A hERG1 potassium channel subdomain is trafficked to the cardiac nucleus: mechanisms and targets of the hERG1NP

Project Summary/Abstract KCNH2 encodes the pore-forming subunits of the voltage-gated potassium channel called hERG1. Loss-of- function KCNH2 variants cause the cardiac disorder long QT syndrome type II and are linked with intrauterine fetal death and sudden infant death syndrome (SIDS). Clinical data suggest that the mechanisms of sudden cardiac death differ in the immature and mature myocardia, but how KCNH2 variants trigger sudden cardiac death in the immature heart is not fully understood. We identified a novel KCNH2-encoded protein (hERG1NP) that is targeted to the nuclei of neonatal rat cardiomyocytes and cardiomyocytes derived from human pluripotent stem cells. hERG1NP, which mapped to the distal hERG1 C-terminal domain (Distal-CT), is targeted to the nucleus by a nuclear localization sequence (NLS) within the hERG1NP N-terminus. Using hERG1’s distal C- terminal domain (Distal-CT) as a surrogate for hERG1NP, we found that co-expressing the Distal-CT with the full- length hERG1 channel reduces hERG1 current density and alters hERG1 channel gating, compared to GFP controls. Deleting the NLS from the Distal-CT abolished its targeting to the nucleus and its effects on hERG1 currents. These data demonstrate that the putative hERG1NP is a regulator of hERG1 function and that hERG1 regulation by the Distal-CT is dependent upon targeting into the cell nucleus. In this grant we will use molecular biology, site-directed mutagenesis, and confocal microscopy to identify the nascent hERG1NP polypeptide and define the mechanisms that regulate hERG1NP biogenesis and activity in cardiac tissues. We will also use patch clamp electrophysiology, confocal microscopy, and molecular biology to define the mechanisms by which hERG1NP regulates hERG1 channel function and cardiac electrophysiology. Last, we will use two new gene- edited stem cell lines: (1) a cell line where hERG1NP expression is selectively disrupted, and (2) a double knock- in line carrying a KCNH2 mutation within the hERG1 NLS that disrupts nuclear targeting of the hERG1NP. We will use these lines to define the impact of hERG1NP on cardiac currents (INa, ICa, IKs, Ito, & IK1) and identify mechanisms of proarrhythmia subsequent to hERG1NP dysfunction. We will also measure the impact of hERG1NP on cardiomyocyte development and maturation, and identify targets of hERG1NP activity (RNAseq & ChIP-Seq). The overall goal of this work is to define mechanisms regulating cardiac hERG1NP function, the role of hERG1NP in cardiac physiology, and mechanisms by which hERG1NP dysfunction contributes to human disease.