Muscle weakness in Ryr1I4895T/WT knock-in mice as a result of reduced ryanodine receptor Ca2+ ion permeation and release from the sarcoplasmic reticulum

Abstract

The type 1 isoform of the ryanodine receptor (RYR1) is the Ca2+ release channel of the sarcoplasmic reticulum (SR) that is activated during skeletal muscle excitation–contraction (EC) coupling. Mutations in the RYR1 gene cause several rare inherited skeletal muscle disorders, including malignant hyperthermia and central core disease (CCD). The human RYR1I4898T mutation is one of the most common CCD mutations. To elucidate the mechanism by which RYR1 function is altered by this mutation, we characterized in vivo muscle strength, EC coupling, SR Ca2+ content, and RYR1 Ca2+ release channel function using adult heterozygous Ryr1I4895T/+ knock-in mice (IT/+). Compared with age-matched wild-type (WT) mice, IT/+ mice exhibited significantly reduced upper body and grip strength. In spite of normal total SR Ca2+ content, both electrically evoked and 4-chloro-m-cresol–induced Ca2+ release were significantly reduced and slowed in single intact flexor digitorum brevis fibers isolated from 4–6-mo-old IT/+ mice. The sensitivity of the SR Ca2+ release mechanism to activation was not enhanced in fibers of IT/+ mice. Single-channel measurements of purified recombinant channels incorporated in planar lipid bilayers revealed that Ca2+ permeation was abolished for homotetrameric IT channels and significantly reduced for heterotetrameric WT:IT channels. Collectively, these findings indicate that in vivo muscle weakness observed in IT/+ knock-in mice arises from a reduction in the magnitude and rate of RYR1 Ca2+ release during EC coupling that results from the mutation producing a dominant-negative suppression of RYR1 channel Ca2+ ion permeation.

Type 1 ryanodine receptor knock-in mutation causing central core disease of skeletal muscle also displays a neuronal phenotype

“…However, as studied in bilayers and in cultured myotubes from IT/+ mutant mice, the IT mutation is not a leaky mutation: the presence of more than two IT mutant subunits in a tetramer has been shown to render RyR1 nonpermeant to Ca 2+ (15). We found no increase in global [Ca 2+ ] at rest, so the increase in syntilla frequency is not sufficient to affect the detectable global [Ca 2+ ] (Fig.…”

“…4A) and rate (Fig. 4B) of RyR1-mediated Ca 2+ release during EC coupling (15). Although the RyR1 I4895T CCD mutation causes a core myopathy in our mouse line (14), we do not yet have evidence for a neuropathy in these same mice.…”

“…Loy et al (15) showed that single skeletal muscle fibers from IT/+ mice exhibited a significant reduction in both the magnitude and maximum rate of RyR1-mediated Ca 2+ release during EC coupling. To determine whether a similar slowing exists in nerve terminals, we compared the kinetics of Ca 2+ release in WT and IT/+ terminals during the long depolarization that was able to elicit VICaR in WT (Fig.…”

“…One of these is the human Ryr I4898T mutation, which is among the most frequent of those RyR1 mutations that are associated with CCD and other core myopathies (10)(11)(12). The mutation is located in the selectivity filter of the Ca 2+ pore (13) and disrupts Ca 2+ permeation without affecting the overall structural and supramolecular organization of RyR1 (14)(15)(16).…”

“…Thus, the difference in the global transients does not appear to be caused by changes in Ca 2+ influx but rather indicates a defect in Ca 2+ release from intracellular stores. Because these experiments were carried out under the same conditions in which the mutant was studied in skeletal muscle, a comparison is possible, and the results in the nerve terminal are comparable to those in skeletal fibers (15).…”