STRUCTURE-FUNCTION OF PHOSPHOLAMBAN IN HEART

Project: Research project

Description

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.
StatusFinished
Effective start/end date1/1/936/30/14

Funding

  • National Institutes of Health: $518,243.00
  • National Institutes of Health: $396,609.00
  • National Institutes of Health: $533,420.00
  • National Institutes of Health: $386,491.00
  • National Institutes of Health: $538,809.00
  • National Institutes of Health: $466,800.00
  • National Institutes of Health: $440,006.00
  • National Institutes of Health: $137,438.00
  • National Institutes of Health: $533,420.00
  • National Institutes of Health: $453,208.00
  • National Institutes of Health: $206,419.00
  • National Institutes of Health: $128,593.00
  • National Institutes of Health: $533,790.00
  • National Institutes of Health: $469,571.00
  • National Institutes of Health: $469,509.00
  • National Institutes of Health: $376,669.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $231,706.00
  • National Institutes of Health

Fingerprint

Sarcoplasmic Reticulum
Nucleotides
phospholamban
Protein Kinases
Binding Sites
Adenosine Triphosphate
Thapsigargin
Active Biological Transport
Drug Design
Phosphorylation
Protein Isoforms
Heart Failure
Proteins
Phosphoproteins
Research
Adrenergic Agents
Insects
Membranes
Adenosine Triphosphatases
Amino Acids

ASJC

  • Medicine(all)