Limits...
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+ transients in cells expressing WT and mutant RyR1s.A–D. HEK293 cells expressing WT or mutant RyR1 channels were loaded with fluo-4 AM and stimulated by different concentrations (0.1–10 mM) of caffeine. Measurements were carried out at room temperature (RT). A. Representative traces of fluo-4 signals for WT and three mutants (R164L, Y523S and R615C). Caffeine was applied at the time points indicated by the short horizontal bars. Fluo-4 signals were normalized by Fmax (see Materials and Methods). B. The magnitude of the Ca2+ transients were plotted against caffeine concentrations and fitted to the dose-response curve. C and D. The maximum Ca2+ transients (C) and EC50 for caffeine (D) of WT (filled column), MH mutations (open columns) and MH/CCD mutations (hatched columns). Data are means ± SE (n = 78–150). E. HEK293 cells of WT (filled column), MH mutations (open columns) and MH/CCD mutations (hatched columns) were loaded with fura-2 AM and resting [Ca2+]i was determined. Data are means ± SE (n = 207–494). F. The maximum caffeine-induced Ca2+ transients correlate well with resting [Ca2+]i. (R2 = 0.76, dashed line).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4482644&req=5

pone.0130606.g001: Caffeine-induced Ca2+ transients in cells expressing WT and mutant RyR1s.A–D. HEK293 cells expressing WT or mutant RyR1 channels were loaded with fluo-4 AM and stimulated by different concentrations (0.1–10 mM) of caffeine. Measurements were carried out at room temperature (RT). A. Representative traces of fluo-4 signals for WT and three mutants (R164L, Y523S and R615C). Caffeine was applied at the time points indicated by the short horizontal bars. Fluo-4 signals were normalized by Fmax (see Materials and Methods). B. The magnitude of the Ca2+ transients were plotted against caffeine concentrations and fitted to the dose-response curve. C and D. The maximum Ca2+ transients (C) and EC50 for caffeine (D) of WT (filled column), MH mutations (open columns) and MH/CCD mutations (hatched columns). Data are means ± SE (n = 78–150). E. HEK293 cells of WT (filled column), MH mutations (open columns) and MH/CCD mutations (hatched columns) were loaded with fura-2 AM and resting [Ca2+]i was determined. Data are means ± SE (n = 207–494). F. The maximum caffeine-induced Ca2+ transients correlate well with resting [Ca2+]i. (R2 = 0.76, dashed line).

Mentions: To test the phenotypes of RyR1 with disease-associated mutations, we initially examined caffeine-induced Ca2+ transients in HEK293 cells at RT (25°C) (Fig 1). This assay is based on the observation that caffeine increases the sensitivity of RyR1 to activating Ca2+ [1, 2] and the EC50 for caffeine is a marker for detecting the MH phenotype. Typical fluo-4 Ca2+ signals are shown in Fig 1A. In WT, Ca2+ transients were detected at ~0.3 mM caffeine and reached a plateau at 3 mM or higher concentrations. Disease-associated mutants showed divergent responses. R164L produced Ca2+ transients at lower caffeine concentrations than WT with a reduced peak height. The MH/CCD phenotype Y523S exhibited very small Ca2+ transients. R615C showed enhanced caffeine sensitivity with a peak height comparable to WT. Dose-dependent plots of caffeine-induced Ca2+ transients revealed increased caffeine sensitivity and/or reduced peak amplitude in disease-associated mutations (Fig 1B). We determined the maximum Ca2+ transients (Fig 1C) and the EC50 for caffeine (Fig 1D) from the dose-dependent curves of each mutation. The peak Ca2+ transients were smaller and varied to a degree for some mutants (R164C, R164L, G342R, R402H, Y523C, Y523S and R615L) when compared with WT, but showed no change in other mutants (C36R, G249R, R402C and R615C). All the mutants except for C36R, Y523C and Y523S exhibited a significant reduction in the EC50 value.


