Central core disease mutations R4892W, I4897T and G4898E in the ryanodine receptor isoform 1 reduce the Ca2+ sensitivity and amplitude of Ca2+-dependent Ca2+ release

Three CCD (central core disease) mutants, R4892W (Arg4892→Trp), I4897T and G4898E, in the pore region of the skeletal-muscle Ca2+-release channel RyR1 (ryanodine receptor 1) were characterized using a newly developed assay that monitored Ca2+ release in the presence of Ca2+ uptake in microsomes isolated from HEK-293 cells (human embryonic kidney 293 cells), co-expressing each of the three mutants together with SERCA1a (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 1a). Both Ca2+ sensitivity and peak amplitude of Ca2+ release were either absent from or sharply decreased in homotetrameric mutants. Co-expression of wild-type RyR1 with mutant RyR1 (heterotetrameric mutants) restored Ca2+ sensitivity partially, in the ratio 1:2, or fully, in the ratio 1:1. Peak amplitude was restored only partially in the ratio 1:2 or 1:1. Reduced amplitude was not correlated with maximum Ca2+ loading or the amount of expressed RyR1 protein. High-affinity [3H]ryanodine binding and caffeine-induced Ca2+ release were also absent from the three homotetrameric mutants. These results indicate that decreased Ca2+ sensitivity is one of the serious defects in these three excitation–contraction uncoupling CCD mutations. In CCD skeletal muscles, where a mixture of wild-type and mutant RyR1 is expressed, these defects are expected to decrease Ca2+-induced Ca2+ release, as well as orthograde Ca2+ release, in response to transverse tubular membrane depolarization.

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Functional Characterization of C-terminal Ryanodine Receptor 1 Variants Associated with Central Core Disease or Malignant Hyperthermia

Journal of Neuromuscular Diseases ◽

10.3233/jnd-170210 ◽

2017 ◽

Vol 4 (2) ◽

pp. 147-158 ◽

Cited By ~ 3

Author(s):

Remai Parker ◽

Anja H. Schiemann ◽

Elaine Langton ◽

Terasa Bulger ◽

Neil Pollock ◽

...

Keyword(s):

Malignant Hyperthermia ◽

Ryanodine Receptor ◽

Functional Characterization ◽

Central Core ◽

Central Core Disease ◽

Ryanodine Receptor 1

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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

PLoS ONE ◽

10.1371/journal.pone.0130606 ◽

2015 ◽

Vol 10 (6) ◽

pp. e0130606 ◽

Cited By ~ 23

Author(s):

Takashi Murayama ◽

Nagomi Kurebayashi ◽

Toshiko Yamazawa ◽

Hideto Oyamada ◽

Junji Suzuki ◽

...

Keyword(s):

Malignant Hyperthermia ◽

Ryanodine Receptor ◽

Terminal Region ◽

Central Core ◽

Central Core Disease ◽

Amino Terminal ◽

Disease Mutations

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Identification of four novel mutations in the C-terminal membrane spanning domain of the ryanodine receptor 1: association with central core disease and alteration of calcium homeostasis

Human Molecular Genetics ◽

10.1093/hmg/10.25.2879 ◽

2001 ◽

Vol 10 (25) ◽

pp. 2879-2887 ◽

Cited By ~ 98

Author(s):

N. Tilgen

Keyword(s):

Ryanodine Receptor ◽

Calcium Homeostasis ◽

Central Core ◽

Central Core Disease ◽

Novel Mutations ◽

Ryanodine Receptor 1 ◽

Membrane Spanning

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Functional Effects of Central Core Disease Mutations in the Cytoplasmic Region of the Skeletal Muscle Ryanodine Receptor

The Journal of General Physiology ◽

10.1085/jgp.118.3.277 ◽

2001 ◽

Vol 118 (3) ◽

pp. 277-290 ◽

Cited By ~ 102

Author(s):

Guillermo Avila ◽

Robert T. Dirksen

Keyword(s):

