Abstract 1680 Investigation of PKA Phosphorylation on the Plasma Membrane Expression of the K+ Channel α-subunits hERG and KvLQT1

hERG and KvLQT1 are the α-subunit proteins of the voltage-gated cardiac potassium channels Kv11.1 and Kv7.1, respectively. They pass the IKr and IKs currents, which are responsible for ventricular repolarization and maintaining a normal heartbeat. Mutations in hERG and KvLQT1 are associated with Long QT Syndrome (LQTS) which can manifest in arrhythmia and sudden cardiac death. Site-directed phosphorylation by protein kinase A (PKA) regulates the trafficking and expression of hERG and KvLQT1, although it is unknown whether PKA phosphorylation promotes or diminishes hERG biogenesis. We seek to understand whether the PKA-mediated phosphorylation patterns of hERG and KvLQT1 play a role in their plasma membrane expression. We investigated plasma membrane expression using phosphomimetic and phosphonull mutant constructs of hERG and KvLQT1 and separated plasma membrane-expressed and intracellular proteins with affinity chromatography to compare semi-quantitatively via western blot analysis. In our heterologous system, we have successfully separated membrane-expressed and intracellular hERG, allowing for further investigations about phosphorylation patterns and membrane expression. Our data shows that phosphomimetic hERG exhibits the highest plasma membrane expression. These results suggest that complete PKA phosphorylation of hERG at the four putative sites increases its plasma membrane expression. Our work is adding to the body of knowledge about how PKA-mediated phosphorylation affects the expression and cellular localization of these channel proteins in the plasma membrane where they are required for homeostatic cardiac electrophysiology. This work was funded by the American Heart Association Institutional Research Enhancement Award (AIREA) 23AIREA1051613 Investigating interactions between the cardiac voltage- gated potassium channel proteins hERG and KvLQT1.