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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+ of CSQ2-stripped and CSQ2-added RyR2 channels. The CSQ2-added channels here refer to channels that were first CSQ2 stripped before 0.5 μg/ml CSQ2-WT was added to the luminal solution. Cytosolic free Ca2+ concentration was constant (1 μM). The luminal Ca2+ or Mg2+ concentration was titrated from 10 μM to 10 mM. Holding potential was 0 mV. The luminal solution contained 100 mM Cs+ and net unit current was always in the lumen-to-cytosolic direction. (A) Example single channel recordings from a CSQ2-stripped channel (left) and a CSQ2-added channel (right). Zero current levels are marked. Bar, 2 pA.(B) Summary Po results. Luminal Ca2+ sensitivity of the CSQ2-added data (black circles) was collected from 14 different channels. Luminal Ca2+ sensitivity of the CSQ2-stripped data (open diamonds) was collected from six different channels. Dotted line represents control Po result presented in Fig. 1 B.
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fig2: Luminal Ca2+ of CSQ2-stripped and CSQ2-added RyR2 channels. The CSQ2-added channels here refer to channels that were first CSQ2 stripped before 0.5 μg/ml CSQ2-WT was added to the luminal solution. Cytosolic free Ca2+ concentration was constant (1 μM). The luminal Ca2+ or Mg2+ concentration was titrated from 10 μM to 10 mM. Holding potential was 0 mV. The luminal solution contained 100 mM Cs+ and net unit current was always in the lumen-to-cytosolic direction. (A) Example single channel recordings from a CSQ2-stripped channel (left) and a CSQ2-added channel (right). Zero current levels are marked. Bar, 2 pA.(B) Summary Po results. Luminal Ca2+ sensitivity of the CSQ2-added data (black circles) was collected from 14 different channels. Luminal Ca2+ sensitivity of the CSQ2-stripped data (open diamonds) was collected from six different channels. Dotted line represents control Po result presented in Fig. 1 B.

Mentions: Example single channel recordings illustrating the luminal Ca sensitivity of a CSQ2 stripped channel are shown in Fig. 2 A (left). Summary Po results from several CSQ stripped channels are shown in Fig. 2 B (open diamonds). The Po of stripped channels did not change over the tested luminal Ca2+ range. The lost luminal Ca2+ sensitivity following the stripping procedure suggests the luminal Ca2+ sensitivity of control channels (see Fig. 1 B) was CSQ2 dependent. This was tested by adding recombinant purified CSQ2 (0.5 μg/ml) to the luminal side of previously CSQ2-stripped channels. Sample recordings from CSQ2-replaced channels are shown in Fig. 2 A (right). The average luminal Ca2+ sensitivity of several CSQ2-replaced channels is shown in Fig. 2 B (filled circles). The dotted line in Fig. 2 B represents the control channel data presented in Fig. 1 B. The CSQ2-replaced and control channels have analogous luminal Ca2+ sensitivities. However, there is a clear difference in the peak Po reached at 1000 μM luminal Ca2+. The reason for this may be that not all of the control channels had CSQ2 associated with them and this possibility is examined further below.


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+ of CSQ2-stripped and CSQ2-added RyR2 channels. The CSQ2-added channels here refer to channels that were first CSQ2 stripped before 0.5 μg/ml CSQ2-WT was added to the luminal solution. Cytosolic free Ca2+ concentration was constant (1 μM). The luminal Ca2+ or Mg2+ concentration was titrated from 10 μM to 10 mM. Holding potential was 0 mV. The luminal solution contained 100 mM Cs+ and net unit current was always in the lumen-to-cytosolic direction. (A) Example single channel recordings from a CSQ2-stripped channel (left) and a CSQ2-added channel (right). Zero current levels are marked. Bar, 2 pA.(B) Summary Po results. Luminal Ca2+ sensitivity of the CSQ2-added data (black circles) was collected from 14 different channels. Luminal Ca2+ sensitivity of the CSQ2-stripped data (open diamonds) was collected from six different channels. Dotted line represents control Po result presented in Fig. 1 B.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2279168&req=5

fig2: Luminal Ca2+ of CSQ2-stripped and CSQ2-added RyR2 channels. The CSQ2-added channels here refer to channels that were first CSQ2 stripped before 0.5 μg/ml CSQ2-WT was added to the luminal solution. Cytosolic free Ca2+ concentration was constant (1 μM). The luminal Ca2+ or Mg2+ concentration was titrated from 10 μM to 10 mM. Holding potential was 0 mV. The luminal solution contained 100 mM Cs+ and net unit current was always in the lumen-to-cytosolic direction. (A) Example single channel recordings from a CSQ2-stripped channel (left) and a CSQ2-added channel (right). Zero current levels are marked. Bar, 2 pA.(B) Summary Po results. Luminal Ca2+ sensitivity of the CSQ2-added data (black circles) was collected from 14 different channels. Luminal Ca2+ sensitivity of the CSQ2-stripped data (open diamonds) was collected from six different channels. Dotted line represents control Po result presented in Fig. 1 B.
Mentions: Example single channel recordings illustrating the luminal Ca sensitivity of a CSQ2 stripped channel are shown in Fig. 2 A (left). Summary Po results from several CSQ stripped channels are shown in Fig. 2 B (open diamonds). The Po of stripped channels did not change over the tested luminal Ca2+ range. The lost luminal Ca2+ sensitivity following the stripping procedure suggests the luminal Ca2+ sensitivity of control channels (see Fig. 1 B) was CSQ2 dependent. This was tested by adding recombinant purified CSQ2 (0.5 μg/ml) to the luminal side of previously CSQ2-stripped channels. Sample recordings from CSQ2-replaced channels are shown in Fig. 2 A (right). The average luminal Ca2+ sensitivity of several CSQ2-replaced channels is shown in Fig. 2 B (filled circles). The dotted line in Fig. 2 B represents the control channel data presented in Fig. 1 B. The CSQ2-replaced and control channels have analogous luminal Ca2+ sensitivities. However, there is a clear difference in the peak Po reached at 1000 μM luminal Ca2+. The reason for this may be that not all of the control channels had CSQ2 associated with them and this possibility is examined further below.

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