Disease relevance of PLN

• Both the native phospholamban and fusion proteins produced extensive cell lysis upon induction of gene expression, but only the native protein underwent spontaneous pentamer formation in E. coli [1].
• A synthetic phospholamban gene has been cloned and expressed in Escherichia coli, producing both native phospholamban and a fusion protein with 81 amino acids of the influenza virus NS1 protein [1].
• In addition, ischemia induced a decrease in the pentameric form of phospholamban and in the content of ryanodine-receptor Ca(2+)-release channel protein [2].
• In contrast, the mRNAs of calsequestrin and phospholamban were unchanged in dogs with either congestive heart failure or norepinephrine infusion [3].
• Evaluation of the phospholamban gene in purebred large-breed dogs with dilated cardiomyopathy [4].

High impact information on PLN

• Nature and site of phospholamban regulation of the Ca2+ pump of sarcoplasmic reticulum [5].
• The calcium pump is activated by cyclic AMP- and calmodulin-dependent phosphorylation of phospholamban, an integral membrane protein of the sarcoplasmic reticulum [5].
• Complete complementary DNA-derived amino acid sequence of canine cardiac phospholamban [6].
• Complementary DNA (cDNA) clones specific for phospholamban of sarcoplasmic reticulum membranes have been isolated from a canine cardiac cDNA library [6].
• Localization of phospholamban in smooth muscle using immunogold electron microscopy [7].

Biological context of PLN

• The inhibitory effects of PLN 1-31 and PLN 8-47 on the ATPase were reversed by cAMP-dependent phosphorylation of the peptides (Ser16) [8].
• An acceleration of E2P decomposition upon PLN phosphorylation may contribute to the increased Vmax(Ca) of Ca uptake at 25 degrees C but not at 37 degrees C, based on measurement of the kinetics of E2P decomposition and steady-state E2P formation from Pi at different temperatures [9].
• Investigators previously, however, have been unsuccessful in demonstrating an effect of PLN phosphorylation or anti-PLN monoclonal antibody (mAb), which is considered to mimic phosphorylation's well-known effect on Km(Ca), on microsomal Ca uptake at the (high) Ca2+ concentrations found intracellularly at peak systole [9].
• Phospholamban in the reconstituted vesicles was phosphorylated with added catalytic subunit of cAMP-dependent protein kinase to almost the same extent as that in original vesicles [10].
• The substrate specificity of the purified phosphatase for cardiac phosphoproteins was determined using troponin I, phospholamban, and highly enriched sarcolemmal and sarcoplasmic reticulum preparations, phosphorylated by the cAMP-dependent protein kinase [11].

Anatomical context of PLN

• Role of leucine 31 of phospholamban in structural and functional interactions with the Ca2+ pump of cardiac sarcoplasmic reticulum [12].
• Leu(31) of PLB, located at the cytoplasmic membrane boundary, is a critical amino acid shown previously to be essential for Ca(2+)-ATPase inhibition [12].
• By contrast, the Ca(2+)-pumping activity of skeletal muscle SR, which lacks endogenous PLN, was unaffected [13].
• METHODS: Ca-uptake and Ca(2+)-ATPase activities in purified phosphorylated and control canine cardiac microsomes, incubated at increasing concentrations of NaOCl or NH2Cl, were related to the extent of PLN phosphorylation by protein kinase A, which was quantitated by PhosphorImager analysis [14].
• From 1 g cells we typically purified 13.5 mg fusion protein with a PLN content of 2.8 mg [15].

Associations of PLN with chemical compounds

• Although L31C-PLB failed to cross-link to any Cys or Lys residue of wild-type SERCA2a, L31C did cross-link with high efficiency to T317C of SERCA2a with use of the homobifunctional sulfhydryl cross-linking reagent, 1,6-bismaleimidohexane [12].
• The ability of two loss-of-function mutants, L31A and L31C, of phospholamban (PLB) to bind to and inhibit the Ca(2+) pump of cardiac sarcoplasmic reticulum (SERCA2a) was investigated using a molecular cross-linking approach [12].
• No comparable protection against similar oxidative damage of the Ca pump is observed when light fast skeletal muscle microsomes, which lack PLN, are incubated under conditions favorable for phosphorylation nor when PLN's inhibition of the cardiac Ca pump is relieved by proteolytic cleavage of its cytoplasmic domain [14].
• The specific binding of tannin reverses the inhibition that PLN exerts on cardiac SR ATPase [16].
• Levels of mRNA encoding beta-myosin heavy chain, mitochondrial creatine kinase, phospholamban, and sarcoplasmic reticulum Ca(2+)-ATPase did not significantly change from baseline, despite development of heart failure [17].

Other interactions of PLN

• 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 [18].

Analytical, diagnostic and therapeutic context of PLN

• Circular dichroism experiments have shown that the peptide had a predominantly random-coil conformation but adopted a higher proportion of secondary structure in the presence of a synthetic 32 amino acid peptide corresponding to the hydrophilic portion of PLN [13].
• Sequence analysis of phospholamban. Identification of phosphorylation sites and two major structural domains [19].
• To investigate these effects in detail, we have purified Ca2+-ATPase from cardiac sarcoplasmic reticulum using affinity chromatography and reconstituted it with purified, recombinant phospholamban [20].
• The presence of phospholamban in microsomal fractions isolated from canine superficial digitalis flexor (89 +/- 3% Type I) and extensor carpi radialis skeletal muscle (14 +/- 6% Type I) was confirmed by immunoblotting [21].
• PLB migrates with a sedimentation coefficient of 4.8 S in sedimentation velocity ultracentrifugation experiments, whereas Cys-to-Ser PLB does not sediment, consistent with a monomeric state [22].

References