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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

Determination of resting [Ca2+]ER in cells expressing WT or mutant RyR1s.HEK293 cells expressing WT or mutant RyR1 channels were transfected with G-GECO1.1 and R-CEPIA1er to determine [Ca2+]i and [Ca2+]ER, respectively. Measurements were carried out at RT. A. Typical traces for [Ca2+]i (upper) and [Ca2+]ER (lower) signals. Caffeine was initially applied to deplete Ca2+ in the ER (white bars) and then Ca2+ in the ER was determined at the plateau after the removal of caffeine. Finally, Fmax for indicators was determined by the application of 20 μM ionomycin and 20 mM Ca2+ (black bars). B. [Ca2+]ER of WT (filled column), MH mutations (open columns) and MH/CCD mutations (hatched columns). Data are means ± SE (n = 35–99). *p < 0.05 compared with WT. C and D. Peak caffeine-induced Ca2+ transients (C) and resting [Ca2+]i(D) of WT (filled circle), MH mutations (open circles) and MH/CCD mutations (crosses) were plotted against [Ca2+]ER. Note that the peak caffeine-induced Ca2+ transients (R2 = 0.79) and resting [Ca2+]i (R2 = 0.82) correlated strongly with [Ca2+]ER (dashed lines).
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pone.0130606.g002: Determination of resting [Ca2+]ER in cells expressing WT or mutant RyR1s.HEK293 cells expressing WT or mutant RyR1 channels were transfected with G-GECO1.1 and R-CEPIA1er to determine [Ca2+]i and [Ca2+]ER, respectively. Measurements were carried out at RT. A. Typical traces for [Ca2+]i (upper) and [Ca2+]ER (lower) signals. Caffeine was initially applied to deplete Ca2+ in the ER (white bars) and then Ca2+ in the ER was determined at the plateau after the removal of caffeine. Finally, Fmax for indicators was determined by the application of 20 μM ionomycin and 20 mM Ca2+ (black bars). B. [Ca2+]ER of WT (filled column), MH mutations (open columns) and MH/CCD mutations (hatched columns). Data are means ± SE (n = 35–99). *p < 0.05 compared with WT. C and D. Peak caffeine-induced Ca2+ transients (C) and resting [Ca2+]i(D) of WT (filled circle), MH mutations (open circles) and MH/CCD mutations (crosses) were plotted against [Ca2+]ER. Note that the peak caffeine-induced Ca2+ transients (R2 = 0.79) and resting [Ca2+]i (R2 = 0.82) correlated strongly with [Ca2+]ER (dashed lines).

Mentions: The reduced peak value of caffeine-induced Ca2+ transients and corresponding increased resting [Ca2+]i suggest a reduction of Ca2+ in the ER Ca2+ store ([Ca2+]ER). To test this possibility, we simultaneously measured [Ca2+]i and [Ca2+]ER in HEK293 cells (Fig 2). Fig 2A shows typical traces of [Ca2+]i and [Ca2+]ER signals. We initially treated the cells with 3 mM caffeine to deplete Ca2+ in the ER (open bars). After removal of the caffeine, [Ca2+]ER gradually recovered and achieved a steady-state level. Resting [Ca2+]ER was measured as F/Fmax, in which fluorescence intensity of the steady-state level (F) was normalized by the maximum fluorescence intensity (Fmax) of the indicator, which was determined by treating cells with high Ca2+ plus ionomycin at the end of experiments (filled bars). R164L and Y523S showed markedly lower [Ca2+]ER than WT, whereas R615C exhibited a [Ca2+]ER level similar to that of WT (Fig 2A). The F/Fmax values were converted to Ca2+ concentrations using parameters obtained in the in situ titration [24] and are summarized in Fig 2B. Two MH/CCD mutations, R164C and Y523S, exhibited severe depletion of [Ca2+]ER, which is consistent with previous reports [14, 15, 30, 31]. The other mutations, except for C36R, also showed significant reduction in [Ca2+]ER. The [Ca2+]ER strongly correlated with the peak value of caffeine-induced Ca2+ transients (R2 = 0.79) (Fig 2C) and resting [Ca2+]i (R2 = 0.82) (Fig 2D), indicating that small peak values of caffeine-induced Ca2+ transients are due to low [Ca2+]ER levels.


