Abstract 1672 Validating the use of acceptor photobleach FRET in quantifying protein-protein interactions between K+ channel alpha-subunits hERG and KvLQT1

hERG and KvLQT1 are the respective alpha-subunits of the voltage-gated potassium channels Kv11.1 and Kv7.1 that contribute to repolarization currents and, therefore, regulation of proper action potential durations (APDs) in cardiomyocytes. Mutations to hERG and KvLQT1 are associated with Long QT syndrome (LQTS) and can result in arrhythmias and sudden cardiac death (SCD). Previous data establish that hERG and KvLQT1 undergo direct and dynamic protein-protein interactions which are thought to be regulated by PKA-mediated phosphorylation as activated by β-adrenergic signaling. However, it remains unclear how different states of subunit phosphorylation impact these interactions. Previously, the Darling Lab performed acceptor photobleach FRET (apFRET) analyses to gather quantitative information on hERG-KvLQT1 interactions. Before employing this technique in further studies on the role of site-specific phosphorylation, we revisited the reliability and tractability of apFRET in providing both robust and non-variable data within our heterologous model system. By implementing an intra-complex, inter-molecular positive control achieved through ratiometric hERG-mECFP and hERG-mEYFP mixing, we were able to compare analysis approaches and make recommendations for our future apFRET experiments. In upcoming work, the Darling Lab will be able to further characterize protein-protein interactions between hERG and KvLQT1, which may hold significant clinical relevance. 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.