High Throughput Functional Evaluation of KCNQ1 Decrypts Variants of Unknown Significance

Vanoye, Carlos G.; Fabre, Katarina L.; Potet, F; DeKeyser, Jean-Marc; Macaya, Daniela; Meiler, Jens; Sanders, Charles R.; George, Alfred L.

doi:10.1101/223206

Cited by 34 publications

(66 citation statements)

References 39 publications

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“…Previous studies showed feasibility of this approach to study channel properties of K V 2.1 and the closely related K V 7.1 channel encoded by KCNQ1. 14,25,26 In addition, we also tested on this platform the T374I variant that we previously characterized by manual patch clamp recording, 7 and reproduced the loss-of-function phenotype (see Fig 2B; p < 0.001, unpaired t test @ +60mV vs WT). This further validates use of this platform to screen for K V 2.1 dysfunction.…”

Section: Consequences Of K V 21 Variants On Current Densitymentioning

confidence: 98%

Spectrum of K_V2.1 Dysfunction in KCNB1‐Associated Neurodevelopmental Disorders

Kang

Vanoye

Misra

et al. 2019

Annals of Neurology

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Objective Pathogenic variants in KCNB1, encoding the voltage‐gated potassium channel KV2.1, are associated with developmental and epileptic encephalopathy (DEE). Previous functional studies on a limited number of KCNB1 variants indicated a range of molecular mechanisms by which variants affect channel function, including loss of voltage sensitivity, loss of ion selectivity, and reduced cell‐surface expression. Methods We evaluated a series of 17 KCNB1 variants associated with DEE or other neurodevelopmental disorders (NDDs) to rapidly ascertain channel dysfunction using high‐throughput functional assays. Specifically, we investigated the biophysical properties and cell‐surface expression of variant KV2.1 channels expressed in heterologous cells using high‐throughput automated electrophysiology and immunocytochemistry–flow cytometry. Results Pathogenic variants exhibited diverse functional defects, including altered current density and shifts in the voltage dependence of activation and/or inactivation, as homotetramers or when coexpressed with wild‐type KV2.1. Quantification of protein expression also identified variants with reduced total KV2.1 expression or deficient cell‐surface expression. Interpretation Our study establishes a platform for rapid screening of KV2.1 functional defects caused by KCNB1 variants associated with DEE and other NDDs. This will aid in establishing KCNB1 variant pathogenicity and the mechanism of dysfunction, which will enable targeted strategies for therapeutic intervention based on molecular phenotype. ANN NEUROL 2019;86:899–912

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Section: Consequences Of K V 21 Variants On Current Densitymentioning

confidence: 98%

Spectrum of K_V2.1 Dysfunction in KCNB1‐Associated Neurodevelopmental Disorders

Kang

Vanoye

Misra

et al. 2019

Annals of Neurology

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“…Western blotting or confocal imaging, which are not scalable to thousands of variants. 17,43 Recent advances in automated patch clamp electrophysiology have enabled the rapid characterization of dozens of voltage-gated potassium channel variants, 31,44 and induced pluripotent stem cell derived cardiomyocytes show promise to characterizing KCNH2 variants in a non-heterologous system. However, the throughput of these methods does not allow for the rapid screening of the thousands of possible variants in KCNH2.…”

Section: Approaches To Studying Kcnh2 Variantsmentioning

confidence: 99%

High-throughput discovery of trafficking-deficient variants in the cardiac potassium channelKCNH2: Deep mutational scan ofKCNH2trafficking

Ka¹,

Am²,

Ng³

et al. 2020

Preprint

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Background: KCHN2 encodes the K V 11.1 potassium channel responsible for I Kr , a major repolarization current during the cardiomyocyte action potential. Variants in KCNH2 that decrease I Kr 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.Objective: 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 KCNH2.Methods: We 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.Results: For 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 I Kr , but that some variants which traffic normally may still be functionally compromised. Conclusions:Here, 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.

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“…While progress in the functional characterization of LQTS-associated mutations has been made (12)(13)(14) the mechanistic molecular basis of channel dysfunction for most KCNQ1 mutations is still unclear. The dearth of direct experimental data that can help to determine how mutations alter KCNQ1 structure and function has prompted computational modeling approaches (16,59).…”

Section: Rosetta-predicted Stability Changes In Kcnq1 Models Correlatmentioning

confidence: 99%

“…About 50% of the genetic cases of long QT syndrome (LQTS), which predispose children and young adults to sudden cardiac death, are associated with dominant mutations in KCNQ1 (type 1 LQTS) (11). While progress in the functional characterization of LQTS-associated mutations has been made (12)(13)(14)(15), the molecular mechanisms underlying channel dysfunction remain difficult to assess without the availability of high-accuracy structural data. A detailed molecular understanding is needed to improve decision making for new unclassified KCNQ1 mutations and to support the development of new anti-arrhythmic therapeutics.…”

