title: Identification of hyperreactive cysteines within ryanodine receptor type 1 by mass spectrometry.
abstract: The skeletal-type ryanodine receptor (RyR1) undergoes covalent adduction by nitric oxide (NO), redox-induced shifts in cation regulation, and non-covalent interactions driven by the transmembrane redox potential that enable redox sensing. Tight redox regulation of RyR1 is thought to be primarily mediated through highly reactive (hyperreactive) cysteines. Of the 100 cysteines per subunit of RyR1, approximately 25-50 are reduced, with 6-8 considered hyperreactive. Thus far, only Cys-3635, which undergoes selective adduction by NO, has been identified. In this report, RyR1-enriched junctional sarcoplasmic reticulum is labeled with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM, 1 pmol/microg of protein) in the presence of 10 mm Mg(2+), conditions previously shown to selectively label hyperreactive sulfhydryls and eliminate redox sensing. The CPM-adducted RyR1 is separated by gel electrophoresis and subjected to in-gel tryptic digestion. Isolation of CPM-adducted peptides is achieved by analytical and microbore high-performance liquid chromatography utilizing fluorescence and UV detection. Subsequent analysis using two direct and one tandem mass spectrometry methods results in peptide masses and sequence data that, compared with the known primary sequence of RyR1, enable unequivocal identification of CPM-adducted cysteines. This work is the first to directly identify seven hyperreactive cysteines: 1040, 1303, 2436, 2565, 2606, 2611, and 3635 of RyR1. In addition to Cys-3635, the nitrosylation site, six additional cysteines may contribute toward redox regulation of the RyR1 complex.
authors.affiliation: School of Veterinary Medicine, Department of Molecular Biosciences, University of California, Davis, CA 95616, USA.
authors.forename: Andrew A
authors.initials: AA
authors.lastname: Voss
authors.forename: Jozsef
authors.initials: J
authors.lastname: Lango
authors.forename: Michael
authors.initials: M
authors.lastname: Ernst-Russell
authors.forename: Dexter
authors.initials: D
authors.lastname: Morin
authors.forename: Isaac N
authors.initials: IN
authors.lastname: Pessah
mesh_terms: D000818:Animals; D002412:Cations; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003374:Coumarins / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000494:pharmacology; D008274:Magnesium / Q000737:chemistry; D008297:Male; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D009569:Nitric Oxide / Q000378:metabolism; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D014466:Ultraviolet Rays
chemical_list: D002412:Cations; D003374:Coumarins; D005456:Fluorescent Dyes; D010455:Peptides; D019837:Ryanodine Receptor Calcium Release Channel; D009569:Nitric Oxide; C030860:N-(4-(7-diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide; D014357:Trypsin; D008274:Magnesium; D003545:Cysteine