Imperatoxin A enhances Ca2+ release in developing skeletal muscle containing ryanodine receptor type 3

Most adult mammalian skeletal muscles contain only one isoform of ryanodine receptor (RyR1), whereas neonatal muscles contain two isoforms (RyR1 and RyR3). Membrane depolarization fails to evoke calcium release in muscle cells lacking RyR1, demonstrating an essential role for this isoform in excitation-contraction coupling. In contrast, the role of RyR3 is unknown. We studied the participation of RyR3 in calcium release in wild type (containing both RyR1 and RyR3 isoforms) and RyR3-/- (containing only RyR1) myotubes in the presence or absence of imperatoxin A (IpTxa), a high-affinity agonist of ryanodine receptors. IpTxa significantly increased the amplitude and the rate of release only in wild-type myotubes. Calcium currents, recorded simultaneously with the transients, were not altered with IpTxa treatment. [H-3]ryanodine binding to RyR1 or RyR3 was significantly increased in the presence of IpTxa. Additionally, IpTxa. modified the gating and conductance level of single RyR1 or RyR3 channels when studied in lipid bilayers. Our data show that IpTxa can interact with both RyRs and that RyR3 is functional in myotubes and it can amplify the calcium release signal initiated by RyR1, perhaps through a calcium-induced mechanism. In addition, our data indicate that when RyR3-/- myotubes are voltage-clamped, the effect of IpTxa is not detected because RyR1s are under the control of the dihydropyridine receptor.