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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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Related in: MedlinePlus

Normality testing. Data collected in the presence of 1 μM cytosolic Ca2+ and 1 mM lumenal Ca2+ in three different experimental situations. Control represents channels that were not subjected to the CSQ2 stripping process. Stripped represents channels that underwent the stripping process. Replaced represents stripped channels after CSQ2 (0.5 μg/ml) was added to the luminal bath. Open symbols are individual determinations. Filled symbols are means (±SEM). Outcome of the Anderson-Darling and Shapiro-Wilk normality tests are shown at the bottom.
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fig3: Normality testing. Data collected in the presence of 1 μM cytosolic Ca2+ and 1 mM lumenal Ca2+ in three different experimental situations. Control represents channels that were not subjected to the CSQ2 stripping process. Stripped represents channels that underwent the stripping process. Replaced represents stripped channels after CSQ2 (0.5 μg/ml) was added to the luminal bath. Open symbols are individual determinations. Filled symbols are means (±SEM). Outcome of the Anderson-Darling and Shapiro-Wilk normality tests are shown at the bottom.

Mentions: The results shown in Fig. 3 were collected in the presence of 1 μM cytosolic Ca2+ and 1 mM luminal Ca2+ in three different experimental situations. The first situation is labeled control where channels had never been exposed to the CSQ2 stripping procedure. The second is stripped where channels were exposed to the stripping procedure. The third is replaced where CSQ2 (0.5 μg/ml) was added back to previously stripped channels. In Fig. 3 (top), the mean (filled symbols) and corresponding individual determinations (open symbols) for each situation are plotted. The normality of the distributions about their means was tested using the Anderson-Darling and Shapiro-Wilk normality tests. These tests indicate a distribution is statistically different than normal if the statistic P is less than 0.05. These tests indicated that the control channel data population was the only one that was statistically different than normal (Fig. 3, bottom). This supports the contention that the control channel population contains two classes of channels (i.e., those with and without CSQ2 attached).


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)

Normality testing. Data collected in the presence of 1 μM cytosolic Ca2+ and 1 mM lumenal Ca2+ in three different experimental situations. Control represents channels that were not subjected to the CSQ2 stripping process. Stripped represents channels that underwent the stripping process. Replaced represents stripped channels after CSQ2 (0.5 μg/ml) was added to the luminal bath. Open symbols are individual determinations. Filled symbols are means (±SEM). Outcome of the Anderson-Darling and Shapiro-Wilk normality tests are shown at the bottom.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2279168&req=5

fig3: Normality testing. Data collected in the presence of 1 μM cytosolic Ca2+ and 1 mM lumenal Ca2+ in three different experimental situations. Control represents channels that were not subjected to the CSQ2 stripping process. Stripped represents channels that underwent the stripping process. Replaced represents stripped channels after CSQ2 (0.5 μg/ml) was added to the luminal bath. Open symbols are individual determinations. Filled symbols are means (±SEM). Outcome of the Anderson-Darling and Shapiro-Wilk normality tests are shown at the bottom.
Mentions: The results shown in Fig. 3 were collected in the presence of 1 μM cytosolic Ca2+ and 1 mM luminal Ca2+ in three different experimental situations. The first situation is labeled control where channels had never been exposed to the CSQ2 stripping procedure. The second is stripped where channels were exposed to the stripping procedure. The third is replaced where CSQ2 (0.5 μg/ml) was added back to previously stripped channels. In Fig. 3 (top), the mean (filled symbols) and corresponding individual determinations (open symbols) for each situation are plotted. The normality of the distributions about their means was tested using the Anderson-Darling and Shapiro-Wilk normality tests. These tests indicate a distribution is statistically different than normal if the statistic P is less than 0.05. These tests indicated that the control channel data population was the only one that was statistically different than normal (Fig. 3, bottom). This supports the contention that the control channel population contains two classes of channels (i.e., those with and without CSQ2 attached).

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