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

• Published in: Heart rhythm Vol. 17; no. 12; pp. 2180 - 2189
• Main Authors: Kozek, Krystian A., Glazer, Andrew M., Ng, Chai-Ann, Blackwell, Daniel, Egly, Christian L., Vanags, Loren R., Blair, Marcia, Mitchell, Devyn, Matreyek, Kenneth A., Fowler, Douglas M., Knollmann, Bjorn C., Vandenberg, Jamie I., Roden, Dan M., Kroncke, Brett M.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.12.2020
• Subjects: Deep mutational scanning; hERG; KCNH2; KV11.1; Membrane trafficking; KCNH2; KV11.1; Membrane trafficking; Deep mutational scanning; hERG
• ISSN: 1547-5271; 1556-3871
• DOI: 10.1016/j.hrthm.2020.05.041

KCHN2 encodes the KV11.1 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 KV11.1 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. 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 KV11.1 channel. We developed a method to quantitate KV11.1 variant trafficking on a pilot region of 11 residues in the S5 helix. 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 IKr. Conversely, but expectedly, some variants that traffic normally were still functionally compromised. We describe a new method for detecting KV11.1 trafficking-deficient variants in a multiplexed assay. This new method accurately generated trafficking data for variants in KV11.1 and is extendable both to all residues in KV11.1 and to other cell surface proteins.