Ryanodine Receptor-Ankyrin Interaction Regulates Internal Ca Release in Mouse T-lymphoma Cells

doi:10.1074/jbc.270.30.17917

Ryanodine Receptor-Ankyrin Interaction Regulates Internal Ca Release in Mouse T-lymphoma Cells (*)

From the (1) Department of Cell Biology and Anatomy and the
(2) Department of Molecular Pharmacology, University of Miami Medical School, Miami, Florida 33101

^§ To whom correspondence and reprint requests should be addressed:
Dept. of Cell Biology and Anatomy, University of Miami Medical School, 1600 N. W. 10th Ave., Miami, FL 33101.
Tel: 305-243-6985; Fax: 305-545-7166.

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 (K) 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 (K ≈ 40 nM). In addition, cADP-ribose appears to be a potent activator of internal Ca release in T-lymphoma cells and is capable of overriding ryanodine-mediated inhibition of internal Ca release.

Immunoblot analyses using a monoclonal mouse anti-ryanodine 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 (K = 0.075 nM). Most importantly, the binding of ankyrin to the light density vesicles significantly blocks ryanodine binding and ryanodine-mediated inhibition of internal Ca release. These findings suggest that the cytoskeleton plays a pivotal role in the regulation of ryanodine receptor-mediated internal Ca release during lymphocyte activation.

Footnotes

↵* This work was supported by National Institutes of Health Grants GM 36353 and CA 66163, a Department of Defense grant, and American Heart Association-Florida Affiliate grants. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

RYR

ryanodine receptor

IP₃

inositol 1,4,5-trisphosphate

cADPR

cADP-ribose

PMSF

phenylmethylsulfonyl fluoride

ConA

concanavalin A

PAGE

polyacrylamide gel electrophoresis

MOPS

4-morpholinepropanesulfonic acid

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
- Received January 13, 1995.
- Revision received May 24, 1995.