Hypochlorous acid modifies calcium release channel function from skeletal muscle sarcoplasmic reticulum

We have previously demonstrated that H₂O₂ at millimolar concentrations induces Ca²⁺ release from actively loaded sarcoplasmic reticulum (SR) vesicles and induces biphasic [³H]ryanodine binding behavior. Considering that hypochlorous acid (HOCl) is a related free radical and has been demonstrated to be a more effective oxidant of proteins, we evaluated the effects of HOCl on sarcoplasmic reticulum Ca²⁺-channel release mechanism. In a concentration-dependent manner, HOCl activates the SR Ca²⁺ release channel and induces rapid release of Ca from actively loaded vesicles. HOCl-induced Ca²⁺ release is inhibited in the presence of millimolar concentrations of DMSO. High-affinity [³H]ryanodine binding is also enhanced at concentrations from 10 to 100 μM. At HOCl concentrations of >100 μM, equilibrium binding is inhibited. HOCl stimulation of binding is inhibited by the addition of dithiothreitol. The direct interaction between HOCl and the Ca²⁺ release mechanism was further demonstrated in single-channel reconstitution experiments. HOCl, at 20 μM, activated the Ca²⁺ release channel after fusion of a SR vesicle to a bilayer lipid membrane. At 40 μM, Ca²⁺-channel activity was inhibited. Pretreatment of SR vesicles with HOCl inhibited the fluorescence development of a fluorogenic probe specific to thiol groups critical to channel function. These results suggest that HOCl at micromolar concentrations can modify SR Ca²⁺ handling.