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 [H-3]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 [H-3]ryanodine binding is also enhanced at concentrations from 10 to 100 muM. At HOCl concentrations of >100 muM, 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 muM, activated the Ca2+ release channel after fusion of a SR vesicle to a bilayer lipid membrane. At 40 muM, 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.