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.
Highlights
The type 1 ryanodine receptor (RyR1) is a Ca2+ release channel in the sarcoplasmic reticulum (SR) of skeletal muscle and plays an important role in excitation–contraction (E-C) coupling [1, 2]
Our results provide a reasonable explanation for the mechanisms of malignant hyperthermia (MH) and MH/central core disease (CCD) phenotypes caused by specific mutations in the N-terminal region of the RyR1 channel
Each disease-associated mutation in the N-terminal region corresponding to C36R, R164C, R164L, G249R, G342R, R402C, R402H, Y523C, Y523S, R615C and R615L was introduced by inverse polymerase chain reaction (PCR) using a HindIII-SalI fragment or a SalI-Bsu36I fragment as the PCR template
Summary
The type 1 ryanodine receptor (RyR1) is a Ca2+ release channel in the sarcoplasmic reticulum (SR) of skeletal muscle and plays an important role in excitation–contraction (E-C) coupling [1, 2]. More than 150 different point mutations for MH have been identified in the RyR1 gene and the majority of mutations cluster in three 'hotspots': Nterminal (35–614) and central (2129–2458) regions located in the cytoplasm, and carboxyl (C)terminal region (4637–4973) near or within channel forming segments [11]. Some CCD mutations in the C-terminal region, in contrast, cause loss-of-function phenotype, in which Ca2+ release triggered by depolarization is strongly suppressed (E-C uncoupling) [16,17,18]. These CCD mutations appear to be insusceptible to MH
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