Cardiac ryanodine receptor activation by a high Ca ²⁺ store load is reversed in a reducing cytoplasmic redox environment

Here, we report the impact of redox potential on isolated cardiac ryanodine receptor (RyR2) channel activity and its response to physiological changes in luminal [Ca ²⁺ ]. Basal leak from the sarcoplasmic reticulum is required for normal Ca ²⁺ handling, but excess diastolic Ca ²⁺ leak attributed to oxidative stress is thought to lower the threshold of RyR2 for spontaneous sarcoplasmic reticulum Ca ²⁺ release, thus inducing arrhythmia in pathological situations. Therefore, we examined the RyR2 response to luminal [Ca ²⁺ ] under reducing or oxidising cytoplasmic redox conditions. Unexpectedly, as luminal [Ca ²⁺ ] increased from 0.1 to 1.5 mM, RyR2 activity declined when pretreated with cytoplasmic 1 mM DTT or buffered with GSH:GSSG to a normal reduced cytoplasmic redox potential (-220 mV). Conversely, with 20 μM cytoplasmic 4,4'-DTDP or buffering of the redox potential to an oxidising value (-180 mV), RyR2 activity increased with increasing luminal [Ca ²⁺ ]. The luminal redox potential was constant at -180 mV in each case. These responses to luminal [Ca ²⁺ ] were maintained with cytoplasmic 2 mM Na ₂ ATP or 5 mM MgATP (1 mM free Mg ²⁺ ). Overall, the results suggest that the redox potential in the RyR2 junctional microdomain is normally more oxidised than that of the bulk cytoplasm.