title: Calsequestrin is an inhibitor of skeletal muscle ryanodine receptor calcium release channels.
abstract: We provide novel evidence that the sarcoplasmic reticulum calcium binding protein, calsequestrin, inhibits native ryanodine receptor calcium release channel activity. Calsequestrin dissociation from junctional face membrane was achieved by increasing luminal (trans) ionic strength from 250 to 500 mM with CsCl or by exposing the luminal side of ryanodine receptors to high [Ca(2+)] (13 mM) and dissociation was confirmed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Calsequestrin dissociation caused a 10-fold increase in the duration of ryanodine receptor channel opening in lipid bilayers. Adding calsequestrin back to the luminal side of the channel after dissociation reversed this increased activity. In addition, an anticalsequestrin antibody added to the luminal solution reduced ryanodine receptor activity before, but not after, calsequestrin dissociation. A population of ryanodine receptors (approximately 35%) may have initially lacked calsequestrin, because their activity was high and was unaffected by increasing ionic strength or by anticalsequestrin antibody: their activity fell when purified calsequestrin was added and they then responded to antibody. In contrast to native ryanodine receptors, purified channels, depleted of triadin and calsequestrin, were not inhibited by calsequestrin. We suggest that calsequestrin reduces ryanodine receptor activity by binding to a coprotein, possibly to the luminal domain of triadin.
authors.affiliation: Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra, ACT 0200, Australia.
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Beard
authors.forename: Magdalena M
authors.initials: MM
authors.lastname: Sakowska
authors.forename: Angela F
authors.initials: AF
authors.lastname: Dulhunty
authors.forename: Derek R
authors.initials: DR
authors.lastname: Laver
mesh_terms: D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002122:Calcium Chloride / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002155:Calsequestrin / Q000276:immunology / Q000494:pharmacology / Q000502:physiology*; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D008051:Lipid Bilayers; D018482:Muscle, Skeletal / Q000502:physiology*; D010714:Phosphatidylethanolamines; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects / Q000502:physiology*; D012519:Sarcoplasmic Reticulum / Q000502:physiology
chemical_list: D000906:Antibodies; D002155:Calsequestrin; D008051:Lipid Bilayers; D010714:Phosphatidylethanolamines; D019837:Ryanodine Receptor Calcium Release Channel; D004533:Egtazic Acid; D000252:Calcium-Transporting ATPases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002122:Calcium Chloride