Investigating Phosphorylation of the Cardiac Potassium Channel hERG using MALDI-TOF Mass Spectrometry.

Mutations in the voltage-gated cardiac potassium channel proteins hERG and KvLQT1 are known to be associated with a cardiac condition called Long QT Syndrome (LQTS), which can result in arrhythmia, fainting, and/or sudden cardiac death. Previous research from the Darling lab suggests that hERG and KvLQT1, alpha subunits of two distinct ion channels, are capable of direct protein-protein interactions, which can be regulated by PKA-mediated phosphorylation.. They also found that phospho-null mutants of hERG do not interact with KvLQT1 to the same extent as wild-type hERG, leading to the hypothesis that the phosphorylation state of hERG dominates the regulation of interactions between hERG and KvLQT1. Thus, understanding the phosphorylation pattern of wild-type hERG would help us characterize these protein-protein interactions as well as increase our knowledge about the complex biogenesis and trafficking of hERG. In order to elucidate this baseline phosphorylation state, the goal of this project is to use matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) to characterize which of the four known PKA-phosphorylation sites of hERG are in fact modified when the protein is synthesized in heterologous model cells. In order to do this, the project aims to determine whether MALDI-TOF MS can sensitively and reproducibly detect phosphorylation patterns in hERG that is expressed in model cell systems.