Cardiac ryanodine receptor activation by a high Ca²⁺ 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 [Ca2+]. Basal leak from the sarcoplasmic reticulum is required for normal Ca2+ handling, but excess diastolic Ca2+ leak attributed to oxidative stress is thought to lower the threshold of RyR2 for spontaneous sarcoplasmic reticulum Ca2+ release, thus inducing arrhythmia in pathological situations. Therefore, we examined the RyR2 response to luminal [Ca2+] under reducing or oxidising cytoplasmic redox conditions. Unexpectedly, as luminal [Ca2+] 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 [Ca2+]. The luminal redox potential was constant at −180 mV in each case. These responses to luminal [Ca2+] were maintained with cytoplasmic 2 mM Na2ATP or 5 mM MgATP (1 mM free Mg2+). Overall, the results suggest that the redox potential in the RyR2 junctional microdomain is normally more oxidised than that of the bulk cytoplasm