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)

Determination of resting [Ca2+]ER in cells expressing WT or mutant RyR1s.HEK293 cells expressing WT or mutant RyR1 channels were transfected with G-GECO1.1 and R-CEPIA1er to determine [Ca2+]i and [Ca2+]ER, respectively. Measurements were carried out at RT. A. Typical traces for [Ca2+]i (upper) and [Ca2+]ER (lower) signals. Caffeine was initially applied to deplete Ca2+ in the ER (white bars) and then Ca2+ in the ER was determined at the plateau after the removal of caffeine. Finally, Fmax for indicators was determined by the application of 20 μM ionomycin and 20 mM Ca2+ (black bars). B. [Ca2+]ER of WT (filled column), MH mutations (open columns) and MH/CCD mutations (hatched columns). Data are means ± SE (n = 35–99). *p < 0.05 compared with WT. C and D. Peak caffeine-induced Ca2+ transients (C) and resting [Ca2+]i(D) of WT (filled circle), MH mutations (open circles) and MH/CCD mutations (crosses) were plotted against [Ca2+]ER. Note that the peak caffeine-induced Ca2+ transients (R2 = 0.79) and resting [Ca2+]i (R2 = 0.82) correlated strongly with [Ca2+]ER (dashed lines).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130606.g002: Determination of resting [Ca2+]ER in cells expressing WT or mutant RyR1s.HEK293 cells expressing WT or mutant RyR1 channels were transfected with G-GECO1.1 and R-CEPIA1er to determine [Ca2+]i and [Ca2+]ER, respectively. Measurements were carried out at RT. A. Typical traces for [Ca2+]i (upper) and [Ca2+]ER (lower) signals. Caffeine was initially applied to deplete Ca2+ in the ER (white bars) and then Ca2+ in the ER was determined at the plateau after the removal of caffeine. Finally, Fmax for indicators was determined by the application of 20 μM ionomycin and 20 mM Ca2+ (black bars). B. [Ca2+]ER of WT (filled column), MH mutations (open columns) and MH/CCD mutations (hatched columns). Data are means ± SE (n = 35–99). *p < 0.05 compared with WT. C and D. Peak caffeine-induced Ca2+ transients (C) and resting [Ca2+]i(D) of WT (filled circle), MH mutations (open circles) and MH/CCD mutations (crosses) were plotted against [Ca2+]ER. Note that the peak caffeine-induced Ca2+ transients (R2 = 0.79) and resting [Ca2+]i (R2 = 0.82) correlated strongly with [Ca2+]ER (dashed lines).
Mentions: The reduced peak value of caffeine-induced Ca2+ transients and corresponding increased resting [Ca2+]i suggest a reduction of Ca2+ in the ER Ca2+ store ([Ca2+]ER). To test this possibility, we simultaneously measured [Ca2+]i and [Ca2+]ER in HEK293 cells (Fig 2). Fig 2A shows typical traces of [Ca2+]i and [Ca2+]ER signals. We initially treated the cells with 3 mM caffeine to deplete Ca2+ in the ER (open bars). After removal of the caffeine, [Ca2+]ER gradually recovered and achieved a steady-state level. Resting [Ca2+]ER was measured as F/Fmax, in which fluorescence intensity of the steady-state level (F) was normalized by the maximum fluorescence intensity (Fmax) of the indicator, which was determined by treating cells with high Ca2+ plus ionomycin at the end of experiments (filled bars). R164L and Y523S showed markedly lower [Ca2+]ER than WT, whereas R615C exhibited a [Ca2+]ER level similar to that of WT (Fig 2A). The F/Fmax values were converted to Ca2+ concentrations using parameters obtained in the in situ titration [24] and are summarized in Fig 2B. Two MH/CCD mutations, R164C and Y523S, exhibited severe depletion of [Ca2+]ER, which is consistent with previous reports [14, 15, 30, 31]. The other mutations, except for C36R, also showed significant reduction in [Ca2+]ER. The [Ca2+]ER strongly correlated with the peak value of caffeine-induced Ca2+ transients (R2 = 0.79) (Fig 2C) and resting [Ca2+]i (R2 = 0.82) (Fig 2D), indicating that small peak values of caffeine-induced Ca2+ transients are due to low [Ca2+]ER levels.

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