Malignant hyperthermia-associated mutations in the S2-S3 cytoplasmic loop of type 1 ryanodine receptor calcium channel impair calcium-dependent inactivation

Abstract

Channel activities of skeletal muscle ryanodine receptor (RyR1) are activated by micromolar Ca²⁺ and inactivated by higher (∼1 mM) Ca²⁺ To gain insight into a mechanism underlying Ca²⁺-dependent inactivation of RyR1 and its relationship with skeletal muscle diseases, we constructed nine recombinant RyR1 mutants carrying malignant hyperthermia or centronuclear myopathy-associated mutations and determined RyR1 channel activities by [³H]ryanodine binding assay. These mutations are localized in or near the RyR1 domains which are responsible for Ca²⁺-dependent inactivation of RyR1. Four RyR1 mutations (F4732D, G4733E, R4736W, and R4736Q) in the cytoplasmic loop between the S2 and S3 transmembrane segments (S2-S3 loop) greatly reduced Ca²⁺-dependent channel inactivation. Activities of these mutant channels were suppressed at 10-100 μM Ca²⁺, and the suppressions were relieved by 1 mM Mg²⁺ The Ca²⁺- and Mg²⁺-dependent regulation of S2-S3 loop RyR1 mutants are similar to those of the cardiac isoform of RyR (RyR2) rather than wild-type RyR1. Two mutations (T4825I and H4832Y) in the S4-S5 cytoplasmic loop increased Ca²⁺ affinities for channel activation and decreased Ca²⁺ affinities for inactivation, but impairment of Ca²⁺-dependent inactivation was not as prominent as those of S2-S3 loop mutants. Three mutations (T4082M, S4113L, and N4120Y) in the EF-hand domain showed essentially the same Ca²⁺-dependent channel regulation as that of wild-type RyR1. The results suggest that nine RyR1 mutants associated with skeletal muscle diseases were differently regulated by Ca²⁺ and Mg²⁺ Four malignant hyperthermia-associated RyR1 mutations in the S2-S3 loop conferred RyR2-type Ca²⁺- and Mg²⁺-dependent channel regulation.

Keywords: Ca2+-dependent inactivation; central core disease; centronuclear myopathy; malignant hyperthermia; type-1 ryanodine receptor.

Fig. 1.

Skeletal disease-associated mutations in RyR1. Amino acid sequences of three domains are shown. Underlines indicate EF-hand motifs. Bold letters denote MH, CCD, or CNM-linked mutation sites (https://cardiodb.org/Paralogue_Annotation/). Mutation sites characterized in this study are highlighted in gray, and mutations are shown in italic below the sequence.

Fig. 2.

Ca²⁺-dependent activities of MH/CNM-associated RyR1 mutants. Ca²⁺-dependent changes in the activities of wild-type (WT) and mutant RyR1 were determined by the [³H]ryanodine binding method. Activities of mutant RyR1s carrying disease-associated mutations in EF-hand domain (A), S2–S3 loop (B), and S4–S5 loop (C) are shown together with that of WT-RyR1 (dotted line, mean values). A dashed line in B represents a mean value of WT-RyR2. D: IC₅₀ values of wild-type and mutant RyRs. E: activities at 40 nM free Ca²⁺ were normalized to the peak activities for wild-type and each mutant RyR1. *P < 0.05 compared with WT-RyR1 by one-way ANOVA followed by Tukey's test among 10 sample groups (wild-type and mutant RyR1s). Data are shown as means ± SE (n = 4–7).

Fig. 3.

Ca²⁺-dependent activities of MH-associated RyR1 mutants in the presence of 1 mM Mg²⁺. A–H: Ca²⁺-dependent activities with and without 1 mM Mg²⁺ were determined for WT-RyR1, WT-RyR2, and six MH-associated RyR1 mutants. Data of Ca²⁺-dependent activities without 1 mM Mg²⁺ are from Fig. 2. Data are shown as means ± SE (n = 4–8). I: IC₅₀ values of wild-type and mutant RyRs in the presence of 1 mM Mg²⁺ are shown. *P < 0.05 compared with WT-RyR1 by one-way ANOVA followed by Tukey's test among seven sample groups (wild-type and mutant RyR1s). Data are shown as means ± SE (n = 4–8).

Fig. 4.

Effect of Mg²⁺ on MH/CNM-associated RyR1 mutants. Mg²⁺-dependent changes in the activities of wild-type and mutant RyR were determined at 100 μM Ca²⁺ by the [³H]ryanodine binding method. Activities of wild-type RyRs (A), S2–S3 loop mutants (B), S4–S5 loop mutants (C), and EF-hand mutants (D) are shown. Dotted and dashed lines in B, C, and D are mean values of WT-RyR1 and WT-RyR2, respectively. The smaller dotted line indicates the level at 100%. *P < 0.05 compared with control (-Mg²⁺) by one-way ANOVA. In B, *P < 0.05 for F4732D, G4733E, R4736W, and R4736Q mutants; **P < 0.05 for G4733E mutant. Data are shown as means ± SE (n = 4–6). E: IC₅₀ values of WT-RyR1, EF-hand mutants, and S4–S5 loop mutants are shown. *P < 0.05 compared with WT-RyR1 by one-way ANOVA followed by Tukey’s test. Data are shown as means ± SE (n = 4–5).

Fig. 5.

Effect of caffeine on activities of G4733E and R4736W RyR1 mutants. Caffeine-dependent changes in the activities of wild-type, G4733E, and R4736W RyR1s were determined at 0.4 μM Ca²⁺ by [³H]ryanodine binding assays. Changes in the amount of bound [³H]ryanodine were normalized and fit by Eq. 1 as described in materials and methods. Data are shown as means ± SE (n = 4–15). There are no significant differences among three types of RyR1 at any tested caffeine concentrations by one-way ANOVA. Maximal amounts of bound [³H]ryanodine are 121 ± 9, 109 ± 9, and 111 ± 9% (10 mM caffeine as 100%); activation Hill constants of caffeine are 2.2 ± 0.5, 1.4 ± 0.4, and 1.4 ± 0.3 mM, and Hill coefficients are 1.1 ± 0.2, 1.0 ± 0.2, and 1.0 ± 0.2 for WT-RyR1, G4733E, and R4736W, respectively (fitted value ± regression SE).

Fig. 6.

A possible model of Ca²⁺-dependent inactivation of RyR1. Disease-associated RyR1 mutations in the EF-hand region, S2–S3 loop, and S4–S5 loop are shown. The regions involved in Ca²⁺-dependent channel inactivation are highlighted in gray.