Disease relevance of PLN

• This review focuses on advances in the understanding of molecular regulation of SERCA2a by PLN and SLN, and their implications for cardiac hypertrophy and failure in vivo [1].
• Studies with knockout and transgenic mice show that gain of inhibitory function of phospholamban alters cardiac contractility and could be a causal feature in some cardiomyopathies [2].
• Briefly, recombinant wild-type PLB and SLN were over-produced in Escherichia coli as maltose binding protein fusion proteins [3].
• METHODS AND RESULTS: Mutants of PLB (Adv/mPLB) or antisense RNA of PLB (Adv/asPLB) was expressed in cardiac myocytes by recombinant adenovirus, and their effects on SERCA2 activity and myocyte contractility were studied [4].
• METHODS: We determined mRNA and/or protein levels for PLB and SERCA2 in the same left ventricular tissue of patients undergoing heart transplantation due to idiopathic dilated cardiomyopathy (IDC) or ischemic cardiomyopathy (ICM) in comparison to left ventricular tissue from nonfailing human hearts (NF) [5].

High impact information on PLN

• A comprehensive discussion is presented of advances in understanding the structure and function of phospholamban (PLB), the principal regulator of the Ca2+-ATPase of cardiac sarcoplasmic reticulum [6].
• The oligomerization domain of COMP has marked similarities with proposed models of the pentameric transmembrane ion channels in phospholamban and the acetylcholine receptor [7].
• Impairments in blood circulation that accompany heart failure can be traced, in part, to alterations in the activity of the sarcoplasmic reticulum Ca2+ pump that are induced by its interactions with phospholamban, a reversible inhibitor [8].
• The phosphorylation of phospholamban is initiated by beta-adrenergic stimulation, identifying phospholamban as an important component in the stimulation of cardiac activity by beta-agonists [9].
• The structural properties of phospholamban have been studied by mutagenesis, modeling, and spectroscopy, resulting in a new view of the organization of this key molecule in membranes [9].

Chemical compound and disease context of PLN

• Regulation of sarco(endo)plasmic reticulum Ca2+ adenosine triphosphatase by phospholamban and sarcolipin: implication for cardiac hypertrophy and failure [1].
• Increased capacity of SR to take up Ca(2+) can arise from increased density of active SR pumping sites or in slow-twitch fibers from phosphorylation of phospholamban, whereas impaired coupling between ATP hydrolysis and Ca(2+) transport into the SR or intracellular acidosis reduces SR Ca(2+) pump activity [10].
• Subjects ingested an energy bar during each trial; on one cold trial (TYR) the bar contained tyrosine (300 mg/kg body weight), and on the other cold trial (PLB) and on CON the bar contained no tyrosine [11].

Biological context of PLN

• Basal PLN phosphorylation was reduced [12].
• Site-directed mutagenesis was carried out in the cytoplasmic loop (L67) between M6 and M7 in SERCA1a to detect other SERCA-PLN binding sites [13].
• SLN and PLN genes resemble each other in having two small exons, with their entire coding sequences lying in exon 2 and a large intron separating the two segments [14].
• Recent studies using genetically engineered animals revealed the implication of PLN for the development of cardiomyopathic phenotypes [1].
• While PLB protein expression was not different, SERCA2a protein expression as well as SERCA2a/PLB ratio was significantly higher in group A vs. group B. Transfection of SERCA1 increased shortening amplitude and enhanced relaxation kinetics in failing human myocytes [15].

Anatomical context of PLN

• Phospholamban (PLN), a regulator of sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs), interacts with both the cytosolic N domain and transmembrane helices M2, M4, M6, and M9 of SERCA [16].
• Isoproterenol largely restored contractility in papillary muscle and stimulated PLN phosphorylation to wild-type levels in intact hearts [12].
• Like SERCA1, SLN is highly expressed in rabbit fast-twitch skeletal muscle, but it is expressed to a lower extent in slow-twitch muscle and to an even lower extent in cardiac muscle, where SERCA2a and PLN are highly expressed [14].
• Structural and functional properties of SLN were analyzed and compared with the homologous region of human phospholamban (PLB) comprising residues Ala(24)-Leu(52) (PLB-(24-52)), the regulatory protein of the cardiac sarcoplasmic reticulum Ca-ATPase [17].
• Phospholamban was first identified as a phosphoprotein in cardiac myocytes [18].

