Insights into the regulation of the ryanodine receptor: Differential effects of calcium(2+), magnesium(2+) and pharmacological agents on ATP binding

The ryanodine receptors (RyRs) are proteins involved in the release of calcium ions from the sarcoplasmic reticulum. In this study, we have investigated the effects of main cellular effectors of RyR1, Ca 2+ and Mg2+ as well as pharmacologically important agents on the binding characteristics of a third cellular effector of the channel, ATP. Employing an innovative approach using ESR spectroscopy and an ATP analog that carries an ESR-active reporter group, spin-labeled ATP, we directly observed for the first time a total of eight ATP binding sites present on the tetrameric RyR1. We showed that the number of ATP binding sites accessible to the ATP analog and the respective dissociation constants directly depended on the presence and concentrations of Ca2+ and Mg 2+ ions. The RyR1 inhibitions induced by the presence of Ca 2+ or Mg2+ ions resulted in the binding of respectively four and eight ATP analog to the channel, pointing to the existence of two different inhibition mechanisms. Our results also implied that the accessibility of 8 sites seems to correspond to an open channel, while only 4 sites seemed to indicate a stably closed channel. ATP binding modulation to RyR1 therefore appears to play a central role in stabilizing the different conformations adopted by the channel during opening and closing events.; Ryanodine, caffeine, dantrolene and tetracaine are exogenous compounds known or suggested to affect the activity of RyR1. By using ESR spectroscopy, we investigated the ATP binding characteristics to the channel in the presence of these pharmacological agents. We demonstrated that different classes of exogenous effectors act differently on RyR1 by interfering with the negative feedback action of calcium ions on the channel with or without decreasing the ATP binding affinity. We also showed that dantrolene, the unique compound used to treat patients with maligniant hyperthermia, may be able to restore the missing negative feedback action of calcium ions on RyR1. ATP binding modulation to RyR1 appears to be crucial and in correlation with the modified activity of the channel induced by pharmacological agents. We demonstrated that ATP binding studies are a major tool to determine the mode of action of pharmacological agents on RyR1.; We attempted to localize the ATP binding sites of RyR1 by photoaffinity labeling using 2-N3-ATP. Our efforts were not successful, potentially due a low photoaffinity labeling efficiency and a low yield of recovery of the labeled peptide. Therefore the precise location of these ATP binding sites on RyR1 is still to be discovered.; Attempt to clone the cDNA of the human RyR1 into a high expression yeast strain were unfortunately also unsuccessful, most of the problems being due to the large size of the gene coding for the 550000 Da protein monomer of RyR1.