Molecular insights into the disease-causing mechanisms of human phospholamban mutations

  • Author / Creator
    Ceholski, Delaine K
  • The movement of calcium across sarcoplasmic reticulum (SR) membranes is essential in the contraction-relaxation cycle of muscle. An influx of calcium into the muscle cell from the SR triggers muscle contraction and its removal by the sarco(endo)plasmic reticulum calcium ATPase (SERCA) causes muscle relaxation. Phospholamban (PLN) reversibly regulates SERCA by inhibiting its apparent calcium affinity and inhibition is reversed by phosphorylation of PLN by protein kinase A (PKA). The role of SERCA in heart disease has been underscored by the identification of hereditary mutations in the cytoplasmic domain of PLN that have been linked to heart disease: Arg9-to-Cys (R9C), Arg9-to-Leu (R9L), Arg9-to-His (R9H), and deletion of Arg14 (R14del). This thesis aims to provide functional insight into the disease-causing mechanisms of these mutations. We wanted to examine how can mutations in the cytoplasmic domain of PLN cause such severe phenotypes when it is well documented that most of the inhibitory capacity of PLN comes from the transmembrane domain. Using alanine-scanning followed by more selective mutagenesis of the cytoplasmic domain of PLN, we were able to reveal mechanistic insight into how these hereditary mutations alter regulation of SERCA. R14del PLN resulted in constitutive inhibition of SERCA caused by lack of response to β-adrenergic stimulation. While hydrophobic mutation of Arg9, including R9C and R9L, eliminated both SERCA inhibition and PLN phosphorylation, an aromatic substitution (R9H) selectively disrupted phosphorylation. It has been hypothesized that R9C PLN has an altered oligomeric state, which contributes to its disease phenotype. Using a monomeric variant of PLN, we examined the role of PLN oligomerization in disease-causing and -mimicking mutations. We can conclude that the role of Arg9 in PLN is complex – it is not only important for inhibition of SERCA and efficient phosphorylation of the PLN monomer, it is also critical for PKA recognition in the context of the PLN pentamer. Considering the multifaceted role of Arg9 in SERCA inhibition and PKA-mediated phosphorylation, it is not surprising that Arg9 is a hotspot for disease-associated mutations in PLN.

  • Subjects / Keywords
  • Graduation date
    Fall 2012
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
  • Supervisor / co-supervisor and their department(s)
  • Examining committee members and their departments
    • Alexander, Todd (Physiology and Pediatrics)
    • Fliegel, Larry (Biochemistry)
    • Holmes, Charles (Biochemistry)
    • Rao, Rajini (Physiology, Johns Hopkins School of Medicine)