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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

Ca2+-dependent [3H]ryanodine binding of WT and mutant RyR1s.A–D. Ca2+-dependent [3H]ryanodine binding was determined at 25°C in 0.17 M NaCl, 20 mM MOPSO, pH 6.8, 2 mM dithiothreitol, 1 mM AMP and various concentrations of Ca2+ buffered with 10 mM EGTA. Curves without data points in B–D indicate WT. Data are means ± SE (n = 3–5). Note that the mutants show greater [3H]ryanodine binding than WT. E–H. Activity profiles of the mutant channels. The three parameters, Amax, KA and KI, were obtained by fitting analysis (see Materials and Methods) and plotted on the radar charts relative to WT. 1/KA was used as the parameter for activating Ca2+ dissociation constants, in which a larger value represents higher sensitivity. Note that the size of the triangle represents the magnitude of the channel activity.
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pone.0130606.g004: Ca2+-dependent [3H]ryanodine binding of WT and mutant RyR1s.A–D. Ca2+-dependent [3H]ryanodine binding was determined at 25°C in 0.17 M NaCl, 20 mM MOPSO, pH 6.8, 2 mM dithiothreitol, 1 mM AMP and various concentrations of Ca2+ buffered with 10 mM EGTA. Curves without data points in B–D indicate WT. Data are means ± SE (n = 3–5). Note that the mutants show greater [3H]ryanodine binding than WT. E–H. Activity profiles of the mutant channels. The three parameters, Amax, KA and KI, were obtained by fitting analysis (see Materials and Methods) and plotted on the radar charts relative to WT. 1/KA was used as the parameter for activating Ca2+ dissociation constants, in which a larger value represents higher sensitivity. Note that the size of the triangle represents the magnitude of the channel activity.

Mentions: To quantitatively evaluate the activity of mutant RyR1 channels, we determined Ca2+-dependent [3H]ryanodine binding using microsomes isolated from HEK293 cells. Since ryanodine specifically binds to the open channel, [3H]ryanodine binding is a useful measure for functional state of the RyR channel [33–35]. The [3H]ryanodine binding value at the specified [Ca2+] was expressed as B/Bmax, which represents the ratio of active to total channels under the conditions employed [8]. WT exhibited biphasic Ca2+-dependent [3H]ryanodine binding with the peak near 30 μM Ca2+ (Fig 4A). The peak B/Bmax value was ≈0.03, which is consistent with RyR1 from skeletal muscle [36, 37]. All the disease-associated mutants showed greater binding than WT (Fig 4A–4D). To our surprise, there was a large variation in Ca2+-dependent curves between mutants.


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)

Ca2+-dependent [3H]ryanodine binding of WT and mutant RyR1s.A–D. Ca2+-dependent [3H]ryanodine binding was determined at 25°C in 0.17 M NaCl, 20 mM MOPSO, pH 6.8, 2 mM dithiothreitol, 1 mM AMP and various concentrations of Ca2+ buffered with 10 mM EGTA. Curves without data points in B–D indicate WT. Data are means ± SE (n = 3–5). Note that the mutants show greater [3H]ryanodine binding than WT. E–H. Activity profiles of the mutant channels. The three parameters, Amax, KA and KI, were obtained by fitting analysis (see Materials and Methods) and plotted on the radar charts relative to WT. 1/KA was used as the parameter for activating Ca2+ dissociation constants, in which a larger value represents higher sensitivity. Note that the size of the triangle represents the magnitude of the channel activity.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130606.g004: Ca2+-dependent [3H]ryanodine binding of WT and mutant RyR1s.A–D. Ca2+-dependent [3H]ryanodine binding was determined at 25°C in 0.17 M NaCl, 20 mM MOPSO, pH 6.8, 2 mM dithiothreitol, 1 mM AMP and various concentrations of Ca2+ buffered with 10 mM EGTA. Curves without data points in B–D indicate WT. Data are means ± SE (n = 3–5). Note that the mutants show greater [3H]ryanodine binding than WT. E–H. Activity profiles of the mutant channels. The three parameters, Amax, KA and KI, were obtained by fitting analysis (see Materials and Methods) and plotted on the radar charts relative to WT. 1/KA was used as the parameter for activating Ca2+ dissociation constants, in which a larger value represents higher sensitivity. Note that the size of the triangle represents the magnitude of the channel activity.
Mentions: To quantitatively evaluate the activity of mutant RyR1 channels, we determined Ca2+-dependent [3H]ryanodine binding using microsomes isolated from HEK293 cells. Since ryanodine specifically binds to the open channel, [3H]ryanodine binding is a useful measure for functional state of the RyR channel [33–35]. The [3H]ryanodine binding value at the specified [Ca2+] was expressed as B/Bmax, which represents the ratio of active to total channels under the conditions employed [8]. WT exhibited biphasic Ca2+-dependent [3H]ryanodine binding with the peak near 30 μM Ca2+ (Fig 4A). The peak B/Bmax value was ≈0.03, which is consistent with RyR1 from skeletal muscle [36, 37]. All the disease-associated mutants showed greater binding than WT (Fig 4A–4D). To our surprise, there was a large variation in Ca2+-dependent curves between mutants.

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