RyR1 modulation by oxidation and calmodulin.
Alteration of skeletal muscle function by reactive oxygen species and nitric oxide (NO) may involve regulation of the activity of the skeletal muscle Ca2+ release channel (also known as RyR1). We have shown that oxidants can activate RyR1 and produce inter-subunit disulfide bonds. Both effects are prevented by pretreatment with either NO donors or N-ethylmaleimide under conditions that modify less than 5% of the total sulfhydryls on RyR1. Oxidation-induced intersubunit crosslinking can also be prevented by the binding of either Ca2+ calmodulin or apocalmodulin to RyR1. Also, both Ca2+ calmodulin and apocalmodulin binding are blocked by oxidation of RyR1. In contrast, alkylation with N-ethylmaleimide or reaction with NO donors preferentially blocks apocalmodulin binding to RyR1, suggesting the existence of a regulatory cysteine within the apocalmodulin binding site. We have demonstrated that Ca2+ calmodulin and apocalmodulin bind to overlapping, but nonidentical, sites on RyR1 and that cysteine 3635 is close to or within the apocalmodulin-binding site on RyR1. This cysteine is also one of the cysteines that form the intersubunit disulfide bonds, suggesting that calmodulin binds at an intersubunit contact site. Our findings are consistent with a model in which oxidants regulate the activity of RyR1 directly by altering subunit-subunit interactions and indirectly by preventing the binding of either Ca2+-bound calmodulin or apocalmodulin. NO also has both a direct and an indirect effect: it blocks the ability of oxidants to generate intersubunit disulfide bonds and prevents apocalmodulin binding.
Hamilton
S L
SL
Department of Molecular Physiology & Biophysics, Baylor College of Medicine, Houston, TX 77030, USA. susanh@bcm.tmc.edu
Reid
M B
MB
D000478:Alkylation; D000818:Animals; D001665:Binding Sites; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology*; D003545:Cysteine / Q000502:physiology; D003553:Cystine / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D017136:Ion Transport / Q000187:drug effects; D009119:Muscle Contraction / Q000187:drug effects; D009124:Muscle Proteins / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism / Q000648:ultrastructure; D009200:Myocardial Contraction / Q000187:drug effects; D009206:Myocardium / Q000378:metabolism / Q000648:ultrastructure; D020030:Nitric Oxide Donors / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D020033:Protein Isoforms / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D017382:Reactive Oxygen Species; D019163:Reducing Agents / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D002147:Calmodulin; D009124:Muscle Proteins; D020030:Nitric Oxide Donors; D016877:Oxidants; D020033:Protein Isoforms; D017382:Reactive Oxygen Species; D019163:Reducing Agents; D019837:Ryanodine Receptor Calcium Release Channel; D013438:Sulfhydryl Compounds; D003553:Cystine; D006861:Hydrogen Peroxide; D003545:Cysteine; D005033:Ethylmaleimide