Inability of Sphingosine and Calmodulin to Control Ryanodine Receptor in Malignant Hyperthermia

Sarcoplasmic reticulum Ca release of malignant hyperthermia-susceptible (MHS) skeletal muscle is hypersensitive to both agonists and antagonists of the ryanodine receptor channel. This study examined whether sphingosine and calmodulin, endogenous modulators of the channel, play a role in the abnormal responsiveness of pathological muscle. Sphingosine caused a dose dependent inhibition of [H]ryanodine binding to MHS and normal terminal cisterns (TC) membranes of the sarcoplasmic reticulum (SR). The sphingosine concentration capable of inhibiting 50% of the binding (IC50) was 1.5-times higher for MHS compared to membranes isolated form normal animals. Calmodulin also caused a dose dependent inhibition of [H]ryanodine binding. However, at variance with sphingosine and other inhibitors, the calmodulin effect was incomplete in that 1 μM calmodulin inhibited only 50% of the [H]ryanodine binding to MHS membranes. Sphingosine’s inhibitory action was particularly effective at activating pCa levels (5 to 4) in normal membranes, while in MHS membranes the ability of sphingosine to block ryanodine receptor was minimal. Similarly, Calmodulin inhibition of [H]ryanodine binding was maximal in normal membranes at pCa 4 but less potent in MHS membranes. Both sphingosine and calmodulin exert the highest inhibitory action to the ryanodine receptor at activating pCa levels in normal membranes. Similarly, at the same pCa values, both drugs are less effective in blocking the mutated channel. i.e., particularly at pCa levels that maximally activate muscle cells. These results indicate that the lower sensitivity to inhibition of the MHS Ca-release channel by endogenous antagonists, such as sphingosine and calmodulin, may play an important role in the abnormal response of the mutated channel.