Hypochlorous acid modifies calcium release channel function from skeletal muscle sarcoplasmic reticulum.
We have previously demonstrated that H2O2 at millimolar concentrations induces Ca(2+) release from actively loaded sarcoplasmic reticulum (SR) vesicles and induces biphasic [(3)H]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 Ca(2+)-channel release mechanism. In a concentration-dependent manner, HOCl activates the SR Ca(2+) release channel and induces rapid release of Ca from actively loaded vesicles. HOCl-induced Ca(2+) release is inhibited in the presence of millimolar concentrations of DMSO. High-affinity [(3)H]ryanodine binding is also enhanced at concentrations from 10 to 100 microM. At HOCl concentrations of >100 microM, equilibrium binding is inhibited. HOCl stimulation of binding is inhibited by the addition of dithiothreitol. The direct interaction between HOCl and the Ca(2+) release mechanism was further demonstrated in single-channel reconstitution experiments. HOCl, at 20 microM, activated the Ca(2+) release channel after fusion of a SR vesicle to a bilayer lipid membrane. At 40 microM, Ca(2+)-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 Ca(2+) handling.
Favero
Terence G
TG
Department of Biology, University of Portland, Portland, Oregon 97203, USA. favero@up.edu
Webb
Jason
J
Papiez
Maria
M
Fisher
Erin
E
Trippichio
Robert J
RJ
Broide
Michael
M
Abramson
Jonathan J
JJ
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000187:drug effects* / Q000378:metabolism*; D006997:Hypochlorous Acid / Q000494:pharmacology*; D066298:In Vitro Techniques; D018482:Muscle, Skeletal / Q000378:metabolism; D011817:Rabbits; D012433:Ryanodine / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism
D015220:Calcium Channels; D013438:Sulfhydryl Compounds; D012433:Ryanodine; D006997:Hypochlorous Acid; D000251:Adenosine Triphosphatases; D002118:Calcium