Linda Groom

Triadin Binding to the C-Terminal Luminal Loop of the Ryanodine Receptor is Important for Skeletal Muscle Excitation–Contraction Coupling

Ca2+ release from intracellular stores is controlled by complex interactions between multiple proteins. Triadin is a transmembrane glycoprotein of the junctional sarcoplasmic reticulum of striated muscle that interacts with both calsequestrin and the type 1 ryanodine receptor (RyR1) to communicate changes in luminal Ca2+ to the release machinery. However, the potential impact of the triadin association with RyR1 in skeletal muscle excitation–contraction coupling remains elusive. Here we show that triadin binding to RyR1 is critically important for rapid Ca2+ release during excitation–contraction coupling. To assess the functional impact of the triadin-RyR1 interaction, we expressed RyR1 mutants in which one or more of three negatively charged residues (D4878, D4907, and E4908) in the terminal RyR1 intraluminal loop were mutated to alanines in RyR1-null (dyspedic) myotubes. Coimmunoprecipitation revealed that triadin, but not junctin, binding to RyR1 was abolished in the triple (D4878A/D4907A/E4908A) mutant and one of the double (D4907A/E4908A) mutants, partially reduced in the D4878A/D4907A double mutant, but not affected by either individual (D4878A, D4907A, E4908A) mutations or the D4878A/E4908A double mutation. Functional studies revealed that the rate of voltage- and ligand-gated SR Ca2+ release were reduced in proportion to the degree of interruption in triadin binding. Ryanodine binding, single channel recording, and calcium release experiments conducted on WT and triple mutant channels in the absence of triadin demonstrated that the luminal loop mutations do not directly alter RyR1 function. These findings demonstrate that junctin and triadin bind to different sites on RyR1 and that triadin plays an important role in ensuring rapid Ca2+ release during excitation–contraction coupling in skeletal muscle.

Identical de novo Mutation in the Type 1 Ryanodine Receptor Gene Associated with Fatal, Stress-induced Malignant Hyperthermia in Two Unrelated Families

Background: Mutations in the type 1 ryanodine receptor gene (RYR1) result in malignant hyperthermia, a pharmacogenetic disorder typically triggered by administration of anesthetics. However, cases of sudden death during exertion, heat challenge, and febrile illness in the absence of triggering drugs have been reported. The underlying causes of such drug-free fatal “awake” episodes are unknown. Methods: De novo R3983C variant in RYR1 was identified in two unrelated children who experienced fatal, nonanesthetic awake episodes associated with febrile illness and heat stress. One of the children also had a second novel, maternally inherited D4505H variant located on a separate haplotype. Effects of all possible heterotypic expression conditions on RYR1 sensitivity to caffeine-induced Ca2+ release were determined in expressing RYR1-null myotubes. Results: Compared with wild-type RYR1 alone (EC50 = 2.85 ± 0.49 mM), average (±SEM) caffeine sensitivity of Ca2+ release was modestly increased after coexpression with either R3983C (EC50 = 2.00 ± 0.39 mM) or D4505H (EC50 = 1.64 ± 0.24 mM). Remarkably, coexpression of wild-type RYR1 with the double mutant in cis (R3983C-D4505H) produced a significantly stronger sensitization of caffeine-induced Ca2+ release (EC50 = 0.64 ± 0.17 mM) compared with that observed after coexpression of the two variants on separate subunits (EC50 = 1.53 ± 0.18 mM). Conclusions: The R3983C mutation potentiates D4505H-mediated sensitization of caffeine-induced RYR1 Ca2+ release when the mutations are in cis (on the same subunit) but not when present on separate subunits. Nevertheless, coexpression of the two variants on separate subunits still resulted in a ∼2-fold increase in caffeine sensitivity, consistent with the observed awake episodes and heat sensitivity.

Congenital myopathy results from misregulation of a muscle Ca2+ channel by mutant Stac3

