Abstract. The subcellular distribution of sarcolemmal dihydropyridine receptor (DHPR) and sarcoplasmic reticular triadin and Ca 2÷ release channel/ryanodine receptor (RyR) was determined in adult rabbit ventricle and atrium by double labeling immunofluorescence and laser scanning confocal microscopy. In ventricular muscle cells the immunostaining was observed primarily as transversely oriented punctate bands spaced at approximately 2-/zm intervals along the whole length of the muscle fibers. Image analysis demonstrated a virtually complete overlap of the staining patterns of the three proteins, suggesting their close association at or near dyadic couplings that are formed where the sarcoplasmic reticulum (SR) is apposed to the surface membrane or its infoldings, the transverse (T-) tubules. In rabbit atrial cells, which lack an extensive T-tubular system, DHPR-specific staining was observed to form discrete spots along the sarcolemma but was absent from the interior of the fibers. In atrium, punctate triadin-and RyR-specific staining was also observed as spots at the cell periphery and image analysis indicated that the three proteins were co-localized at, or just below, the sarcolemma. In addition, in the atrial cells triadin-and RyR-specific staining was observed to form transverse bands in the interior cytoplasm at regularly spaced intervals of approximately 2 #m. Electron microscopy suggested that this cytoplasmic staining was occurring in regions where substantial amounts of extended junctional SR were present. These data indicate that the DHPR codistributes with triadin and the RyR in rabbit ventricle and atrium, and furthermore suggest that some of the SR Ca 2+ release channels in atrium may be activated in the absence of a close association with the DHPR. IN striated muscle, depolarization of the sarcolemma and transverse (T-) t tubular network induces release of Ca 2÷ from internal stores in the sarcoplasmic reticulum (SR) by a process commonly referred to as excitation-contraction (EC) coupling. In skeletal muscle it is generally accepted that the electrical signal is transduced to a release of Ca 2+ at specialized triad junctions formed between a central T-tubule flanked by two elements of closely apposed junctional SR (jSR). The junctional T-tubules contain DHPR which act as voltage sensors (36,45,(56)(57)(58) 1. Abbreviations used in this paper: DHP, dihydropyridine; DHPR, DHP receptor; jSR, junctional sarcoplasmic reticulum; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; T-tubule, transverse tubule; TC, terminal cisternae.rows of "feet" (23) that have been identified as ryanodine receptors (RyR), the SR Ca 2÷ release channels (35,55,61). The exact mechanism of skeletal muscle EC coupling is not yet completely understood; however, it is thought that the release of Ca 2÷ from the SR is a depolarization-induced mechanism without the necessity of Ca 2÷ flow (46). In contrast, excitation contraction coupling in cardiac muscle requires an influx of Ca z÷ through L-type Ca ~÷ channels (the cardiac i...

A monoclonal antibody, GE 4.90, has been produced following immunization of mice with the 95-kDa protein (triadin) of terminal cisternae of rabbit fast skeletal muscle isolated in nondenaturing detergent. The antibody binds to a protein of Mr95K in Western blots of microsomal vesicles electrophoresed in the presence of mercaptoethanol. The greatest intensity of the immunoblot reaction is to enriched terminal cisternae vesicles while little binding is seen to longitudinal reticulum and transverse tubules. The content of antigen in different microsomal subfractions has been estimated by immunoassay: terminal cisternae/triads contain 5.6 micrograms/mg of protein while heavy terminal cisternae contain 32 micrograms/mg. The molar content of triadin in vesicles is approximately the same as that of the ryanodine receptor. When Western blots of gels of terminal cisternae are run in nonreducing conditions, little protein of Mr95K is visible. A number of bands, however, forming a ladder of higher molecular weight are discerned, indicating that the 95-kDa protein forms a disulfide-linked homopolymer. A biotinylated aromatic disulfide reagent (biotin-HPDP) labels the 95-kDa protein, the junctional foot protein, and the Mr 106K protein described by others as a Ca(2+)-release channel (SG 106). This latter protein migrates in gel electrophoresis under nonreducing conditions at a molecular weight different from that of the 95-kDa protein. We did not detect any alteration of binding of the 95-kDa protein to the dihydropyridine receptor or junctional foot protein dependent on the state of oxidation of cysteine residues of either triadin or receptor protein used as the overlay probe.

Isolated triadic proteins were employed to investigate the molecular architecture of the triad junction in skeletal muscle. Immunoaffinity-purified junctional foot protein (JFP), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), aldolase and partially purified dihydropyridine (DHP) receptor were employed to probe protein-protein interactions using affinity chromatography, protein overlay and crosslinking techniques. The JFP, an integral protein of the sarcoplasmic reticulum (SR) preferentially binds to GAPDH and aldolase, peripheral proteins of the transverse (T)-tubule. No direct binding of JFP to the DHP receptor was detected. The interactions of JFP with GAPDH and aldolase appear to be specific since other glycolytic enzymes associated with membranes do not bind to the JFP. The DHP receptor, an integral protein of the T-tubule, also binds GAPDH and aldolase. A ternary complex between the JFP and the DHP receptor can be formed in the presence of GAPDH. In addition, the DHP receptor binds to a previously undetected Mr 95 K protein which is distinct from the SR Ca2+ pump and phosphorylase b. The Mr 95 K protein is an integral protein of the junctional domain of the SR terminal cisternae. It is also present in the newly identified "strong triads" (accompanying paper). From these findings, we propose a new model for the triad junction.

The isolated dihydropyridine receptor and junctional foot protein were employed as protein ligands in overlay experiments to investigate the mode of interaction of these two proteins. As previously demonstrated by Brandt et al. [Brandt et al. (1990) J. Membr. Biol. 113, 237-251], the DHP receptor directly binds to an intrinsic terminal cisterna protein of Mr 95,000 (95-kDa protein). The junctional foot protein also binds to an Mr 95,000 protein showing similar organelle distribution to the 95-kDa protein which binds to the dihydropyridine receptor. The 95-kDa protein which binds to the dihydropyridine receptor was isolated to over 85% purity employing sequential column chromatography. Junctional foot protein and dihydropyridine receptor overlays of the column fractions at successive stages of isolation show an identical pattern of distribution, indicating that both probes bind to the same protein. When CHAPS-solubilized terminal cisterna/triads were passed through Sepharose with attached 95-kDa protein, the junctional foot protein was specifically retained, as evidenced by ryanodine binding. The junctional foot protein was incompletely released by 1 M NaCl. The alpha 1 subunit but not the beta subunit of the dihydropyridine receptor was also specifically retained, as evidenced by immunoblotting employing dihydropyridine receptor subunit-specific antibodies. A 170-kDa Stains-all blue staining protein, which appears to be bound to the luminal side of the terminal cisterna, was also retained on the 95-kDa protein column. From these findings, a model for the triad junction is proposed.

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.