@article {Diszhazimol.119.116475, author = {Gyula Diszhazi and Zsuzsanna Edua Magyar and Janos Andras Motyan and Laszlo Csernoch and Istvan Jona and Peter Nanasi Pal Nanasi and Janos Almassy}, title = {Dantrolene requires Mg2+ and ATP to inhibit the ryanodine receptor}, elocation-id = {mol.119.116475}, year = {2019}, doi = {10.1124/mol.119.116475}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {Dantrolene is a ryanodine receptor (RyR) inhibitor, which is used to relax muscles in malignant hyperthermia syndrome. Although dantrolene binds to the RyR protein, its mechanism of action is unknown, mainly because of the controversial data showing that dantrolene inhibited Ca2+-release from intact fibers and sarcoplasmic reticulum (SR) vesicles, but failed to inhibit single ryanodine receptor (RyR) channel currents in bilayers. Accordingly, it was concluded that an important factor for dantrolene{\textquoteleft}s action was lost during the purification procedure of RyR. Recently, Mg2+ was demonstrated to be the essential factor for dantrolene to inhibit Ca2+- release in skinned muscle fibers. The aim of the present study was to confirm these results in Ca2+ release- and bilayer experiments, using SR vesicles and solubilized channels, respectively. Our Ca2+-release experiments demonstrated that the effect of dantrolene and Mg2+ was cooperative; and that ATP enhanced the inhibiting effect of dantrolene. Namely, 10 {\textmu}M dantrolene reduced RyR channel open probability by ~50\% in the presence of 3 mM free Mg2+ and 1 mM ATP, while channel activity further decreased to ~20\% of control when [ATP] was increased to 2 mM. Our data provide important complementary information that support the direct, Mg2+-dependent mechanism of dantrolene{\textquoteright}s action and suggest that dantrolene also requires ATP to inhibit RyR.SIGNIFICANCE STATEMENT The muscle relaxant dantrolene acts on ryanodine receptor in the presence of magnesium and ATP, which should be considered in therapy.}, issn = {0026-895X}, URL = {https://molpharm.aspetjournals.org/content/early/2019/07/23/mol.119.116475}, eprint = {https://molpharm.aspetjournals.org/content/early/2019/07/23/mol.119.116475.full.pdf}, journal = {Molecular Pharmacology} }