Significance Skeletal muscle contractions are regulated by a process called excitation–contraction (EC) coupling, and defects in it are associated with numerous human myopathies. Recently, stac3 (SH3 and cysteine-rich domain 3) was identified as a key regulator of EC coupling and a STAC3 mutation as causal for the debilitating Native American myopathy (NAM). We now show that Stac3 controls EC coupling by regulating Ca2+ channels in muscles. Both the NAM mutation and a mutation that leads to the loss of Stac3 decrease the amount, organization, stability, and voltage sensitivity of Ca2+ channels. Furthermore, we find evidence that the NAM allele of STAC3 is linked to malignant hyperthermia, a common pharmacogenic disorder. These findings define critical roles for Stac3 in muscle contraction and human disease. Skeletal muscle contractions are initiated by an increase in Ca2+ released during excitation–contraction (EC) coupling, and defects in EC coupling are associated with human myopathies. EC coupling requires communication between voltage-sensing dihydropyridine receptors (DHPRs) in transverse tubule membrane and Ca2+ release channel ryanodine receptor 1 (RyR1) in the sarcoplasmic reticulum (SR). Stac3 protein (SH3 and cysteine-rich domain 3) is an essential component of the EC coupling apparatus and a mutation in human STAC3 causes the debilitating Native American myopathy (NAM), but the nature of how Stac3 acts on the DHPR and/or RyR1 is unknown. Using electron microscopy, electrophysiology, and dynamic imaging of zebrafish muscle fibers, we find significantly reduced DHPR levels, functionality, and stability in stac3 mutants. Furthermore, stac3NAM myofibers exhibited increased caffeine-induced Ca2+ release across a wide range of concentrations in the absence of altered caffeine sensitivity as well as increased Ca2+ in internal stores, which is consistent with increased SR luminal Ca2+. These findings define critical roles for Stac3 in EC coupling and human disease.

Characterization of ryanodine receptor type 1 single channel activity using “on-nucleus” patch clamp

In this study, we provide the first description of the biophysical and pharmacological properties of ryanodine receptor type 1 (RyR1) expressed in a native membrane using the on-nucleus configuration of the patch clamp technique. A stable cell line expressing rabbit RyR1 was established (HEK-RyR1) using the FLP-in 293 cell system. In contrast to untransfected cells, RyR1 expression was readily demonstrated by immunoblotting and immunocytochemistry in HEK-RyR1 cells. In addition, the RyR1 agonists 4-CMC and caffeine activated Ca(2+) release that was inhibited by high concentrations of ryanodine. On nucleus patch clamp was performed in nuclei prepared from HEK-RyR1 cells. Raising the [Ca(2+)] in the patch pipette resulted in the appearance of a large conductance cation channel with well resolved kinetics and the absence of prominent subconductance states. Current versus voltage relationships were ohmic and revealed a chord conductance of ∼750pS or 450pS in symmetrical 250mM KCl or CsCl, respectively. The channel activity was markedly enhanced by caffeine and exposure to ryanodine resulted in the appearance of a subconductance state with a conductance ∼40% of the full channel opening with a Po near unity. In total, these properties are entirely consistent with RyR1 channel activity. Exposure of RyR1 channels to cyclic ADP ribose (cADPr), nicotinic acid adenine dinucleotide phosphate (NAADP) or dantrolene did not alter the single channel activity stimulated by Ca(2+), and thus, it is unlikely these molecules directly modulate RyR1 channel activity. In summary, we describe an experimental platform to monitor the single channel properties of RyR channels. We envision that this system will be influential in characterizing disease-associated RyR mutations and the molecular determinants of RyR channel modulation.

Defects in Ca2+ release associated with local expression of pathological ryanodine receptors in mouse muscle fibres

Non‐Technical Summary  Calcium ions flowing through the type 1 ryanodine receptor (RyR1) calcium channel trigger contraction of skeletal muscle cells. Close to 300 mutations of the gene encoding RyR1 are responsible for several muscular diseases in human. Properties of pathological mutant RyR1s have so far been essentially assessed from studies in cultured cells and in differentiated native muscle fibres from a few available transgenic mouse models. We show that functional properties of mutant RyR1s can be reliably assessed following in vivo expression in adult mouse muscles. The Y523S, R615C and R2163H RyR1 mutants produce a similar over‐sensitive activation of the calcium flux whereas I4897T RyR1 mutants are responsible for a depressed Ca2+ flux. The alterations appear to result from inherent modifications of RyR1 channel function and not from indirect changes in the muscle fibre homeostasis. The present strategy will help understand the physio‐pathological defects underlying alterations of muscle function in affected patients.

Ca2+ release in muscle fibers expressing R4892W and G4896V type 1 ryanodine receptor disease mutants.

The large and rapidly increasing number of potentially pathological mutants in the type 1 ryanodine receptor (RyR1) prompts the need to characterize their effects on voltage-activated sarcoplasmic reticulum (SR) Ca2+ release in skeletal muscle. Here we evaluated the function of the R4892W and G4896V RyR1 mutants, both associated with central core disease (CCD) in humans, in myotubes and in adult muscle fibers. For both mutants expressed in RyR1-null (dyspedic) myotubes, voltage-gated Ca2+ release was absent following homotypic expression and only partially restored following heterotypic expression with wild-type (WT) RyR1. In muscle fibers from adult WT mice, both mutants were expressed in restricted regions of the fibers with a pattern consistent with triadic localization. Voltage-clamp-activated confocal Ca2+ signals showed that fiber regions endowed with G4896V-RyR1s exhibited an ∼30% reduction in the peak rate of SR Ca2+ release, with no significant change in SR Ca2+ content. Immunostaining revealed no associated change in the expression of either α1S subunit (Cav1.1) of the dihydropyridine receptor (DHPR) or type 1 sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA1), indicating that the reduced Ca2+ release resulted from defective RyR1 function. Interestingly, in spite of robust localized junctional expression, the R4892W mutant did not affect SR Ca2+ release in adult muscle fibers, consistent with a low functional penetrance of this particular CCD-associated mutant.

