title: Ryanodine receptor-ankyrin interaction regulates internal Ca2+ release in mouse T-lymphoma cells.
abstract: In this study, we have identified and partially characterized a mouse T-lymphoma ryanodine receptor on a unique type of internal vesicle which bands at the relatively light density of 1.07 g/ml. Analysis of the binding of [3H]ryanodine to these internal vesicles reveals the presence of a single, low affinity binding site with a dissociation constant (Kd) of 200 nM. The second messenger, cyclic ADP-ribose, was found to increase the binding affinity of [3H]ryanodine to its vesicle receptor at least 5-fold (Kd approximately 40 nM). In addition, cADP-ribose appears to be a potent activator of internal Ca2+ release in T-lymphoma cells and is capable of overriding ryanodine-mediated inhibition of internal Ca2+ release. Immunoblot analyses using a monoclonal mouse antiryanodine receptor antibody indicate that mouse T-lymphoma cells contain a 500-kDa polypeptide similar to the ryanodine receptor found in skeletal muscle, cardiac muscle, and brain tissues. Double immunofluorescence staining and laser confocal microscopic analysis show that the ryanodine receptor is preferentially accumulated underneath surface receptor-capped structures. T-lymphoma ryanodine receptor was isolated (with an apparent sedimentation coefficient of 30 S) by extraction of the light density vesicles with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) in 1 M NaCl followed by sucrose gradient centrifugation. Further analysis indicates that specific, high affinity binding occurs between ankyrin and this 30 S lymphoma ryanodine receptor (Kd = 0.075 nM). Most importantly, the binding of ankyrin to the light density vesicles significantly blocks ryanodine binding and ryanodine-mediated inhibition of internal Ca2+ release. These findings suggest that the cytoskeleton plays a pivotal role in the regulation of ryanodine receptor-mediated internal Ca2+ release during lymphocyte activation.
authors.affiliation: Department of Cell Biology and Anatomy, University of Miami Medical School, Florida 33101, USA.
authors.forename: L Y
authors.initials: LY
authors.lastname: Bourguignon
authors.forename: A
authors.initials: A
authors.lastname: Chu
authors.forename: H
authors.initials: H
authors.lastname: Jin
authors.forename: N R
authors.initials: NR
authors.lastname: Brandt
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D017487:Ankyrins / Q000378:metabolism*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D036563:Cyclic ADP-Ribose; D006801:Humans; D016399:Lymphoma, T-Cell / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D009124:Muscle Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel; D013347:Subcellular Fractions / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D017487:Ankyrins; D015220:Calcium Channels; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D036563:Cyclic ADP-Ribose; D012433:Ryanodine; D000246:Adenosine Diphosphate Ribose; D002118:Calcium