Key result
Integrated proteomic method maps five novel phosphorylation sites on the Kir2.1 potassium channel.
Why the study?
Characterizing post-translational modifications of membrane proteins like Kir2.1 remains challenging, but understanding its phosphorylation is important for arrhythmia prevention.
A novel proteomic approach successfully mapped five new phosphorylation sites on the Kir2.1 channel, providing a foundation for understanding its regulation in cardiac arrhythmias.
Maps Kir2.1 sites in animals; leaves open arrhythmia relevance in humans.
Membrane proteins represent a large family of proteins that perform vital physiological roles and represent key drug targets. Despite their importance, bioanalytical methods aiming to comprehensively characterize the post-translational modification (PTM) of membrane proteins remain challenging compared to other classes of proteins in part because of their inherent low expression and hydrophobicity. The inward rectifier potassium channel (Kir) 2.1, an integral membrane protein, is critical for the maintenance of the resting membrane potential and phase-3 repolarization of the cardiac action potential in the heart. The importance of this channel to cardiac physiology is highlighted by the recognition of several sudden arrhythmic death syndromes, Andersen-Tawil and short QT syndromes, which are associated with loss or gain of function mutations in Kir2.1, often triggered by changes in the β-adrenergic tone. Therefore, understanding the PTMs of this channel (particularly β-adrenergic tone-driven phosphorylation) is important for arrhythmia prevention. Here, we developed a proteomic method, integrating both top-down (intact protein) and bottom-up (after enzymatic digestion) proteomic analyses, to characterize the PTMs of recombinant wild-type and mutant Kir2.1, successfully mapping five novel sites of phosphorylation and confirming a sixth site. Our study provides a framework for future work to assess the role of PTMs in regulating Kir2.1 functions.
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Brown et al. (2021) studied Sudden arrhythmic death syndromes. Proteomic analysis (top-down and bottom-up) was evaluated on Identification of post-translational modifications (phosphorylation sites). A novel proteomic method integrating top-down and bottom-up analyses successfully mapped five novel phosphorylation sites and confirmed a sixth site on the inward rectifier potassium channel Kir2.1.
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