Murine Fig4 is dispensable for muscle development but required for muscle function

BackgroundPhosphatidylinositol phosphates (PIPs) are low-abundance phospholipids that participate in a range of cellular processes, including cell migration and membrane traffic. PIP levels and subcellular distribution are regulated by a series of lipid kinases and phosphatases. In skeletal muscle, PIPs and their enzymatic regulators serve critically important functions exemplified by mutations of the PIP phosphatase MTM1 in myotubular myopathy (MTM), a severe muscle disease characterized by impaired muscle structure and abnormal excitation–contraction coupling. FIG4 functions as a PIP phosphatase that participates in both the synthesis and breakdown of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). Mutation of FIG4 results in a severe neurodegenerative disorder in mice and a progressive peripheral polyneuropathy in humans. The effect of FIG4 mutation on skeletal muscle has yet to be examined.MethodsHerein we characterize the impact of FIG4 on skeletal muscle development and function using the spontaneously occurring mouse mutant pale tremor (plt), a mouse line with a loss of function mutation in Fig4.ResultsIn plt mice, we characterized abnormalities in skeletal muscle, including reduced muscle size and specific force generation. We also uncovered ultrastructural abnormalities and increased programmed cell death. Conversely, we detected no structural or functional abnormalities to suggest impairment of excitation–contraction coupling, a process previously shown to be influenced by PI(3,5)P2 levels. Conditional rescue of Fig4 mutation in neurons prevented overt muscle weakness and the development of obvious muscle abnormalities, suggesting that the changes observed in the plt mice were primarily related to denervation of skeletal muscle. On the basis of the ability of reduced FIG4 levels to rescue aspects of Mtmr2-dependent neuropathy, we evaluated the effect of Fig4 haploinsufficiency on the myopathy of Mtm1-knockout mice. Male mice with a compound Fig4+/−/Mtm1–/Y genotype displayed no improvements in muscle histology, muscle size or overall survival, indicating that FIG4 reduction does not ameliorate the Mtm1-knockout phenotype.ConclusionsOverall, these data indicate that loss of Fig4 impairs skeletal muscle function but does not significantly affect its structural development.

The Functional Impact of Cis Acting Ryanodine Receptor Type 1 Mutations in a Child with a Fatal Spontaneous MH Event

Mutations in the RYR1 gene result in MH, a pharmacogenetic disorder of skeletal muscle typically triggered by administration of anesthetics. However, cases of sudden death during exertion, heat challenge, and febrile illness in the absence of triggering drugs have been reported in individuals with RYR1 gene variants. We describe the clinical history and genetic analysis of a child that suffered a fatal, non-anesthetic MH episode associated with febrile illness who was heterozygous for two novel RYR1 variants where one variant, R3983C, occurred de-novo and another, D4505H, was inherited. Effects of the two variants on RYR1 sensitivity to activation by caffeine were assessed following expression in RYR1-null myotubes. The single (R3983C and D4505H) and double (R3983C-D4505H) variants were engineered into a full-length rabbit RYR1 cDNA and introduced into RyR1-null myotubes via nuclear microinjection. Effects of the different heterotypic expression conditions (WT+R3983C, WT+D4505H, R3983C+D4505H, and WT+R3983C-D4505H) on RYR1 activation by caffeine were determined in indo-1-loaded myotubes. Compared to WT RYR1 alone (EC50 = 1.5 mM), the caffeine sensitivity of RYR1 Ca2+ release was only modestly increased following co-expression of either R3983C (EC50 = 1.3 mM) or D4505H (EC50 = 0.9 mM). Remarkably, co-expression of WT RYR1 with the double mutant in cis (R3983C-D4505H) produced a much stronger sensitization of caffeine-induced release (EC50 = 0.3) than that observed following co-expression of the two variants on separate subunits (EC50 = 0.9). Thus, the R3893C mutation potentiates D4505H-mediated sensitization of caffeine-induced RYR1 Ca2+ release when these mutations are in cis or present on the same subunit, but not when present on separate subunits. These results indicate that allelic segregation can be a critical, and heretofore unappreciated, pathogenic factor in compound heterozygous MH individuals.