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+ transients in cells expressing WT and mutant RyR1s.A–D. HEK293 cells expressing WT or mutant RyR1 channels were loaded with fluo-4 AM and stimulated by different concentrations (0.1–10 mM) of caffeine. Measurements were carried out at room temperature (RT). A. Representative traces of fluo-4 signals for WT and three mutants (R164L, Y523S and R615C). Caffeine was applied at the time points indicated by the short horizontal bars. Fluo-4 signals were normalized by Fmax (see Materials and Methods). B. The magnitude of the Ca2+ transients were plotted against caffeine concentrations and fitted to the dose-response curve. C and D. The maximum Ca2+ transients (C) and EC50 for caffeine (D) of WT (filled column), MH mutations (open columns) and MH/CCD mutations (hatched columns). Data are means ± SE (n = 78–150). E. HEK293 cells of WT (filled column), MH mutations (open columns) and MH/CCD mutations (hatched columns) were loaded with fura-2 AM and resting [Ca2+]i was determined. Data are means ± SE (n = 207–494). F. The maximum caffeine-induced Ca2+ transients correlate well with resting [Ca2+]i. (R2 = 0.76, dashed line).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130606.g001: Caffeine-induced Ca2+ transients in cells expressing WT and mutant RyR1s.A–D. HEK293 cells expressing WT or mutant RyR1 channels were loaded with fluo-4 AM and stimulated by different concentrations (0.1–10 mM) of caffeine. Measurements were carried out at room temperature (RT). A. Representative traces of fluo-4 signals for WT and three mutants (R164L, Y523S and R615C). Caffeine was applied at the time points indicated by the short horizontal bars. Fluo-4 signals were normalized by Fmax (see Materials and Methods). B. The magnitude of the Ca2+ transients were plotted against caffeine concentrations and fitted to the dose-response curve. C and D. The maximum Ca2+ transients (C) and EC50 for caffeine (D) of WT (filled column), MH mutations (open columns) and MH/CCD mutations (hatched columns). Data are means ± SE (n = 78–150). E. HEK293 cells of WT (filled column), MH mutations (open columns) and MH/CCD mutations (hatched columns) were loaded with fura-2 AM and resting [Ca2+]i was determined. Data are means ± SE (n = 207–494). F. The maximum caffeine-induced Ca2+ transients correlate well with resting [Ca2+]i. (R2 = 0.76, dashed line).
Mentions: To test the phenotypes of RyR1 with disease-associated mutations, we initially examined caffeine-induced Ca2+ transients in HEK293 cells at RT (25°C) (Fig 1). This assay is based on the observation that caffeine increases the sensitivity of RyR1 to activating Ca2+ [1, 2] and the EC50 for caffeine is a marker for detecting the MH phenotype. Typical fluo-4 Ca2+ signals are shown in Fig 1A. In WT, Ca2+ transients were detected at ~0.3 mM caffeine and reached a plateau at 3 mM or higher concentrations. Disease-associated mutants showed divergent responses. R164L produced Ca2+ transients at lower caffeine concentrations than WT with a reduced peak height. The MH/CCD phenotype Y523S exhibited very small Ca2+ transients. R615C showed enhanced caffeine sensitivity with a peak height comparable to WT. Dose-dependent plots of caffeine-induced Ca2+ transients revealed increased caffeine sensitivity and/or reduced peak amplitude in disease-associated mutations (Fig 1B). We determined the maximum Ca2+ transients (Fig 1C) and the EC50 for caffeine (Fig 1D) from the dose-dependent curves of each mutation. The peak Ca2+ transients were smaller and varied to a degree for some mutants (R164C, R164L, G342R, R402H, Y523C, Y523S and R615L) when compared with WT, but showed no change in other mutants (C36R, G249R, R402C and R615C). All the mutants except for C36R, Y523C and Y523S exhibited a significant reduction in the EC50 value.

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