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Divergent Activity Profiles of Type 1 Ryanodine Receptor Channels Carrying Malignant Hyperthermia and Central Core Disease Mutations in the Amino-Terminal Region.

Murayama T, Kurebayashi N, Yamazawa T, Oyamada H, Suzuki J, Kanemaru K, Oguchi K, Iino M, Sakurai T - PLoS ONE (2015)

Bottom Line: Most MH and CCD mutations cause accelerated Ca2+ release, resulting in abnormal Ca2+ homeostasis in skeletal muscle.The gain was consistently higher in both MH and MH/CCD mutations.This approach should be useful for diagnosis and treatment of diseases with mutations in RyR1.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Pharmacology, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan.

ABSTRACT
The type 1 ryanodine receptor (RyR1) is a Ca2+ release channel in the sarcoplasmic reticulum of skeletal muscle and is mutated in several diseases, including malignant hyperthermia (MH) and central core disease (CCD). Most MH and CCD mutations cause accelerated Ca2+ release, resulting in abnormal Ca2+ homeostasis in skeletal muscle. However, how specific mutations affect the channel to produce different phenotypes is not well understood. In this study, we have investigated 11 mutations at 7 different positions in the amino (N)-terminal region of RyR1 (9 MH and 2 MH/CCD mutations) using a heterologous expression system in HEK293 cells. In live-cell Ca2+ imaging at room temperature (~25 °C), cells expressing mutant channels exhibited alterations in Ca2+ homeostasis, i.e., an enhanced sensitivity to caffeine, a depletion of Ca2+ in the ER and an increase in resting cytoplasmic Ca2+. RyR1 channel activity was quantitatively evaluated by [3H]ryanodine binding and three parameters (sensitivity to activating Ca2+, sensitivity to inactivating Ca2+ and attainable maximum activity, i.e., gain) were obtained by fitting analysis. The mutations increased the gain and the sensitivity to activating Ca2+ in a site-specific manner. The gain was consistently higher in both MH and MH/CCD mutations. Sensitivity to activating Ca2+ was markedly enhanced in MH/CCD mutations. The channel activity estimated from the three parameters provides a reasonable explanation to the pathological phenotype assessed by Ca2+ homeostasis. These properties were also observed at higher temperatures (~37 °C). Our data suggest that divergent activity profiles may cause varied disease phenotypes by specific mutations. This approach should be useful for diagnosis and treatment of diseases with mutations in RyR1.

No MeSH data available.


Related in: MedlinePlus

Comparison of the activity profiles between 25 and 37°C.The three parameters, Amax (A), KA (B) and KI, (C) for WT (filled circles), MH (open circles), MH/CCD (crosses) and C36R (triangles) at 25 and 37°C are plotted. Note that high linear correlations (R2 > 0.8) were found between mutants for all parameters. D. Estimated ryanodine binding at pCa 7.
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pone.0130606.g007: Comparison of the activity profiles between 25 and 37°C.The three parameters, Amax (A), KA (B) and KI, (C) for WT (filled circles), MH (open circles), MH/CCD (crosses) and C36R (triangles) at 25 and 37°C are plotted. Note that high linear correlations (R2 > 0.8) were found between mutants for all parameters. D. Estimated ryanodine binding at pCa 7.

Mentions: We initially determined the Ca2+-dependent [3H]ryanodine binding of WT and mutant channels. Since the rate of [3H]ryanodine binding at 37°C was faster than at 25°C [39, 40], the incubation period was shortened to 3 h. Therefore, B/Bmax values obtained at 37°C cannot be directly compared with those at 25°C. All the mutants showed greater binding than the WT and some mutants exhibited higher sensitivity to Ca2+ for activation at 37°C (Fig 6 and S1 Table). The effect of temperature on the activity profiles was examined by comparing the three parameters (Amax, KA, and KI) for each mutant at 25 and 37°C (Fig 7A–7C). High linear correlations (R2 > 0.8) were obtained between the mutants for the three parameters and no specific effects on C36R were detected. A good correlation was also observed for the estimated ryanodine binding at pCa 7 with the obtained parameters (Fig 7D). These findings indicate that temperature dependence of the activity profiles are similar between the N-terminal mutations examined.


