSQ2-stripped (open circles; n = 8) and CSQ2-replaced (filled circles; n = 6) channels. The CSQ2-replaced channels were associated with CSQ2-WT (0.5 μg/ml in luminal chamber). Luminal free Ca2+ concentration was 1 mM and cytosolic Ca2+ was titrated from 0.1 to 100 μM. The curve fit to the filled circles has an EC50 of 1.04 ± 0.17 μM and a 3.4 Hill coefficient. The curve fit to the CSQ2-stripped data has an EC50 of 2.01 ± 0.34 μM and a 2.6 Hill coefficient. An unpaired t test was used to determine if the Po between CSQ2-replaced and stripped channels at each Ca2+ concentration was statistically different (**, P < 0.01; *, P < 0.05). Dotted curve represents the cytosolic Ca2+ sensitivity of CSQ2-stripped channels when 10 mM luminal Ca2+ was present. (B) Luminal Ca2+ and Mg2+ sensitivity of CSQ2-stripped channels. These stripped channels were maximally activated by high cytosolic Ca2+ (100 μM) and then luminal Ca2+ (open diamond; n = 13) or Mg2+ (open square; n = 16) was varied. The curve fit to the Ca2+ data has an EC50 of 379 ± 247 μM and a 0.70 Hill coefficient. The curve fit to the Mg2+ data has an EC50 of 972 ± 208 μM and a 0.77 Hill coefficient.
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Related In: Results  -  Collection

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fig4: CSQ2 shifted the cytosolic Ca2+ sensitivity of single RyR2 channels. Holding potential was 0 mV and the luminal solution contained 100 mM Cs+. (A) Summary Po results from CSQ2-stripped (open circles; n = 8) and CSQ2-replaced (filled circles; n = 6) channels. The CSQ2-replaced channels were associated with CSQ2-WT (0.5 μg/ml in luminal chamber). Luminal free Ca2+ concentration was 1 mM and cytosolic Ca2+ was titrated from 0.1 to 100 μM. The curve fit to the filled circles has an EC50 of 1.04 ± 0.17 μM and a 3.4 Hill coefficient. The curve fit to the CSQ2-stripped data has an EC50 of 2.01 ± 0.34 μM and a 2.6 Hill coefficient. An unpaired t test was used to determine if the Po between CSQ2-replaced and stripped channels at each Ca2+ concentration was statistically different (**, P < 0.01; *, P < 0.05). Dotted curve represents the cytosolic Ca2+ sensitivity of CSQ2-stripped channels when 10 mM luminal Ca2+ was present. (B) Luminal Ca2+ and Mg2+ sensitivity of CSQ2-stripped channels. These stripped channels were maximally activated by high cytosolic Ca2+ (100 μM) and then luminal Ca2+ (open diamond; n = 13) or Mg2+ (open square; n = 16) was varied. The curve fit to the Ca2+ data has an EC50 of 379 ± 247 μM and a 0.70 Hill coefficient. The curve fit to the Mg2+ data has an EC50 of 972 ± 208 μM and a 0.77 Hill coefficient.
Mentions: To this point, RyR2 luminal Ca2+ sensitivity has been defined in the presence of a constant 1 μM cytosolic bath Ca2+ concentration. Fig. 4 A shows how CSQ2 affects RyR2 cytosolic Ca2+ sensitivity at a constant luminal Ca2+ concentration (1 mM). The cytosolic Ca2+ sensitivity of CSQ2-stripped (Fig. 4 A, open diamonds) and CSQ2-replaced channels (Fig. 4 A, filled circles) is compared. The EC50 of cytosolic Ca2+ activation was 2.01 ± 0.34 μM (maximum Po ∼0.6) when no CSQ2 was associated with the channel. It was 1.04 ± 0.17 μM (maximum Po ∼0.8) when CSQ2 was present. An unpaired T test was used to determine if mean Po's of the CSQ2-replaced and stripped channels at each of the different cytosolic Ca2+ levels were statistical different. The Po was significantly different at all cytosolic Ca2+ levels >0.5 μM. Note that the lumen-to-cytosolic Ca2+ flux was essentially constant in these experiments because luminal Ca2+ was always 1 mM. Thus, this CSQ2-dependent change in cytosolic RyR2 Ca2+ sensitivity was not the result of feed-through Ca2+ modulation.

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