Immunoelectron microscopical localization of phospholamban in adult canine ventricular muscle.

Abstract

Abstract. The subcellular distribution of phospholamban in adult canine ventricular myocardial cells was determined by the indirect immunogold-labeling technique. The results presented suggest that phospholamban, like the Ca2+-ATPase, is uniformly distributed in the network sarcoplasmic reticulum but absent from the junctional portion of the junctional sarcoplasmic reticulum. Unlike the Ca2÷-ATPase, but like cardiac calsequestrin, phospholamban also appears to be present in the corbular sarcoplasmic reticulum. Comparison of the relative distribution of phospholamban immunolabeling in the sarcoplasmic reticulum with that of the sarcolemma showed that the density of phospholamban in the network sarcoplasmic reticulum was •35-fold higher than that of the cytoplasmic side of the sarcolemma, which in turn was found to be threeto fourfold higher than the density of the background labeling. However, a majority of the specific phospholamban labeling within 30 nm of the cytoplasmic side of the sarcolemma was clustered and present over the sarcoplasmic reticulum in the subsarcolemmal region of the myocardial cells, suggesting that phospholamban is confined to the junctional regions between the sarcolemma and the sarcoplasmic reticulum, but absent from the nonjunctional portion of the sarcolemma. Although the resolution of the immunogoldlabeling technique used (60 nm) does not permit one to determine whether the specific labeling within 30 nm of the cytoplasmic side of the sarcolemma is associated with the sarcolemma and/or the junctional sarcoplasmic reticulum, it is likely that the low amount of labeling in this region represents phospholamban associated with sarcoplasmic reticulum. These results suggest that phospholamban is absent from the sarcolemma and confined to the sarcoplasmic reticulum in cardiac muscle.

Purification, Primary Structure, and Immunological Characterization of the 26-kDa Calsequestrin Binding Protein (Junctin) from Cardiac Junctional Sarcoplasmic Reticulum

“…Monoclonal antibody 2A7-A1 was produced to the Ca 2ϩ -ATPase in canine cardiac SR vesicles (37). Immunofluorescence Labeling-Immunofluorescence labeling with affinity-purified rabbit antibodies to the intact 26-kDa CSQ binding protein was performed as described previously (38,39). To localize the fast (type II) isoform of the SR Ca 2ϩ -ATPase, mouse monoclonal antibody IIH11 was used (40).…”

Co-reconstitution of Phospholamban Mutants with the Ca-ATPase Reveals Dependence of Inhibitory Function on Phospholamban Structure

“…Upon phosphorylation of PLB by cAMP-dependent protein kinase A or Ca 2ϩ / calmodulin-dependent protein kinase II, this inhibition is relieved (4, 6, 10). Although PLB is not present in fast-twitch muscle (8,11,12), it has been shown to be capable of regulating the fast-twitch Ca-ATPase isoform (SERCA1) in both reconstitution (13-15) and coexpression (16) experiments.…”

“…Upon phosphorylation of PLB by cAMP-dependent protein kinase A or Ca 2ϩ / calmodulin-dependent protein kinase II, this inhibition is relieved (4, 6, 10). Although PLB is not present in fast-twitch muscle (8,11,12), it has been shown to be capable of regulating the fast-twitch Ca-ATPase isoform (SERCA1) in both reconstitution (13-15) and coexpression (16) experiments.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and low angle laser light scattering in SDS solutions (17, 18) showed that PLB is predominantly a homopentamer in equilibrium with a small fraction as monomer. Systematic replacement of single hydrophobic amino acid residues, Leu or Ile, by Ala in the membrane-spanning region transformed PLB from pentameric to monomeric form on SDS-PAGE (19).…”

Biochemical Characterization and Molecular Cloning of Cardiac Triadin

“…Immunofluorescence Labeling of Papillary Muscle Cryosections-Dissection, cryofixation, and cryosectioning (6 -8 m) of atrial and ventricular muscle tissues of anesthetized rabbits and dogs were performed as described previously (24,26). Indirect immunofluorescence labeling of cardiac muscle was carried out as described previously (26).…”

“…Indirect immunofluorescence labeling of cardiac muscle was carried out as described previously (26). Affinitypurified GP58 antibodies to H-triadin were used for primary labeling.…”