Phospholamban (PLB) is the principal membrane protein of the heart phosphorylated in response to P-adrenergic stimulation. The protein is localized to cardiac sarcoplasmic reticulum (SR), where it exists as a pentamer of small, tightly associated, 52-amino acid subunits. When dephosphorylated, PLB inhibits the Ca pump and Ca transport by cardiac SR. Phosphorylation of PLB at Ser16 and Thr17 disinhibits the Ca pump, stimulates active Ca sequestration, and increases the rate of myocardial relaxation during beta adrenergic activation. Precisely how this detailed regulation occurs is currently unknown. In this application we propose structure/function studies on PLB to define a molecular mechanism of action.
Aim 1 will address protein structure in native SR vesicles. The membrane protein topology, pentameric organization, and molar stoichiometry with the Ca pump will be determined. These studies will lay the framework for expression studies correlating protein structure with function.
In Aim 2, PLB will be expressed in atrial tumor cells, which contain Ca pumps, but no PLB. Both wild-type and mutated subunits will be expressed. Using this cellular reconstitution system, we will determine which amino acids stabilize the pentamer, and if the pentameric structure is required for Ca pump regulation. Whether the Ca channel activity of PLB is involved in this process will be investigated, and the mechanism of regulation of active Ca transport by the phosphorylation sites will be determined.
In Aim 3, mg quantities of PLB will be expressed and purified from insect cells. The goal here is to produce sufficient material for detailed biochemical studies, including protein crystallization to determine the three dimensional structure. Co- expression of PLB with the Ca pump in insect cells will also be attempted.
In Aim 4, expression of wild-type and mutant PLB subunits will be targeted to mouse atrium and ventricle in transgenic animals. By overexpressing wild-type PLB, the role of the PLB/Ca pump stoichiometry in controlling the rate of myocardial relaxation will be assessed. By expressing mutant PLB subunits in mouse hearts, we hope to identify transdominant mutations that disrupt the function of the wild- type pentamer. Mutations that destabilize the pentamer and/or alter the phosphorylation sites will be investigated. Thus, the studies will address the cellular and molecular physiology of PLB, from the purified protein level to the whole animal. By correlating experimental results from Aims 1-4 a complete picture of PLB structure and function will emerge. The results will be important for a precise understanding of catecholamine regulation of the heart.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL049428-01
Application #
3368546
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1993-01-01
Project End
1996-12-31
Budget Start
1993-01-01
Budget End
1993-12-31
Support Year
1
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Type
Schools of Medicine
DUNS #
005436803
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
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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