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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

Caffeine-induced Ca2+ oscillations by monitoring ER Ca2+.ER Ca2+ of HEK293 cells expressing WT or mutant RyR1 channels (C36R, G249R and R615C) was monitored with R-CEPIA1er as described in Fig 2. A. Typical traces for ER Ca2+ signals. Cells were incubated for 2 min with normal Krebs solution containing 2 mM Ca2+ and 2 mM caffeine (grey bar). Ca2+ in Krebs solution was then increased to 5 mM (black bars). After depletion of Ca2+ by 30 mM caffeine, Fmax for indicators was determined by the application of 20 μM ionomycin and 20 mM Ca2+. B. Ca2+ oscillation frequencies determined with normal (2 mM Ca2+) and 5 mM Ca2+ Krebs in the presence of 2 mM caffeine. C. Caffeine-induced Ca2+ oscillations by monitoring ER Ca2+. ER [Ca2+]ER levels in normal and 5 mM Ca2+ Krebs solution with 2 mM caffeine. For WT and R615C, upper levels during Ca2+ oscillations (threshold levels for Ca2+ release) were measured.
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pone.0130606.g003: Caffeine-induced Ca2+ oscillations by monitoring ER Ca2+.ER Ca2+ of HEK293 cells expressing WT or mutant RyR1 channels (C36R, G249R and R615C) was monitored with R-CEPIA1er as described in Fig 2. A. Typical traces for ER Ca2+ signals. Cells were incubated for 2 min with normal Krebs solution containing 2 mM Ca2+ and 2 mM caffeine (grey bar). Ca2+ in Krebs solution was then increased to 5 mM (black bars). After depletion of Ca2+ by 30 mM caffeine, Fmax for indicators was determined by the application of 20 μM ionomycin and 20 mM Ca2+. B. Ca2+ oscillation frequencies determined with normal (2 mM Ca2+) and 5 mM Ca2+ Krebs in the presence of 2 mM caffeine. C. Caffeine-induced Ca2+ oscillations by monitoring ER Ca2+. ER [Ca2+]ER levels in normal and 5 mM Ca2+ Krebs solution with 2 mM caffeine. For WT and R615C, upper levels during Ca2+ oscillations (threshold levels for Ca2+ release) were measured.

Mentions: It has been reported that caffeine or halothane induces Ca2+ oscillations in HEK cells expressing WT RyR1 and that a MH-associated mutation (R615C) reduced a threshold level of luminal Ca2+ for the spontaneous Ca2+ release, which is referred to as store-overload-induced Ca2+ release (SOICR) level [32]. We therefore tested whether this is also the case with the other disease-associated mutations by determining [Ca2+]ER (Fig 3). Unlike RyR2 expressing cells, no Ca2+ oscillations were detected in WT nor any mutant RyR1 cells during 8-min observations in the absence of caffeine. In the presence of low concentration of caffeine, Ca2+ oscillations were detected in WT cells (9 out of 42 cells) (Fig 3A). R615C also exhibited Ca2+ oscillations (16 out of 36 cells) with higher frequency than that of WT (Fig 3B). These results are in agreement with the previous report [32].


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)

Caffeine-induced Ca2+ oscillations by monitoring ER Ca2+.ER Ca2+ of HEK293 cells expressing WT or mutant RyR1 channels (C36R, G249R and R615C) was monitored with R-CEPIA1er as described in Fig 2. A. Typical traces for ER Ca2+ signals. Cells were incubated for 2 min with normal Krebs solution containing 2 mM Ca2+ and 2 mM caffeine (grey bar). Ca2+ in Krebs solution was then increased to 5 mM (black bars). After depletion of Ca2+ by 30 mM caffeine, Fmax for indicators was determined by the application of 20 μM ionomycin and 20 mM Ca2+. B. Ca2+ oscillation frequencies determined with normal (2 mM Ca2+) and 5 mM Ca2+ Krebs in the presence of 2 mM caffeine. C. Caffeine-induced Ca2+ oscillations by monitoring ER Ca2+. ER [Ca2+]ER levels in normal and 5 mM Ca2+ Krebs solution with 2 mM caffeine. For WT and R615C, upper levels during Ca2+ oscillations (threshold levels for Ca2+ release) were measured.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130606.g003: Caffeine-induced Ca2+ oscillations by monitoring ER Ca2+.ER Ca2+ of HEK293 cells expressing WT or mutant RyR1 channels (C36R, G249R and R615C) was monitored with R-CEPIA1er as described in Fig 2. A. Typical traces for ER Ca2+ signals. Cells were incubated for 2 min with normal Krebs solution containing 2 mM Ca2+ and 2 mM caffeine (grey bar). Ca2+ in Krebs solution was then increased to 5 mM (black bars). After depletion of Ca2+ by 30 mM caffeine, Fmax for indicators was determined by the application of 20 μM ionomycin and 20 mM Ca2+. B. Ca2+ oscillation frequencies determined with normal (2 mM Ca2+) and 5 mM Ca2+ Krebs in the presence of 2 mM caffeine. C. Caffeine-induced Ca2+ oscillations by monitoring ER Ca2+. ER [Ca2+]ER levels in normal and 5 mM Ca2+ Krebs solution with 2 mM caffeine. For WT and R615C, upper levels during Ca2+ oscillations (threshold levels for Ca2+ release) were measured.
Mentions: It has been reported that caffeine or halothane induces Ca2+ oscillations in HEK cells expressing WT RyR1 and that a MH-associated mutation (R615C) reduced a threshold level of luminal Ca2+ for the spontaneous Ca2+ release, which is referred to as store-overload-induced Ca2+ release (SOICR) level [32]. We therefore tested whether this is also the case with the other disease-associated mutations by determining [Ca2+]ER (Fig 3). Unlike RyR2 expressing cells, no Ca2+ oscillations were detected in WT nor any mutant RyR1 cells during 8-min observations in the absence of caffeine. In the presence of low concentration of caffeine, Ca2+ oscillations were detected in WT cells (9 out of 42 cells) (Fig 3A). R615C also exhibited Ca2+ oscillations (16 out of 36 cells) with higher frequency than that of WT (Fig 3B). These results are in agreement with the previous report [32].

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