RYANODINE RECEPTOR-ANKYRIN INTERACTION REGULATES INTERNAL CA2-LYMPHOMA CELLS( RELEASE IN MOUSE T)

In this study, we have identified and partially characterized a mouse T-lymphoma ryanodine receptor on a unique type of internal vesicle whi ch bands at the relatively light density of 1.07 g/ml. Analysis of the binding of [H-3]ryanodine to these internal vesicles reveals the pres ence of a single, low affinity binding site with a dissociation consta nt (K-d) of 200 nM. The second messenger, cyclic ADP-ribose, was found to increase the binding affinity of [H-3]ryanodine to its vesicle rec eptor at least 5-fold (K-d approximate to 40 nM). In addition, cADP-ri bose appears to be a potent activator of internal Ca2+ release in T-ly mphoma cells and is capable of overriding ryanodine-mediated inhibitio n of internal Ca2+ release. Immunoblot analyses using a monoclonal mou se anti-ryanodine receptor antibody indicate that mouse T-lymphoma cel ls contain a 500-kDa polypeptide similar to the ryanodine receptor fou nd in skeletal muscle, cardiac muscle, and brain tissues. Double immun ofluorescence staining and laser confocal microscopic analysis show th at the ryanodine receptor is preferentially accumulated underneath sur face receptor-capped structures. T-lymphoma ryanodine receptor was iso lated (with an apparent sedimentation coefficient of 30 S) by extracti on of the light density vesicles with cholamidopropyl)dimethylammonio] -1-propanesulfonic acid (CHAPS) in 1 M NaCl followed by sucrose gradie nt centrifugation. Further analysis indicates that specific, high affi nity binding occurs between ankyrin and this 30 S lymphoma ryanodine r eceptor (K-d = 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.