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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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Luminal Ca2+ regulation of RyR2 channels by the CSQ2-R33Q and CSQ2-L167H mutants. Mutant CSQ2 (0.5 μg/ml) was added to the luminal side of previously CSQ2-stripped channels. Cytosolic free Ca2+ concentration was 1 μM and luminal Ca2+ was titrated from 10 μM to 10 mM. Holding potential was 0 mV and the luminal solution contained 100 mM Cs+. (A) Example channel recordings with CSQ2-R33Q (left) or CSQ2-L167H (right) present are shown (zero current level marked). (B) Summary Po results. The CSQ2-R33Q data (triangle) was collected on eight different channels. The CSQ2-L167H data (inverted triangles) was collected on eight different channels as well. Dotted line represents CSQ2-WT result presented in Fig. 2 B.
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fig5: Luminal Ca2+ regulation of RyR2 channels by the CSQ2-R33Q and CSQ2-L167H mutants. Mutant CSQ2 (0.5 μg/ml) was added to the luminal side of previously CSQ2-stripped channels. Cytosolic free Ca2+ concentration was 1 μM and luminal Ca2+ was titrated from 10 μM to 10 mM. Holding potential was 0 mV and the luminal solution contained 100 mM Cs+. (A) Example channel recordings with CSQ2-R33Q (left) or CSQ2-L167H (right) present are shown (zero current level marked). (B) Summary Po results. The CSQ2-R33Q data (triangle) was collected on eight different channels. The CSQ2-L167H data (inverted triangles) was collected on eight different channels as well. Dotted line represents CSQ2-WT result presented in Fig. 2 B.

Mentions: Several mutants of CSQ2 are linked to the tachyarrhythmic disorder CPVT. How two of these CSQ2 mutants (R33Q and L167H) regulate single RyR2 channels is shown in Fig. 5. Single RyR2 channels were incorporated into the bilayer and then stripped of any endogenous CSQ2 present. With 1 μM cytosolic Ca2+ always present, the luminal Ca2+ concentration was titrated in the presence of either the R33Q or L167H mutant (0.5 μg/ml). Sample single channel recordings are shown in Fig. 5 A. Single RyR2 channels associated with the R33Q mutant were sensitive to luminal Ca2+. Channels associated with the L167H mutant were not. Fig. 5 B shows summary results collected from many different channels. The dashed line represents the luminal Ca2+ sensitivity of channels associated with WT CSQ2 (from Fig. 2 B). Channels associated with the R33Q mutant (filled triangles) had significantly higher Po (t test P < 0.05) at luminal ≤250 μM compared with those channels associated with WT CSQ2. However, their Po was similar to channels associated with WT CSQ2 at 1 mM luminal Ca2+. The Po at 5 and 10 mM luminal Ca2+ were not statistically different (t test P > 0.1) between the CSQ2-R33Q and CSQ2-WT datasets. In the presence of the L167H mutant (Fig. 5 B, filled inverted triangles), the Po did not change as the luminal Ca2+ concentration varied. Indeed, the Po's at 10 μM, 1 mM, and 10 mM luminal Ca2+ were not statistically different (t test P > 0.1) when these data were compared with the stripped (CSQ2-free) channel data.


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)

Luminal Ca2+ regulation of RyR2 channels by the CSQ2-R33Q and CSQ2-L167H mutants. Mutant CSQ2 (0.5 μg/ml) was added to the luminal side of previously CSQ2-stripped channels. Cytosolic free Ca2+ concentration was 1 μM and luminal Ca2+ was titrated from 10 μM to 10 mM. Holding potential was 0 mV and the luminal solution contained 100 mM Cs+. (A) Example channel recordings with CSQ2-R33Q (left) or CSQ2-L167H (right) present are shown (zero current level marked). (B) Summary Po results. The CSQ2-R33Q data (triangle) was collected on eight different channels. The CSQ2-L167H data (inverted triangles) was collected on eight different channels as well. Dotted line represents CSQ2-WT result presented in Fig. 2 B.
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fig5: Luminal Ca2+ regulation of RyR2 channels by the CSQ2-R33Q and CSQ2-L167H mutants. Mutant CSQ2 (0.5 μg/ml) was added to the luminal side of previously CSQ2-stripped channels. Cytosolic free Ca2+ concentration was 1 μM and luminal Ca2+ was titrated from 10 μM to 10 mM. Holding potential was 0 mV and the luminal solution contained 100 mM Cs+. (A) Example channel recordings with CSQ2-R33Q (left) or CSQ2-L167H (right) present are shown (zero current level marked). (B) Summary Po results. The CSQ2-R33Q data (triangle) was collected on eight different channels. The CSQ2-L167H data (inverted triangles) was collected on eight different channels as well. Dotted line represents CSQ2-WT result presented in Fig. 2 B.
Mentions: Several mutants of CSQ2 are linked to the tachyarrhythmic disorder CPVT. How two of these CSQ2 mutants (R33Q and L167H) regulate single RyR2 channels is shown in Fig. 5. Single RyR2 channels were incorporated into the bilayer and then stripped of any endogenous CSQ2 present. With 1 μM cytosolic Ca2+ always present, the luminal Ca2+ concentration was titrated in the presence of either the R33Q or L167H mutant (0.5 μg/ml). Sample single channel recordings are shown in Fig. 5 A. Single RyR2 channels associated with the R33Q mutant were sensitive to luminal Ca2+. Channels associated with the L167H mutant were not. Fig. 5 B shows summary results collected from many different channels. The dashed line represents the luminal Ca2+ sensitivity of channels associated with WT CSQ2 (from Fig. 2 B). Channels associated with the R33Q mutant (filled triangles) had significantly higher Po (t test P < 0.05) at luminal ≤250 μM compared with those channels associated with WT CSQ2. However, their Po was similar to channels associated with WT CSQ2 at 1 mM luminal Ca2+. The Po at 5 and 10 mM luminal Ca2+ were not statistically different (t test P > 0.1) between the CSQ2-R33Q and CSQ2-WT datasets. In the presence of the L167H mutant (Fig. 5 B, filled inverted triangles), the Po did not change as the luminal Ca2+ concentration varied. Indeed, the Po's at 10 μM, 1 mM, and 10 mM luminal Ca2+ were not statistically different (t test P > 0.1) when these data were compared with the stripped (CSQ2-free) channel data.

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