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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

Estimation of the activity of mutant channels at resting [Ca2+]i.A. Ryanodine binding of mutant channels under resting conditions (pCa 7) was estimated by substituting the three parameters (KA and KI and Amax) into eqs (1)–(3) and plotted relative to WT in ascending order. B. [Ca2+]ER for WT (filled circle), MH mutations (open circles) and MH/CCD mutations (crosses) were plotted against their estimated ryanodine binding at pCa 7.
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pone.0130606.g005: Estimation of the activity of mutant channels at resting [Ca2+]i.A. Ryanodine binding of mutant channels under resting conditions (pCa 7) was estimated by substituting the three parameters (KA and KI and Amax) into eqs (1)–(3) and plotted relative to WT in ascending order. B. [Ca2+]ER for WT (filled circle), MH mutations (open circles) and MH/CCD mutations (crosses) were plotted against their estimated ryanodine binding at pCa 7.

Mentions: To consider the pathological phenotype by specific mutations, it is critically important to obtain quantitative information about the activity of the mutant channel at resting [Ca2+]i. However, [3H]ryanodine binding over the resting [Ca2+]i range was too low to be accurately determined (see Fig 4). Instead, we estimated the ryanodine binding of the mutant channel at pCa 7 using the three parameters (KA, KI and Amax) listed in S1 Table (see Materials and Methods). Binding was significantly higher than WT with large variability (4–100-fold increase) (Fig 5A). The rank order was as follows: WT < R615C < C36R < R402H ≈ R402C < G249R < R615L ≈ R164C < G342R < R164L < Y523C < Y523S. To test the validity of these values, we compared the estimated [3H]ryanodine binding with the [Ca2+]ER of HEK293 cells, which reflects Ca2+ leakage via Ca2+ release. A good inverse correlation between the two was observed (Fig 5B).


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)

Estimation of the activity of mutant channels at resting [Ca2+]i.A. Ryanodine binding of mutant channels under resting conditions (pCa 7) was estimated by substituting the three parameters (KA and KI and Amax) into eqs (1)–(3) and plotted relative to WT in ascending order. B. [Ca2+]ER for WT (filled circle), MH mutations (open circles) and MH/CCD mutations (crosses) were plotted against their estimated ryanodine binding at pCa 7.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130606.g005: Estimation of the activity of mutant channels at resting [Ca2+]i.A. Ryanodine binding of mutant channels under resting conditions (pCa 7) was estimated by substituting the three parameters (KA and KI and Amax) into eqs (1)–(3) and plotted relative to WT in ascending order. B. [Ca2+]ER for WT (filled circle), MH mutations (open circles) and MH/CCD mutations (crosses) were plotted against their estimated ryanodine binding at pCa 7.
Mentions: To consider the pathological phenotype by specific mutations, it is critically important to obtain quantitative information about the activity of the mutant channel at resting [Ca2+]i. However, [3H]ryanodine binding over the resting [Ca2+]i range was too low to be accurately determined (see Fig 4). Instead, we estimated the ryanodine binding of the mutant channel at pCa 7 using the three parameters (KA, KI and Amax) listed in S1 Table (see Materials and Methods). Binding was significantly higher than WT with large variability (4–100-fold increase) (Fig 5A). The rank order was as follows: WT < R615C < C36R < R402H ≈ R402C < G249R < R615L ≈ R164C < G342R < R164L < Y523C < Y523S. To test the validity of these values, we compared the estimated [3H]ryanodine binding with the [Ca2+]ER of HEK293 cells, which reflects Ca2+ leakage via Ca2+ release. A good inverse correlation between the two was observed (Fig 5B).

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