Pharmacological clues to calmodulin-mediated activation of skeletal ryanodine receptor using [3H]-ryanodine binding

The hypothesis that calmodulin (CaM) may act as a positive modulator of junctional SR Ca-2+-release channel/ryanodine receptor (RyR1) rests largerly on the demonstrated capacity of CaM to interact structurally and functionally with RyR1 at pCa > 8 (Tripathy et al., 1995). The goal of the present [H-3]-ryanodine binding study was to produce, in isolated terminal cisternae (TC) and in purified junctional face membrane (JFM), CaM-mediated activation of RyR1 at less extreme pCa values, i.e. closer to resting myoplasmic pCa, and to analyze more accurately the corresponding changes in binding affinity for ryanodine of the receptor. We were able to monitor these changes at an optimum pCa of 6.5, following pre-activation of native RyR1 by mM concentrations of caffeine or mu M concentrations of antraquinone compound doxorubicin, and at various doses of these triggers. CaM increased the affinity of ryanodine binding to isolated TC in the presence of 1 mM AMP-PCP as an activator of RyR1; the K-d for ryanodine binding was reduced from 21.8 nM to 13.2 nM by 1 mu M CaM. Similar effects of CaM were seen when AMP-PCP was replaced by either caffeine or doxorubicin. In order to discount the involvement of SR extra-junctional proteins in this effect, the experiments were repeated on purified JFM. Again, CaM increased the affinity of ryanodine binding; the K-d was reduced from 11.1 nM to 7.0 nM by 1 mu M CaM (in the presence of doxorubicin). Pharmacological triggers of CaM-activatory action on native RyR1, like caffeine and doxorubicin, have long been characterized for their ability to activate RyR1 by increasing the Ca-2+-sensitivity of the receptor. We speculate that the triggering effect of these agents on the CaM-mediated mechanism in vitro might mimick one of the early effects of the activation of RyR1 in skeletal muscle, during E-C coupling.