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Frog alpha- and beta-ryanodine receptors provide distinct intracellular Ca2+ signals in a myogenic cell line.

Kashiyama T, Murayama T, Suzuki E, Allen PD, Ogawa Y - PLoS ONE (2010)

Bottom Line: Furthermore, procaine did not affect the peak height of high [K(+)](o)-induced Ca(2+) transients, suggesting minor amplification of Ca(2+) release by beta-RyR via CICR in 1B5 myotubes.These findings suggest that alpha-RyR and beta-RyR provide distinct intracellular Ca(2+) signals in a myogenic cell line.These distinct properties may also occur in frog skeletal muscle and will be important for E-C coupling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Juntendo University School of Medicine, Tokyo, Japan.

ABSTRACT

Background: In frog skeletal muscle, two ryanodine receptor (RyR) isoforms, alpha-RyR and beta-RyR, are expressed in nearly equal amounts. However, the roles and significance of the two isoforms in excitation-contraction (E-C) coupling remains to be elucidated.

Methodology/principal findings: In this study, we expressed either or both alpha-RyR and beta-RyR in 1B5 RyR-deficient myotubes using the herpes simplex virus 1 helper-free amplicon system. Immunological characterizations revealed that alpha-RyR and beta-RyR are appropriately expressed and targeted at the junctions in 1B5 myotubes. In Ca(2+) imaging studies, each isoform exhibited caffeine-induced Ca(2+) transients, an indicative of Ca(2+)-induced Ca(2+) release (CICR). However, the fashion of Ca(2+) release events was fundamentally different: alpha-RyR mediated graded and sustained Ca(2+) release observed uniformly throughout the cytoplasm, whereas beta-RyR supported all-or-none type regenerative Ca(2+) oscillations and waves. alpha-RyR but not beta-RyR exhibited Ca(2+) transients triggered by membrane depolarization with high [K(+)](o) that were nifedipine-sensitive, indicating that only alpha-RyR mediates depolarization-induced Ca(2+) release. Myotubes co-expressing alpha-RyR and beta-RyR demonstrated high [K(+)](o)-induced Ca(2+) transients which were indistinguishable from those with myotubes expressing alpha-RyR alone. Furthermore, procaine did not affect the peak height of high [K(+)](o)-induced Ca(2+) transients, suggesting minor amplification of Ca(2+) release by beta-RyR via CICR in 1B5 myotubes.

Conclusions/significance: These findings suggest that alpha-RyR and beta-RyR provide distinct intracellular Ca(2+) signals in a myogenic cell line. These distinct properties may also occur in frog skeletal muscle and will be important for E-C coupling.

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Ca2+ release properties of 1B5 myotubes co-expressing α-RyR and β-RyR.A. Immunoblot of total lysate from 1B5 myotubes co-expressing α-RyR and β-RyR showing two bands corresponding in size to those observed in preparations of frog SR. B. A representative trace of Ca2+ transients induced by varied concentrations of caffeine. The responses of cells co-expressing both isoforms appeared to be additive and independent of each other: uniform and sustained Ca2+ release characteristic of α-RyR and Ca2+ oscillations characteristic β-RyR were both observed in these cells. C. Representative Ca2+ transients of myotubes expressing either α-RyR alone (upper traces) or α-RyR and β-RyR expressed together (lower traces) induced by increasing concentrations of [K+]o. D. [K+]o dependences of the Ca2+ transients of myotubes expressing α-RyR (open circles) and co-expressing α-RyR and β-RyR (closed circles). (mean ± SE, n = 112 for α-RyR and 121 for α-RyR and β-RyR). There was no statistical difference between the two groups at any [K+]o concentration examined.
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pone-0011526-g005: Ca2+ release properties of 1B5 myotubes co-expressing α-RyR and β-RyR.A. Immunoblot of total lysate from 1B5 myotubes co-expressing α-RyR and β-RyR showing two bands corresponding in size to those observed in preparations of frog SR. B. A representative trace of Ca2+ transients induced by varied concentrations of caffeine. The responses of cells co-expressing both isoforms appeared to be additive and independent of each other: uniform and sustained Ca2+ release characteristic of α-RyR and Ca2+ oscillations characteristic β-RyR were both observed in these cells. C. Representative Ca2+ transients of myotubes expressing either α-RyR alone (upper traces) or α-RyR and β-RyR expressed together (lower traces) induced by increasing concentrations of [K+]o. D. [K+]o dependences of the Ca2+ transients of myotubes expressing α-RyR (open circles) and co-expressing α-RyR and β-RyR (closed circles). (mean ± SE, n = 112 for α-RyR and 121 for α-RyR and β-RyR). There was no statistical difference between the two groups at any [K+]o concentration examined.

