High-throughput discovery of trafficking-deficient variants in the cardiac potassium channel KV11.1

BACKGROUND KCHN2 encodes the K(V)11.1 potassium channel responsible for IKr, a major repolarization current during the cardiomyocyte action potential. Variants in KCNH2 that lead to decreased IKr have been associated with long QT syndrome type 2 (LQT2). The mechanism of LQT2 is most often induced loss of K(V)11.1 trafficking to the cell surface. Accurately discriminating between variants with normal and abnormal trafficking would aid in understanding the deleterious nature of these variants; however, the volume of reported nonsynonymous KCNH2 variants precludes the use of conventional methods for functional study. OBJECTIVE The purpose of this study was to report a high-throughput, multiplexed screening method for KCNH2 genetic variants capable of measuring the cell surface abundance of hundreds of missense variants in the resulting K(V)11.1 channel. METHODS We developed a method to quantitate K(V)11.1 variant trafficking on a pilot region of 11 residues in the S5 helix. RESULTS We generated trafficking scores for 220 of 231 missense variants in the pilot region. For 5 of 5 variants, high-throughput trafficking scores validated when tested in single variant flow cytometry and confocal microscopy experiments. We further explored these results with planar patch electrophysiology and found that loss-of-trafficking variants do not produce I-Kr. Conversely, but expectedly, some variants that traffic normally were still functionally compromised. CONCLUSION We describe a new method for detecting K(V)11.1 trafficking-deficient variants in a multiplexed assay. This new method accurately generated trafficking data for variants in K(V)11.1 and is extendable both to all residues in K(V)11.1 and to other cell surface proteins.