Skeletal Muscle ◽

Ryanodine Receptor ◽

Central Core ◽

Central Core Disease ◽

Calcium Release Channel ◽

Release Channel ◽

Voltage Gated ◽

Channel Function ◽

Store Depletion ◽

The Impact

Central core disease (CCD) is a human myopathy that involves a dysregulation in muscle Ca2+ homeostasis caused by mutations in the gene encoding the skeletal muscle ryanodine receptor (RyR1), the protein that comprises the calcium release channel of the SR. Although genetic studies have clearly demonstrated linkage between mutations in RyR1 and CCD, the impact of these mutations on release channel function and excitation-contraction coupling in skeletal muscle is unknown. Toward this goal, we have engineered the different CCD mutations found in the NH2-terminal region of RyR1 into a rabbit RyR1 cDNA (R164C, I404M, Y523S, R2163H, and R2435H) and characterized the functional effects of these mutations after expression in myotubes derived from RyR1-knockout (dyspedic) mice. Resting Ca2+ levels were elevated in dyspedic myotubes expressing four of these mutants (Y523S > R2163H > R2435H R164C > I404M RyR1). A similar rank order was also found for the degree of SR Ca2+ depletion assessed using maximal concentrations of caffeine (10 mM) or cyclopiazonic acid (CPA, 30 μM). Although all of the CCD mutants fully restored L-current density, voltage-gated SR Ca2+ release was smaller and activated at more negative potentials for myotubes expressing the NH2-terminal CCD mutations. The shift in the voltage dependence of SR Ca2+ release correlated strongly with changes in resting Ca2+, SR Ca2+ store depletion, and peak voltage–gated release, indicating that increased release channel activity at negative membrane potentials promotes SR Ca2+ leak. Coexpression of wild-type and Y523S RyR1 proteins in dyspedic myotubes resulted in release channels that exhibited an intermediate degree of SR Ca2+ leak. These results demonstrate that the NH2-terminal CCD mutants enhance release channel sensitivity to activation by voltage in a manner that leads to increased SR Ca2+ leak, store depletion, and a reduction in voltage-gated Ca2+ release. Two fundamentally distinct cellular mechanisms (leaky channels and EC uncoupling) are proposed to explain how altered release channel function caused by different mutations in RyR1 could result in muscle weakness in CCD.

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The Pore Region of the Skeletal Muscle Ryanodine Receptor Is a Primary Locus for Excitation-Contraction Uncoupling in Central Core Disease

The Journal of General Physiology ◽

10.1085/jgp.200308791 ◽

2003 ◽

Vol 121 (4) ◽

pp. 277-286 ◽

Cited By ~ 51

Author(s):

Guillermo Avila ◽

Kristen M. S. O'Connell ◽

Robert T. Dirksen

Keyword(s):

Skeletal Muscle ◽

Ryanodine Receptor ◽

Central Core ◽

Central Core Disease ◽

Release Channel ◽

Voltage Gated ◽

Ryr1 Gene ◽

Ec Coupling ◽

Store Depletion ◽

Sensor Activation

Human central core disease (CCD) is caused by mutations/deletions in the gene that encodes the skeletal muscle ryanodine receptor (RyR1). Previous studies have shown that CCD mutations in the NH2-terminal region of RyR1 lead to the formation of leaky SR Ca2+ release channels when expressed in myotubes derived from RyR1-knockout (dyspedic) mice, whereas a COOH-terminal mutant (I4897T) results in channels that are not leaky to Ca2+ but lack depolarization-induced Ca2+ release (termed excitation-contraction [EC] uncoupling). We show here that store depletion resulting from NH2-terminal (Y523S) and COOH-terminal (Y4795C) leaky CCD mutant release channels is eliminated after incorporation of the I4897T mutation into the channel (Y523S/I4897T and Y4795C/I4897T). In spite of normal SR Ca2+ content, myotubes expressing the double mutants lacked voltage-gated Ca2+ release and thus exhibited an EC uncoupling phenotype similar to that of I4897T-expressing myotubes. We also show that dyspedic myotubes expressing each of seven recently identified CCD mutations located in exon 102 of the RyR1 gene (G4890R, R4892W, I4897T, G4898E, G4898R, A4905V, R4913G) behave as EC-uncoupled release channels. Interestingly, voltage-gated Ca2+ release was nearly abolished (reduced ∼90%) while caffeine-induced Ca2+ release was only marginally reduced in R4892W-expressing myotubes, indicating that this mutation preferentially disrupts voltage-sensor activation of release. These data demonstrate that CCD mutations in exon 102 disrupt release channel permeation to Ca2+ during EC coupling and that this region represents a primary molecular locus for EC uncoupling in CCD.

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