Altered inhibition of the rat skeletal ryanodine receptor/calcium release channel by magnesium in the presence of ATP

Magnesium-induced inhibition of the skeletal ryanodine receptor/calcium-release channel (RyR) was studied in the presence and absence of ATP under isolated conditions and in situ, by examining the RyR incorporated into a planar lipid bilayer and the calcium release flux (R-rel) in isolated single fibres mounted in the double Vaseline gap system. When the incorporated RyR had been activated by calcium (50 muM) in the absence of ATP, the magnesium-induced inhibition showed co-operativity with a Hill coefficient (N) of 1.83 and a half-inhibitory concentration (IC50) of 635 muM When the open probability was measured in the presence of 5 mM ATP and at a low calcium concentration, the magnesium-induced inhibition was non-cooperative (N=1.1, IC50= 860 muM) In isolated muscle fibres, in the presence of ATP, lowering the intracellular magnesium concentration ([Mg2+](i)) increased the maximal R-rel and shifted its voltage dependence to more negative membrane potentials. Increasing [Mg2+](i) had the opposite effect. The concentration dependence was described with an IC50 Of 174 muM, N=1, under depolarized conditions and showed a tenfold increase in affinity in polarized fibres. At the concentration required for the measurements from isolated fibres, ATP had a full activatory effect on the isolated channel. At a low calcium concentration, the RyR had two ATP-binding sites with half-activatory concentrations of 19 and 350 muM, respectively.