STRUCTURE/FUNCTION OF PHOSPHOLAMBAN IN HEART

磷脂在心脏中的结构/功能

• 批准号: 2901175
• 负责人: LARRY R. JONES
• 金额: $ 20.64万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2901175
• 关键词: active sites; affinity labeling; calcium transporting ATPase; cryoelectron microscopy; crystallization; dogs; electron spin resonance spectroscopy; enzyme inhibitors; enzyme reconstitution; fluorescence resonance energy transfer; genetically modified animals; heart contraction; isozymes; laboratory mouse; laboratory rabbit; liposomes; membrane proteins; phospholamban; phosphorylation; protein engineering; protein sequence; protein structure function; sarcoplasmic reticulum; site directed mutagenesis; tissue /cell culture

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.

本研究的长期目标是阐明其机制