SERCA2a-phospholamban interaction monitored by an interposed circularly permutated green fluorescent protein

Maren E. Arnold, Wolfgang R. Dostmann, Jody Martin, Michael J. Previs, Bradley Palmer, Martin LeWinter, Markus Meyer

Research output: Contribution to journalArticlepeer-review

Abstract

The interaction of phospholamban (PLB) and the sarcoplasmic reticulum Ca2 + -ATPase (SERCA2a) is a key regulator of cardiac contractility and a therapeutic target in heart failure (HF). PLB-mediated increases in SERCA2a activity improve cardiac function and HF. Clinically, this mechanism can only be exploited by a general activation of the proteinkinase A (PKA), which is associated with side effects and adverse clinical outcomes. A selective interference of the PLB-SERCA2a interaction is desirable but will require novel tools that allow for an integrated assessment of this interaction under both physiological and pathophysiological conditions. A circularly permutated green fluorescent protein (cpGFP) was interposed between SERCA2a and PLB to result into a single SERCA2a-cpGFP-PLB recombinant protein (SGP). Expression, phosphorylation, fluorescence, and function of SGP were evaluated. Expression of SGP-cDNA results in a functional recombinant protein at the predicted molecular weight. The PLB domain of SGP retains its ability to polymerize and can be phosphorylated by PKA activation. This increases the fluorescent yield of SGP by between 10% and 165% depending on cell line and conditions. In conclusion, a single recombinant fusion protein that combines SERCA2a, a circularly permutated green fluorescent protein, and PLB can be expressed in cells and can be phosphorylated at the PLB domain that markedly increases the fluorescence yield. SGP is a novel cellular SERCA2a-PLB interaction monitor.

Original languageEnglish (US)
Pages (from-to)H2188-H2200
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume320
Issue number6
DOIs
StatePublished - 2021

Bibliographical note

Funding Information:
This research was supported by National Heart, Lung, and Blood Institute Grant R01 HL-122744 (to M. Meyer). W. R. Dostmann was funded by the Totman Trust for Biomedical Research.

Publisher Copyright:
Copyright © 2021 the American Physiological Society

Keywords

  • Calcium
  • Phospholamban
  • SERCA2a
  • Sarcoplasmic reticulum

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

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