Divergent Activity Profiles of Type 1 Ryanodine Receptor Channels Carrying Malignant Hyperthermia and Central Core Disease Mutations in the Amino-Terminal Region.

Murayama T, Kurebayashi N, Yamazawa T, Oyamada H, Suzuki J, Kanemaru K, Oguchi K, Iino M, Sakurai T - PLoS ONE (2015)

Comparison of the activity profiles between 25 and 37°C.The three parameters, Amax (A), KA (B) and KI, (C) for WT (filled circles), MH (open circles), MH/CCD (crosses) and C36R (triangles) at 25 and 37°C are plotted. Note that high linear correlations (R2 > 0.8) were found between mutants for all parameters. D. Estimated ryanodine binding at pCa 7.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482644&req=5

pone.0130606.g007: Comparison of the activity profiles between 25 and 37°C.The three parameters, Amax (A), KA (B) and KI, (C) for WT (filled circles), MH (open circles), MH/CCD (crosses) and C36R (triangles) at 25 and 37°C are plotted. Note that high linear correlations (R2 > 0.8) were found between mutants for all parameters. D. Estimated ryanodine binding at pCa 7.
Mentions: We initially determined the Ca2+-dependent [3H]ryanodine binding of WT and mutant channels. Since the rate of [3H]ryanodine binding at 37°C was faster than at 25°C [39, 40], the incubation period was shortened to 3 h. Therefore, B/Bmax values obtained at 37°C cannot be directly compared with those at 25°C. All the mutants showed greater binding than the WT and some mutants exhibited higher sensitivity to Ca2+ for activation at 37°C (Fig 6 and S1 Table). The effect of temperature on the activity profiles was examined by comparing the three parameters (Amax, KA, and KI) for each mutant at 25 and 37°C (Fig 7A–7C). High linear correlations (R2 > 0.8) were obtained between the mutants for the three parameters and no specific effects on C36R were detected. A good correlation was also observed for the estimated ryanodine binding at pCa 7 with the obtained parameters (Fig 7D). These findings indicate that temperature dependence of the activity profiles are similar between the N-terminal mutations examined.

Bottom Line: Most MH and CCD mutations cause accelerated Ca2+ release, resulting in abnormal Ca2+ homeostasis in skeletal muscle.The gain was consistently higher in both MH and MH/CCD mutations.This approach should be useful for diagnosis and treatment of diseases with mutations in RyR1.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Pharmacology, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan.

ABSTRACT
The type 1 ryanodine receptor (RyR1) is a Ca2+ release channel in the sarcoplasmic reticulum of skeletal muscle and is mutated in several diseases, including malignant hyperthermia (MH) and central core disease (CCD). Most MH and CCD mutations cause accelerated Ca2+ release, resulting in abnormal Ca2+ homeostasis in skeletal muscle. However, how specific mutations affect the channel to produce different phenotypes is not well understood. In this study, we have investigated 11 mutations at 7 different positions in the amino (N)-terminal region of RyR1 (9 MH and 2 MH/CCD mutations) using a heterologous expression system in HEK293 cells. In live-cell Ca2+ imaging at room temperature (~25 °C), cells expressing mutant channels exhibited alterations in Ca2+ homeostasis, i.e., an enhanced sensitivity to caffeine, a depletion of Ca2+ in the ER and an increase in resting cytoplasmic Ca2+. RyR1 channel activity was quantitatively evaluated by [3H]ryanodine binding and three parameters (sensitivity to activating Ca2+, sensitivity to inactivating Ca2+ and attainable maximum activity, i.e., gain) were obtained by fitting analysis. The mutations increased the gain and the sensitivity to activating Ca2+ in a site-specific manner. The gain was consistently higher in both MH and MH/CCD mutations. Sensitivity to activating Ca2+ was markedly enhanced in MH/CCD mutations. The channel activity estimated from the three parameters provides a reasonable explanation to the pathological phenotype assessed by Ca2+ homeostasis. These properties were also observed at higher temperatures (~37 °C). Our data suggest that divergent activity profiles may cause varied disease phenotypes by specific mutations. This approach should be useful for diagnosis and treatment of diseases with mutations in RyR1.

No MeSH data available.


Related in: MedlinePlus