Serca2a-Phospholamban Interaction Monitored by an Interposed Circularly Permutated Green Fluorescent Protein

Am J Physiol Heart Circ Physiol. 2021 Apr 16. doi: 10.1152/ajpheart.00858.2020. Online ahead of print.ABSTRACTBACKGROUND: 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.METHODS: 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.RESULTS: 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% to 165% depending on cell line and conditions.SUMMARY: A single recombinant fusion protein that combines Serca2a, a circularly permutated green fluorescent protein and PLB can be expressed in cells and can be...
Source: American Journal of Physiology. Heart and Circulatory Physiology - Category: Physiology Authors: Source Type: research