How many cysteine residues regulate ryanodine receptor channel activity?
RyRs contain 80-100 cysteine residues per subunit, of which approximately 25% are free for covalent modification, while the remainder are either modified or form intraprotein disulfides. Oxidizing and nitrosylating reagents have several effects on single RyR channel activity, which depend on the type of modifying reagent, the isoform of the RyR, and ligands bound to the channel. We present evidence here for four major classes of functional cysteine residues associated with RyR channels, i.e., two classes with free -SH groups that either activate or inhibit channels when covalently modified and two classes, with endogenous modification, that either inhibit or activate. Single-channel characteristics provide evidence for four discrete responses within the first activating class, two responses within the second inhibiting class and two types of response within the third endogenously modified class. All but one of these changes in channel properties depend on residues located on the cytoplasmic or membrane-associated domains of the RyR; the remaining response is confined to the luminal domain. If it is assumed that each type of response depends on a separate subclass of cysteine residue and that each subclass contains a minimum of one cysteine per subunit, our results suggest that there are at least nine cysteine residues per subunit with functional connections to the gating mechanism of RyR channels. These cysteine residues may be selectively modified under physiological and pathological conditions to regulate Ca2+ release from the sarcoplasmic reticulum and contraction.
Muscle Research Group, John Curtin School of Medical Research, Australian National University, ACT, Canberra. angela.dulhunty@anu.edu.au
A
A
Dulhunty
C
C
Haarmann
D
D
Green
J
J
Hart
D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D003545:Cysteine / Q000502:physiology*; D003553:Cystine / Q000502:physiology; D004220:Disulfides / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D006321:Heart / Q000187:drug effects; D017136:Ion Transport / Q000187:drug effects; D009119:Muscle Contraction / Q000187:drug effects; D009124:Muscle Proteins / Q000737:chemistry* / Q000187:drug effects / Q000502:physiology; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D009200:Myocardial Contraction / Q000187:drug effects; D009206:Myocardium / Q000378:metabolism / Q000648:ultrastructure; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D011487:Protein Conformation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011725:Pyridines / Q000494:pharmacology; D019163:Reducing Agents / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000187:drug effects / Q000502:physiology; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013849:Thimerosal / Q000494:pharmacology
D004220:Disulfides; D009124:Muscle Proteins; D016877:Oxidants; D021122:Protein Subunits; D011725:Pyridines; D019163:Reducing Agents; D019837:Ryanodine Receptor Calcium Release Channel; D013438:Sulfhydryl Compounds; D013849:Thimerosal; C007542:4,4'-dipyridyl disulfide; D003553:Cystine; D004228:Dithionitrobenzoic Acid; D005978:Glutathione; D003545:Cysteine; D004229:Dithiothreitol