Proteomic Analysis of the Functional Inward Rectifier Potassium Channel (Kir) 2.1 Reveals Several Novel Phosphorylation Sites

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.