Associations of PLN with chemical compounds

• Modeling showed that the SLN/SERCA1a complex closely resembles the PLN/SERCA1a complex, but with the luminal end of SLN extending to the loop connecting M1 and M2, where Tyr-29 and Tyr-31 interact with aromatic residues in SERCA1a [16].
• Elevated Ca(2+) dissociates both PLN and NF-SLN from their complexes with both SERCA1a and SERCA2a, but NF-SLN induced resistance to Ca(2+) dissociation of the PLN.SERCA complex [19].
• The further addition of vanadate and thapsigargin, both of which stabilize the E(2) conformation, did not diminish binding of PLN to SERCA [20].
• We recently solved the three-dimensional structure of chemically synthesized, unphosphorylated, monomeric PLN (C41F) by high-resolution nuclear magnetic resonance spectroscopy in chloroform/methanol [21].
• The amino acid sequence of PLN is highly conserved, and although all species contain asparagine (Asn), human PLN is unique in containing lysine (Lys) at amino acid 27 [22].

Physical interactions of PLN

• Sarcolipin regulates sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) by binding to transmembrane helices alone or in association with phospholamban [16].
• Mutagenesis studies of phospholamban also demonstrated that the hydrophilic, cytoplasmic region of phospholamban contains a potential binding site for SERCA 2 [18].
• A leucine zipper stabilizes the pentameric membrane domain of phospholamban and forms a coiled-coil pore structure [23].

Regulatory relationships of PLN

• Sarcolipin inhibits polymerization of phospholamban to induce superinhibition of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) [19].
• Phospholamban (PLB) inhibits SERCA2 activity and is therefore a potential target to improve the cardiac performance in heart failure [4].
• Therefore, in human myocardium, the Ca(2+)-sensitivity but not the Vmax of SERCA 2a is regulated by cAMP-dependent phosphorylation of phospholamban at position serine-16 [24].
• When the second site of phospholamban is phosphorylated with cAMP-dependent protein kinase both Ca2+ transport and ATPase are stimulated and phospholamban becomes inaccessible to DOC solubilization and trypsin [25].

Other interactions of PLN

• These homologous proteins differ at their N and C termini: the C-terminal Met-Leu-Leu in PLN is replaced by Arg-Ser-Tyr-Gln-Tyr in SLN [26].
• The reduced phosphorylation state of PLB may lead to decreased Ca2+ sensitivity of SERCA2 in failing human myocardium [27].
• We detected the formation of a Ca(2+)-dependent complex of S100A1 with SERCA2a and PLB in the human myocardium [28].
• Coexpression of the transmembrane sequence of phospholamban (Met-PLN28-52) with SERCA1a, SERCA2a, and SERCA3 inhibited Ca2+ transport by lowering apparent Ca2+ affinity [29].
• Replacing Asn34 of PLB in the Leu9 peptide resulted in superinhibition of SERCA [30].

Analytical, diagnostic and therapeutic context of PLN

• Protein expression of SERCA2a and phospholamban (Western blot) was assessed in a subset of failing trabeculae [15].
• In related studies, those PLN mutants that gained inhibitory function also increased levels of co-immunoprecipitation of wild-type SERCA1a and those that lost inhibitory function also reduced association, correlating functional interaction sites with physical interaction sites [31].
• PLB mRNA expression was quantified in human cardiac preparations by Northern blot analysis [32].
• Protein expression of PLB and the sarcoplasmic Ca(2+)-ATPase (SERCA) was measured in homogenates by quantitative immunoblotting using specific antibodies [32].
• Human GM and PLB have been produced in an in vitro transcription/translation system and used for co-immunoprecipitation and biosensor experiments [33].

References