Section: Introductionmentioning

confidence: 99%

Upgraded molecular models of the human KCNQ1 potassium channel

Künze

Duran

Woods

et al. 2019

Preprint

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The voltage-gated potassium channel KCNQ1 (KV7.1) assembles with the KCNE1 accessory protein to generate the slow delayed rectifier current, IKS, which is critical for membrane repolarization as part of the cardiac action potential. Loss-of-function (LOF) mutations in KCNQ1 are the most common cause of congenital long QT syndrome (LQTS), type 1 LQTS, an inherited genetic predisposition to cardiac arrhythmia and sudden cardiac death. A detailed structural understanding of KCNQ1 is needed to elucidate the molecular basis for KCNQ1 LOF in disease and to enable structure-guided design of new anti-arrhythmic drugs. In this work, advanced structural models of human KCNQ1 in the resting/closedand activated/open states were developed by Rosetta homology modeling guided by newly available experimentally-based templates: X. leavis KCNQ1 and resting voltage sensor structures. Using molecular dynamics (MD) simulations, the models' capability to describe experimentally established channel properties including state-dependent voltage sensor gating charge interactions and pore conformations, PIP2 binding sites, and voltage sensorpore domain interactions were validated. Rosetta energy calculations were applied to assess the models' utility in interpreting mutation-evoked KCNQ1 dysfunction by predicting the change in protein thermodynamic stability for 50 characterized KCNQ1 variants with mutations located in the voltage-sensing domain. Energetic destabilization was successfully predicted for folding-defective KCNQ1 LOF mutants whereas wild type-like mutants had no significant energetic frustrations, which supports growing evidence that mutation-induced protein destabilization is an especially common cause of KCNQ1 dysfunction. The new KCNQ1 Rosetta models provide helpful tools in the study of the structural mechanisms of KCNQ1 function and can be used to generate structurebased hypotheses to explain KCNQ1 dysfunction. Author SummaryCardiac rhythm is maintained by synchronized electrical impulses conducted throughout the heart. The potassium ion channel KCNQ1 is important for the repolarization phase of the cardiac action potential that underlies these electrical impulses. Heritable mutations in KCNQ1 can lead to channel loss-offunction (LOF) and predisposition to a life-threatening cardiac arrhythmia. Knowledge of the threedimensional structure of KCNQ1 is important to understand how mutations lead to LOF and to support structurally-guided design of new anti-arrhythmic drugs. In this work, we present the development and validation of molecular models of human KCNQ1 inferred by homology from the structure of frog KCNQ1.Models were developed for the open channel state in which potassium ions can pass through the channel and the closed state in which the channel is not conductive. Using molecular dynamics simulations, interactions in the voltage-sensing and pore domain of KCNQ1 and with the membrane lipid PIP2 were analyzed. Energy calculations for KCNQ1 mutations in the voltage-sensing domain reveled that most of 3 the mutations t...

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High Throughput Functional Evaluation of KCNQ1 Decrypts Variants of Unknown Significance

Abstract: Background -The explosive growth in known human gene variation presents enormous

Cited by 34 publications

References 39 publications

Spectrum of K_V2.1 Dysfunction in KCNB1‐Associated Neurodevelopmental Disorders

Spectrum of K_V2.1 Dysfunction in KCNB1‐Associated Neurodevelopmental Disorders

High-throughput discovery of trafficking-deficient variants in the cardiac potassium channelKCNH2: Deep mutational scan ofKCNH2trafficking

Upgraded molecular models of the human KCNQ1 potassium channel

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High Throughput Functional Evaluation of KCNQ1 Decrypts Variants of Unknown Significance

Abstract: Background -The explosive growth in known human gene variation presents enormous

Cited by 34 publications

References 39 publications

Spectrum of KV2.1 Dysfunction in KCNB1‐Associated Neurodevelopmental Disorders

Spectrum of KV2.1 Dysfunction in KCNB1‐Associated Neurodevelopmental Disorders

High-throughput discovery of trafficking-deficient variants in the cardiac potassium channelKCNH2: Deep mutational scan ofKCNH2trafficking

Upgraded molecular models of the human KCNQ1 potassium channel

Contact Info

Product

Resources

About

Spectrum of K_V2.1 Dysfunction in KCNB1‐Associated Neurodevelopmental Disorders

Spectrum of K_V2.1 Dysfunction in KCNB1‐Associated Neurodevelopmental Disorders