Calexcitin interaction with neuronal ryanodine receptors

Calexcitin (CE), a Ca²⁺- and GTP-binding protein, which is phosphorylated during memory consolidation, is shown here to co-purify with ryanodine receptors (RyRs) and bind to RyRs in a calcium-dependent manner. Nanomolar concentrations of CE released up to 46% of the ⁴⁵Ca label from microsomes preloaded with ⁴⁵CaCl₂. This release was Ca²⁺-dependent and was blocked by antibodies against the RyR or CE, by the RyR inhibitor dantrolene, and by a seven-amino-acid peptide fragment corresponding to positions 4689-4697 of the RyR, but not by heparin, an Ins(1,4,5)P₃-receptor antagonist. Anti-CE antibodies, in the absence of added CE, also blocked Ca²⁺ release elicited by ryanodine, suggesting that the CE and ryanodine binding sites were in relative proximity. Calcium imaging with bis-fura-2 after loading CE into hippocampal CA1 pyramidal cells in hippocampal slices revealed slow, local calcium transients independent of membrane depolarization. Calexcitin also released Ca²⁺ from liposomes into which purified RyR had been incorporated, indicating that CE binding can be a proximate cause of Ca²⁺ release. These results indicated that CE bound to RyRs and suggest that CE may be an endogenous modulator of the neuronal RyR.