Voltage- and ligand-gated ryanodine receptors are functionally separated in developing C2C12 mouse myotubes

Abstract

1. In order to further understand the role of voltage- and ligand-gated ryanodine receptors in the control of intracellular Ca²⁺ signalling during myogenesis, changes in cytosolic free calcium concentration ([Ca²⁺]_i) were investigated by fura-2 videoimaging in C2C12 mouse myotubes developing in vitro.

2. A synchronous [Ca²⁺]_i increase was observed after depolarisation with high [K⁺], while the Ca²⁺ response propagated as a wave following caffeine administration. Application of the two stimuli to the same myotube often revealed the existence of cellular zones that were responsive to depolarisation but not to caffeine.

3. Focal application of high [K⁺] promoted a [Ca²⁺]_i response detectable only in the cellular areas close to the pipette tip, while focal application of caffeine elicited a [Ca²⁺]_i increase which spread as a Ca²⁺ wave. Buffering of [Ca²⁺]_i by BAPTA did not affect the pattern of the depolarisation-induced [Ca²⁺]_i transient but abolished the Ca²⁺ waves elicited by caffeine.

4. When high [K⁺] and caffeine were applied in sequence, reciprocal inhibition of the [Ca²⁺]_i responses was observed.

5. Our results suggest that the different spatial patterns of [Ca²⁺]_i responses are due to uneven distribution of voltage- and ligand-gated ryanodine receptors within the myotube. These two types of receptor control two functionally distinct Ca²⁺ pools which are part of a common intracellular compartment. Finally, the two differently operated ryanodine receptor channels appear to be independently activated, so that a mechanism of Ca²⁺-induced Ca²⁺ release is not required to sustain the global response in C2C12 myotubes.