The long term goal of this research is to elucidate the mechanism by which phospholamban (PLB) regulates the activity of the Ca pump (SERCA2a isoform) in cardiac sarcoplasmic reticulum (SR). PLB is a pentameric phosphoprotein in cardiac SR which is composed of five identical monomers. Dephosphorylated PLB inhibits the Ca pump and Ca transport by SR, suppressing basal myocardial contractility. Phosphorylation of PLB during beta-adrenergic stimulation of the heart reverses Ca pump inhibition, augmenting contractility. For this renewal period, we will test the novel hypothesis that the PLB monomer is the active species inhibiting the Ca pump in the SR membrane. To test this hypothesis, four Specific Aims are proposed, which will examine PLB structure and function from the purified protein level to the level of the live animal.
In Aim 1, the role of the PLB monomer in SERCA2a regulation will be investigated using co-expression of PLB with SERCA2a in Sf21 insect cells. The goal is to correlate the monomeric propensities of PLB mutants with the degree of SERCA2a inhibition. Taking advantage of this high-level expression system, we will resolve the kinetic step(s) regulated by PLB in the ATPase reaction scheme, and correlate changes in ATPase kinetics with PLB's effect on the rotational mobility of SERCA2a in the membrane.
Aim 2 proposes to identify the sites of PLB:SERCA2a interaction in the membrane. This will be done by co- reconstituting SERCA2a into liposomes along with bioengineered PLB containing covalently attached photoaffinity- and spin-label probes. SERCA2a peptide fragments tagged with PLB photoaffinity probes will be sequenced. Residues of spin-labeled PLB in contact with the Ca pump will be identified by EPR spectroscopy.
Aim 3 examines the dynamic equilibrium between PLB pentamers and monomers. The hypothesis will be tested that phosphorylation of PLB stabilizes monomeric mutants of PLB will be overexpressed in transgenic mouse hearts to assess the effects of the monomer on cardiac performance. Overexpression of superinhibitory PLB monomers in myocardium should yield dominant phenotypes exhibiting strong depression of contractility, conceivably leading to new animal models of heart failure. A total picture of PLB structure and function will emerge from the studies proposed. New insights on catecholamine regulation of the strength of the heartbeat will result.
|Sirenko, Syevda; Maltsev, Victor A; Maltseva, Larissa A et al. (2014) Sarcoplasmic reticulum Ca2+ cycling protein phosphorylation in a physiologic Ca2+ milieu unleashes a high-power, rhythmic Ca2+ clock in ventricular myocytes: relevance to arrhythmias and bio-pacemaker design. J Mol Cell Cardiol 66:106-15|
|Akin, Brandy L; Jones, Larry R (2012) Characterizing phospholamban to sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a) protein binding interactions in human cardiac sarcoplasmic reticulum vesicles using chemical cross-linking. J Biol Chem 287:7582-93|
|Chen, Zhenhui; Akin, Brandy L; Jones, Larry R (2010) Ca2+ binding to site I of the cardiac Ca2+ pump is sufficient to dissociate phospholamban. J Biol Chem 285:3253-60|
|Akin, Brandy L; Chen, Zhenhui; Jones, Larry R (2010) Superinhibitory phospholamban mutants compete with Ca2+ for binding to SERCA2a by stabilizing a unique nucleotide-dependent conformational state. J Biol Chem 285:28540-52|
|Chopra, Nagesh; Yang, Tao; Asghari, Parisa et al. (2009) Ablation of triadin causes loss of cardiac Ca2+ release units, impaired excitation-contraction coupling, and cardiac arrhythmias. Proc Natl Acad Sci U S A 106:7636-41|
|Chen, Zhenhui; Akin, Brandy L; Jones, Larry R (2007) Mechanism of reversal of phospholamban inhibition of the cardiac Ca2+-ATPase by protein kinase A and by anti-phospholamban monoclonal antibody 2D12. J Biol Chem 282:20968-76|
|Chen, Zhenhui; Akin, Brandy L; Stokes, David L et al. (2006) Cross-linking of C-terminal residues of phospholamban to the Ca2+ pump of cardiac sarcoplasmic reticulum to probe spatial and functional interactions within the transmembrane domain. J Biol Chem 281:14163-72|
|Chen, Zhenhui; Stokes, David L; Jones, Larry R (2005) Role of leucine 31 of phospholamban in structural and functional interactions with the Ca2+ pump of cardiac sarcoplasmic reticulum. J Biol Chem 280:10530-9|
|Waggoner, Jason R; Huffman, Jamie; Griffith, Brian N et al. (2004) Improved expression and characterization of Ca2+-ATPase and phospholamban in High-Five cells. Protein Expr Purif 34:56-67|
|Chen, Zhenhui; Stokes, David L; Rice, William J et al. (2003) Spatial and dynamic interactions between phospholamban and the canine cardiac Ca2+ pump revealed with use of heterobifunctional cross-linking agents. J Biol Chem 278:48348-56|
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