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 Ca2+-release channel/ ryanodine receptor (RyRl) rests largerly on the demonstrated capacity of CaM to interact structurally and functionally with RyRl at pCa > 8 (Tripathy et al., 1995). The goal of the present [3H]-ryanodine binding study was to produce, in isolated terminal cisternae (TC) and in purified junctional face membrane (JFM), CaM-mediated activation of RyRl 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 RyRl by mM concentrations of caffeine or microM 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 RyRl; the Kd for ryanodine binding was reduced from 21.8 nM to 13.2 nM by 1microM 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 extrajunctional proteins in this effect, the experiments were repeated on purified JFM. Again, CaM increased the affinity of ryanodine binding; the Kd was reduced from 11.1 nM to 7.0 nM by 1 microM CaM (in the presence of doxorubicin). Pharmacological triggers of CaM-activatory action on native RyRl, like caffeine and doxorubicin, have long been characterized for their ability to activate RyRl by increasing the Ca2+-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 RyRl in skeletal muscle, during E-C coupling.
Damiani
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E
Department of Experimnental Biomedical Sciences Physiopathology, University of Padova, Italy.
Margreth
A
A
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000970:Antineoplastic Agents; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism* / Q000494:pharmacology*; D002458:Cell Fractionation; D000697:Central Nervous System Stimulants; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology; D008297:Male; D018482:Muscle, Skeletal / Q000187:drug effects* / Q000378:metabolism*; D011817:Rabbits; D011869:Radioligand Assay; D012433:Ryanodine / Q000494:pharmacology*; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects* / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D013347:Subcellular Fractions; D014316:Tritium
D000970:Antineoplastic Agents; D015220:Calcium Channels; D002147:Calmodulin; D000697:Central Nervous System Stimulants; D019837:Ryanodine Receptor Calcium Release Channel; D014316:Tritium; D012433:Ryanodine; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D002110:Caffeine; D004317:Doxorubicin; D000255:Adenosine Triphosphate; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D002118:Calcium