Egly, C. L. | Search bioRxiv

bioRxiv2024

The proteostasis interactomes of trafficking-deficient KV11.1 variants associated with Long QT Syndrome and pharmacological chaperone rescue

IntroductionThe voltage gated potassium ion channel KV11.1 plays a critical role in cardiac repolarization. Genetic variants that render Kv11.1 dysfunctional cause Long QT Syndrome (LQTS), which is associated with fatal arrhythmias. Approximately 90% of LQTS-associated variants cause intracellular protein transport (trafficking) dysfunction, which can be rescued by pharmacological chaperones like E-4031. Protein folding and trafficking decisions are regulated by chaperones, protein quality control factors, and trafficking machinery, comprising the cellular proteostasis network. Here, we test whether trafficking dysfunction is associated with alterations in the proteostasis network of pathogenic Kv11.1 variants, and whether pharmacological chaperones can normalize the proteostasis network of responsive variants. MethodsWe used affinity-purification coupled with tandem mass tag-based quantitative mass spectrometry to assess protein interaction changes in human embryonic kidney (HEK293) cells expressing wild-type (WT) KV11.1 or trafficking-deficient channel variants in the presence or absence of E-4031. ResultsaWe identified 573 core KV11.1 protein interactors. Both variants KV11.1-G601S and KV11.1-G601S-G965* had significantly increased interactions with proteins responsible for folding, trafficking, and degradation compared to WT. We found that proteasomal degradation is a key component for KV11.1 degradation and that the KV11.1-G601S-G965* variant was more responsive to E-4031 treatment. This suggests a role in the C-terminal domain and the ER retention motif of KV11.1 in regulating trafficking. ConclusionOur report characterizes the proteostasis network of KV11.1, two trafficking deficient KV11.1 variants, and variants treated with a pharmacological chaperone. The identified protein interactions could be targeted therapeutically to improve KV11.1 trafficking and treat Long QT Syndrome.

biochemistry↗

bioRxiv2020

High-throughput discovery of trafficking-deficient variants in the cardiac potassium channel KCNH2

BackgroundKCHN2 encodes the KV11.1 potassium channel responsible for IKr, a major repolarization current during the cardiomyocyte action potential. Variants in KCNH2 that decrease IKr can cause Type 2 Long QT syndrome, usually due to mistrafficking to the cell surface. Accurately discriminating between variants with normal and abnormal trafficking would help clinicians identify and treat individuals at risk of a major cardiac event. The volume of reported non-synonymous KCNH2 variants preclude the use of conventional electrophysiologic methods for functional study. ObjectiveTo report a high-throughput, multiplexed screening method for KCNH2 genetic variants capable of measuring the cell surface abundance of hundreds of missense variants in KCNH2. MethodsWe develop a method to quantitate KCNH2 variant trafficking on a pilot region of 11 residues in the S5 helix, and generate trafficking scores for 220/231 missense variants in this region. ResultsFor 5/5 variants, high-throughput trafficking scores validated when tested in single variant flow cytometry and confocal microscopy experiments. We additionally compare our results with planar patch electrophysiology and find that loss-of-trafficking variants do not produce IKr, but that some variants which traffic normally may still be functionally compromised. ConclusionsHere, we describe a new method for detecting trafficking-deficient variants in KCNH2 in a multiplexed assay. This new method accurately generates trafficking data for variants in KCNH2 and can be readily extended to all residues in Kv11.1 and to other cell surface proteins. CLINICAL IMPLICATIONSHundreds of KCNH2 variants have been observed to date, and thousands more will be found as clinical and population sequencing efforts become increasingly widespread. The major mechanism of KV11.1 loss of function is misfolding and failure to traffic to the cell surface. Deep mutational scanning of KCNH2 trafficking is a scalable, high-throughput method that can help identify new loss of function variants and decipher the large number of KCNH2 variants being found in the population.

genetics↗