High-throughput discovery of trafficking-deficient variants in the cardiac potassium channel K_V11.1

Background: KCHN2 encodes the K_V11.1 potassium channel responsible for I_Kr, a major repolarization current during the cardiomyocyte action potential. Variants in KCNH2 that lead to decreased I_Kr have been associated with long QT syndrome type 2 (LQT2). The mechanism of LQT2 is most often induced loss of K_V11.1 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_V11.1 channel.

Methods: We developed a method to quantitate K_V11.1 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_V11.1 trafficking-deficient variants in a multiplexed assay. This new method accurately generated trafficking data for variants in K_V11.1 and is extendable both to all residues in K_V11.1 and to other cell surface proteins.