Mentions: It has been proposed that CICR of β-RyR may serve to amplify the Ca2+ signals of DICR by α-RyR [33]. To assess this possibility, we expressed both isoforms in 1B5 myotubes and examined their Ca2+ release properties. 1B5 myotubes were transduced with a mixture of α-RyR and β-RyR virions. Immunoblotting of total lysate of the infected myotubes revealed that α-RyR and β-RyR were expressed in nearly equal amounts (Fig. 5A). These myotubes exhibited caffeine-induced Ca2+ transients that consisted of Ca2+ oscillations and the sustained Ca2+ rise, which correspond to the combined phenotype of cells expressing the individual isoforms (Fig. 5B). This confirmed functional expression of both α-RyR and β-RyR in the myotubes. The two isoforms were independent of each other in their Ca2+ release fashion. The overall features of high [K+]o-induced Ca2+ transients in myotubes expressing the two isoforms, however, were similar to those with myotubes expressing α-RyR alone (Fig. 5C). Neither Ca2+ oscillations nor waves observed during the stimuli. The averaged amplitude and [K+]o dependence of the Ca2+ transients were not significantly different from those of myotubes expressing α-RyR alone (Fig. 5D). Thus, co-expression of β-RyR did not affect DICR of α-RyR.


Frog alpha- and beta-ryanodine receptors provide distinct intracellular Ca2+ signals in a myogenic cell line.

Kashiyama T, Murayama T, Suzuki E, Allen PD, Ogawa Y - PLoS ONE (2010)

Ca2+ release properties of 1B5 myotubes co-expressing α-RyR and β-RyR.A. Immunoblot of total lysate from 1B5 myotubes co-expressing α-RyR and β-RyR showing two bands corresponding in size to those observed in preparations of frog SR. B. A representative trace of Ca2+ transients induced by varied concentrations of caffeine. The responses of cells co-expressing both isoforms appeared to be additive and independent of each other: uniform and sustained Ca2+ release characteristic of α-RyR and Ca2+ oscillations characteristic β-RyR were both observed in these cells. C. Representative Ca2+ transients of myotubes expressing either α-RyR alone (upper traces) or α-RyR and β-RyR expressed together (lower traces) induced by increasing concentrations of [K+]o. D. [K+]o dependences of the Ca2+ transients of myotubes expressing α-RyR (open circles) and co-expressing α-RyR and β-RyR (closed circles). (mean ± SE, n = 112 for α-RyR and 121 for α-RyR and β-RyR). There was no statistical difference between the two groups at any [K+]o concentration examined.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2902508&req=5

