Activation of the cardiac ryanodine receptor by sulfhydryl oxidation is modified by Mg²⁺ and ATP

The reactive disulfide 4,4'-dithiodipyridine (4,4'DTDP) was added to single cardiac ryanodine receptors (RyRs) in lipid bilayers. The activity of native RyRs, with cytoplasmic (cis) [Ca²⁺] of 10^-7 M (in the absence of Mg²⁺ and ATP), increased within ~1 min of addition of 1 mM 4,4'-DTDP, and then irreversibly ceased 5 to 6 min after the addition. Channels, inhibited by either 1 mM cis Mg²⁺ (10^-7 M cis Ca²⁺) or by 10 mM cis Mg²⁺ (10^-3 M cis Ca²⁺), or activated by 4 mM ATP (10^-7 M cis Ca²⁺), also responded to 1 mM cis 4,4'-DTDP with activation and then loss of activity, P(o) and mean open time (T(o)) of the maximally activated channels were lower in the presence of Mg²⁺ than in its absence, and the number of openings within the long time constant components of the open time distribution was reduced. In contrast to the reduced activation by 1 mM 4,4'-DTDP in channels inhibited by Mg²⁺, and the previously reported enhanced activation by 4,4'-DTDP in channels activated by Ca²⁺ or caffeine (Eager et al., 1997), the activation produced by 1 mM cis 4,4'-DTDP was the same in the presence and absence of ATP. These results suggest that there is a physical interaction between the ATP binding domain of the cardiac RyR and the SH groups whose oxidation leads to channel activation.