Dataset Information

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

ABSTRACT:

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.

SUBMITTER: Kozek KA

PROVIDER: S-EPMC7704534 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:ATP-sensitive K+ (KATP) channels uniquely link cellular energy metabolism to membrane excitability and are expressed in diverse cell types that range from the endocrine pancreas to neurons and smooth, skeletal, and cardiac muscle. A decrease in the surface expression of KATP channels has been linked to various disorders, including dysregulated insulin secretion, abnormal blood pressure, and impaired resistance to cardiac injury. In contrast, up-regulation of KATP channel surface expression may be protective, for example, by mediating the beneficial effect of ischemic preconditioning. Molecular mechanisms that regulate KATP channel trafficking are poorly understood. Here, we used cellular assays with immunofluorescence, surface biotinylation, and patch clamping to demonstrate that Eps15 homology domain-containing protein 2 (EHD2) is a novel positive regulator of KATP channel trafficking to increase surface KATP channel density. EHD2 had no effect on cardiac Na+ channels (Nav1.5). The effect is specific to EHD2 as other members of the EHD family-EHD1, EHD3, and EHD4-had no effect on KATP channel surface expression. EHD2 did not directly affect KATP channel properties as unitary conductance and ATP sensitivity were unchanged. Instead, we observed that the mechanism by which EHD2 increases surface expression is by stabilizing KATP channel-containing caveolar structures, which results in a reduced rate of endocytosis. EHD2 also regulated KATP channel trafficking in isolated cardiomyocytes, which validated the physiologic relevance of these observations. Pathophysiologically, EHD2 may be cardioprotective as a dominant-negative EHD2 mutant sensitized cardiomyocytes to ischemic damage. Our findings highlight EHD2 as a potential pharmacologic target in the treatment of diseases with KATP channel trafficking defects.-Yang, H. Q., Jana, K., Rindler, M. J., Coetzee, W. A. The trafficking protein, EHD2, positively regulates cardiac sarcolemmal KATP channel surface expression: role in cardioprotection.

Dataset Information

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

Background

Objective

Methods

Results

Conclusion

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

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

Background

Objective

Methods

Results

Conclusion

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

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