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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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Effect of procaine on Ca2+ release properties of 1B5 myotubes co-expressing α-RyR and β-RyR.A. Representative Ca2+ transients induced by increasing concentrations of caffeine in the absence (Control) and presence (Procaine) of 10 mM procaine. Procaine abolished Ca2+ oscillations caused by exposure to lower concentrations (<5 mM) of caffeine. B. The percentage of myotubes showing Ca2+ oscillations at each caffeine concentration tested was plotted in reference to the number of cells responding in the presence of 20 mM caffeine. Open circles, control; filled circles, 10 mM procaine (n = 42). Procaine reduced the caffeine sensitivity. C. Representative traces of Ca2+ transients of myotubes induced by increasing [K+]o in the absence (Control) and presence (Procaine) of 10 mM Procaine. D. Dependence of the amplitude of the Ca2+ transient in response to in exposure to increasing [K+]o in the absence (open circles) and presence (filled circles) of 10 mM Procaine. Values represent the mean ± SE (n = 42). No statistical difference was found between the two groups at all the [K+]o concentrations examined.
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pone-0011526-g006: Effect of procaine on Ca2+ release properties of 1B5 myotubes co-expressing α-RyR and β-RyR.A. Representative Ca2+ transients induced by increasing concentrations of caffeine in the absence (Control) and presence (Procaine) of 10 mM procaine. Procaine abolished Ca2+ oscillations caused by exposure to lower concentrations (<5 mM) of caffeine. B. The percentage of myotubes showing Ca2+ oscillations at each caffeine concentration tested was plotted in reference to the number of cells responding in the presence of 20 mM caffeine. Open circles, control; filled circles, 10 mM procaine (n = 42). Procaine reduced the caffeine sensitivity. C. Representative traces of Ca2+ transients of myotubes induced by increasing [K+]o in the absence (Control) and presence (Procaine) of 10 mM Procaine. D. Dependence of the amplitude of the Ca2+ transient in response to in exposure to increasing [K+]o in the absence (open circles) and presence (filled circles) of 10 mM Procaine. Values represent the mean ± SE (n = 42). No statistical difference was found between the two groups at all the [K+]o concentrations examined.

Mentions: To further evaluate the role of β-RyR on DICR, we examined the effect of procaine, an inhibitor of CICR, on the Ca2+ transients of myotubes co-expressing α-RyR and β-RyR. Procaine (10 mM) abolished the Ca2+ oscillations induced by lower concentrations (≦5 mM) of caffeine (Fig. 6A) and reduced the caffeine sensitivity by nearly fivefold (Fig. 6B), suggesting that CICR via β-RyR is strongly inhibited by procaine. The sustained Ca2+ rises probably caused by α-RyR were also reduced by procaine (Fig. 6A). Similar inhibition of caffeine-induced Ca2+ release by procaine was observed with myotubes expressing α-RyR or β-RyR alone (data not shown). These findings suggest that procaine effectively inhibited CICR of α-RyR and β-RyR. In contrast, procaine did not affect the peak value of high [K+]o-induced Ca2+ transients (Fig. 6C, 6D) but it did accelerate the subsequent decline of the Ca2+ transients (Fig. 6C). This is consistent with the reports that procaine did not inhibit activation of the DHPR voltage sensor and consequent Ca2+ release, but it accelerated the inactivation process [34], [35]. Taken together, these findings suggest that CICR by β-RyR may make only a minor contribution to Ca2+ signals of DICR on membrane depolarization in 1B5 myotubes.


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)

Effect of procaine on Ca2+ release properties of 1B5 myotubes co-expressing α-RyR and β-RyR.A. Representative Ca2+ transients induced by increasing concentrations of caffeine in the absence (Control) and presence (Procaine) of 10 mM procaine. Procaine abolished Ca2+ oscillations caused by exposure to lower concentrations (<5 mM) of caffeine. B. The percentage of myotubes showing Ca2+ oscillations at each caffeine concentration tested was plotted in reference to the number of cells responding in the presence of 20 mM caffeine. Open circles, control; filled circles, 10 mM procaine (n = 42). Procaine reduced the caffeine sensitivity. C. Representative traces of Ca2+ transients of myotubes induced by increasing [K+]o in the absence (Control) and presence (Procaine) of 10 mM Procaine. D. Dependence of the amplitude of the Ca2+ transient in response to in exposure to increasing [K+]o in the absence (open circles) and presence (filled circles) of 10 mM Procaine. Values represent the mean ± SE (n = 42). No statistical difference was found between the two groups at all the [K+]o concentrations examined.
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pone-0011526-g006: Effect of procaine on Ca2+ release properties of 1B5 myotubes co-expressing α-RyR and β-RyR.A. Representative Ca2+ transients induced by increasing concentrations of caffeine in the absence (Control) and presence (Procaine) of 10 mM procaine. Procaine abolished Ca2+ oscillations caused by exposure to lower concentrations (<5 mM) of caffeine. B. The percentage of myotubes showing Ca2+ oscillations at each caffeine concentration tested was plotted in reference to the number of cells responding in the presence of 20 mM caffeine. Open circles, control; filled circles, 10 mM procaine (n = 42). Procaine reduced the caffeine sensitivity. C. Representative traces of Ca2+ transients of myotubes induced by increasing [K+]o in the absence (Control) and presence (Procaine) of 10 mM Procaine. D. Dependence of the amplitude of the Ca2+ transient in response to in exposure to increasing [K+]o in the absence (open circles) and presence (filled circles) of 10 mM Procaine. Values represent the mean ± SE (n = 42). No statistical difference was found between the two groups at all the [K+]o concentrations examined.
Mentions: To further evaluate the role of β-RyR on DICR, we examined the effect of procaine, an inhibitor of CICR, on the Ca2+ transients of myotubes co-expressing α-RyR and β-RyR. Procaine (10 mM) abolished the Ca2+ oscillations induced by lower concentrations (≦5 mM) of caffeine (Fig. 6A) and reduced the caffeine sensitivity by nearly fivefold (Fig. 6B), suggesting that CICR via β-RyR is strongly inhibited by procaine. The sustained Ca2+ rises probably caused by α-RyR were also reduced by procaine (Fig. 6A). Similar inhibition of caffeine-induced Ca2+ release by procaine was observed with myotubes expressing α-RyR or β-RyR alone (data not shown). These findings suggest that procaine effectively inhibited CICR of α-RyR and β-RyR. In contrast, procaine did not affect the peak value of high [K+]o-induced Ca2+ transients (Fig. 6C, 6D) but it did accelerate the subsequent decline of the Ca2+ transients (Fig. 6C). This is consistent with the reports that procaine did not inhibit activation of the DHPR voltage sensor and consequent Ca2+ release, but it accelerated the inactivation process [34], [35]. Taken together, these findings suggest that CICR by β-RyR may make only a minor contribution to Ca2+ signals of DICR on membrane depolarization in 1B5 myotubes.

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