Large-scale functional assessment of variants of the potassium channel Kir2.1 - Clinical and comparative insights

Sep 30, 2026

Upload by Aurelia

Large-scale functional testing of KCNJ2/Kir2.1 variants addresses the clinical challenge posed by variants of uncertain significance (VUS) in Andersen-Tawil syndrome. The study applied complementary assays to 70 variants: flow cytometry for Kir2.1 surface expression, a thermal stability assay for 20 variants, a yeast-based functional assay distinguishing loss-of-function in 10 pathogenic/likely pathogenic variants and most VUS, and whole-cell patch clamp for selected variants. Results show many variants reduce surface expression, substantial disagreement with deep mutational scan datasets, and strong evidence supporting LOF for multiple Kir2.1 VUS, improving guidance for ClinVar classifications.

LanguageEnglish

CategoryResearch & Report

FormatPDF

Pages13

Reading Time33 min

AccessFree to download

Methods

Surface expression, thermal stability, yeast assay, and patch clamp

Results

Discrepancies with deep mutational scans

Loss-of-function patterns across assays

Clinical implications

Supporting ClinVar variant classifications

RESEARCH ARTICLE Large-scale functional assessment of variants of the potassium channel Kir2.1: Clinical and comparative insights Received for publication, March 29, 2025, and in revised form, November 16, 2025 Published, Papers in Press, November 26, 2025 [https://doi.org/10.1016/j.jbc.2025.1](https://doi.org/10.1016/j.jbc.2025.1)10985 Corey L. Anderson 1,2,*, Saba Munuwar 1,2, Janay K. Walters 1,2, Seamus F. McWilliams 1,2, Molly Melnick 1,2, Emma R. Langer 1,2, Igor Bereslavskyy1,2, Maxwell R. Milaitis 1,2, Louise Reilly 1,2, and Lee L. Eckhardt 1,2,* From the 1Department of Medicine and 2Cellular and Molecular Arrhythmias Research Program, University of WisconsinMadison, Madison, Wisconsin, USA Reviewed by members of the JBC Editorial Board. Edited by Roger Colbran Hundreds of KCNJ2 (Kir2.1) variants of uncertain significance (VUS) have been associated with Andersen-Tawil Syndrome (ATS). Remarkably, most Kir2.1 variants’ surface expression and function have been described via deep mutational scans (DMS). These results have provided an unprecedented picture of Kir2.1 structure-function relationships and insights into VUS. However, these studies are limited by the lack of robust validation. We performed a flow cytometrybased Kir2.1 surface expression assay for 70 variants (61 ATS-linked) distributed across the potassium inward rectifier channel, a thermal stability assay of 20 variants with reduced surface expression, a yeast-based functional assay for 20 variants (10 pathogenic or likely pathogenic (P/LP) and 10 VUS), as well as whole-cell patch clamp for 13 variants (4 P/LP and 9 VUS). Kir2.1 cell surface expression results showed that 􀀙 30% of variants have reduced surface expression when co-expressed with WT, and that 􀀙 25% disagreed with the DMS datasets. 􀀙 70% of variants with reduced surface tested had reduced thermal stability. Our yeast assay showed all 10 P/LP variants exhibiting LOF, and 7 out of 10 VUS were LOF, in contrast to the DMS method. Patch clamp data further validated the yeast assay. Our data underscores the limitations to interpreting the Kir2.1 DMS datasets, demonstrates a proof-of-principle yeast assay as a complementary method to better inform ClinVar classifications, and provides several lines of evidence for LOF of 9 Kir2.1 VUS in the process. The finding of variants of uncertain significance (VUS) negatively impacts clinical management of suspected genetically associated conditions, including inherited arrhythmias (1). One major class of proteins linked to inherited arrhythmias are ion channels, which orchestrate the flow of ions across the sarcolemma in response to stimuli to generate cardiac action potentials. The macroscopic current generated across a group of ion channels depends on the number of channels at the surface, open probability, and single-channel current. Mutations can perturb one or more of these properties in a variety of ways, leading to disease, such as * For correspondence: Lee L. Eckhardt, [lle@medicine.wisc.edu](lle@medicine.wisc.edu); Corey L. Anderson, [clanders@medicine.wisc.edu](clanders@medicine.wisc.edu). misfolding and defective trafficking, disrupting ligand binding or disrupting the selectivity filter, respectively (2, 3). Functional analyses targeting one or more of these LOF mechanisms can provide strong evidence for or against variant pathogenicity per the ACSM guidelines in combination with other criteria (4). The strength of support, however, can be improved with proper validation studies and multiple lines of functional evidence (4, 5). Given the expanding number of VUS, deep mutational scans (DMS), also known as multiplex assays of variant effects or MAVEs, have been developed to assess thousands of variants simultaneously, including many proteins identified in inherited arrhythmias (1, 6). KCNJ2, which encodes Kir2.1 and is the dominant protein subunit for the inward rectifying current IK1 sets cardiac resting membrane potential and com

FAQs

Why were Kir2.1 VUS functional assessments needed for clinical decision-making?

VUS in genetically associated conditions can negatively affect clinical management. The work targets functional uncertainty to better evaluate variant pathogenicity.

Which experimental assays were used to evaluate Kir2.1 variants?

The study used flow cytometry for surface expression (70 variants), a thermal stability assay (20 variants), a yeast-based functional assay (20 variants), and whole-cell patch clamp (13 variants).

How did the results compare with deep mutational scan (DMS) datasets?

Approximately 25% of variants disagreed with DMS surface-expression data, and the yeast assay indicated loss-of-function for all pathogenic/likely pathogenic variants and most VUS, with patch clamp data further supporting the yeast findings.

Sep 30, 2026

Upload by Aurelia