pone-0011526-g005: Ca2+ release properties of 1B5 myotubes co-expressing α-RyR and β-RyR.A. Immunoblot of total lysate from 1B5 myotubes co-expressing α-RyR and β-RyR showing two bands corresponding in size to those observed in preparations of frog SR. B. A representative trace of Ca2+ transients induced by varied concentrations of caffeine. The responses of cells co-expressing both isoforms appeared to be additive and independent of each other: uniform and sustained Ca2+ release characteristic of α-RyR and Ca2+ oscillations characteristic β-RyR were both observed in these cells. C. Representative Ca2+ transients of myotubes expressing either α-RyR alone (upper traces) or α-RyR and β-RyR expressed together (lower traces) induced by increasing concentrations of [K+]o. D. [K+]o dependences of the Ca2+ transients of myotubes expressing α-RyR (open circles) and co-expressing α-RyR and β-RyR (closed circles). (mean ± SE, n = 112 for α-RyR and 121 for α-RyR and β-RyR). There was no statistical difference between the two groups at any [K+]o concentration examined.
Mentions: It has been proposed that CICR of β-RyR may serve to amplify the Ca2+ signals of DICR by α-RyR [33]. To assess this possibility, we expressed both isoforms in 1B5 myotubes and examined their Ca2+ release properties. 1B5 myotubes were transduced with a mixture of α-RyR and β-RyR virions. Immunoblotting of total lysate of the infected myotubes revealed that α-RyR and β-RyR were expressed in nearly equal amounts (Fig. 5A). These myotubes exhibited caffeine-induced Ca2+ transients that consisted of Ca2+ oscillations and the sustained Ca2+ rise, which correspond to the combined phenotype of cells expressing the individual isoforms (Fig. 5B). This confirmed functional expression of both α-RyR and β-RyR in the myotubes. The two isoforms were independent of each other in their Ca2+ release fashion. The overall features of high [K+]o-induced Ca2+ transients in myotubes expressing the two isoforms, however, were similar to those with myotubes expressing α-RyR alone (Fig. 5C). Neither Ca2+ oscillations nor waves observed during the stimuli. The averaged amplitude and [K+]o dependence of the Ca2+ transients were not significantly different from those of myotubes expressing α-RyR alone (Fig. 5D). Thus, co-expression of β-RyR did not affect DICR of α-RyR.

Bottom Line: Furthermore, procaine did not affect the peak height of high [K(+)](o)-induced Ca(2+) transients, suggesting minor amplification of Ca(2+) release by beta-RyR via CICR in 1B5 myotubes.These findings suggest that alpha-RyR and beta-RyR provide distinct intracellular Ca(2+) signals in a myogenic cell line.These distinct properties may also occur in frog skeletal muscle and will be important for E-C coupling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Juntendo University School of Medicine, Tokyo, Japan.

ABSTRACT

Background: In frog skeletal muscle, two ryanodine receptor (RyR) isoforms, alpha-RyR and beta-RyR, are expressed in nearly equal amounts. However, the roles and significance of the two isoforms in excitation-contraction (E-C) coupling remains to be elucidated.

Methodology/principal findings: In this study, we expressed either or both alpha-RyR and beta-RyR in 1B5 RyR-deficient myotubes using the herpes simplex virus 1 helper-free amplicon system. Immunological characterizations revealed that alpha-RyR and beta-RyR are appropriately expressed and targeted at the junctions in 1B5 myotubes. In Ca(2+) imaging studies, each isoform exhibited caffeine-induced Ca(2+) transients, an indicative of Ca(2+)-induced Ca(2+) release (CICR). However, the fashion of Ca(2+) release events was fundamentally different: alpha-RyR mediated graded and sustained Ca(2+) release observed uniformly throughout the cytoplasm, whereas beta-RyR supported all-or-none type regenerative Ca(2+) oscillations and waves. alpha-RyR but not beta-RyR exhibited Ca(2+) transients triggered by membrane depolarization with high [K(+)](o) that were nifedipine-sensitive, indicating that only alpha-RyR mediates depolarization-induced Ca(2+) release. Myotubes co-expressing alpha-RyR and beta-RyR demonstrated high [K(+)](o)-induced Ca(2+) transients which were indistinguishable from those with myotubes expressing alpha-RyR alone. Furthermore, procaine did not affect the peak height of high [K(+)](o)-induced Ca(2+) transients, suggesting minor amplification of Ca(2+) release by beta-RyR via CICR in 1B5 myotubes.

Conclusions/significance: These findings suggest that alpha-RyR and beta-RyR provide distinct intracellular Ca(2+) signals in a myogenic cell line. These distinct properties may also occur in frog skeletal muscle and will be important for E-C coupling.

Show MeSH
Related in: MedlinePlus