Chemical and Genetic Knockout Identifies KCNQ2 as the Principal Background Voltage-gated Potassium Current in Human Embryonic Kidney 293 Cells

Patch-clamp electrophysiology has transformed functional assessment of genotype-phenotype relationships in inherited arrhythmia syndromes. These methods have traditionally relied on in vitro analysis of ion channels transiently or stably expressed in heterologous cell models. Voltage-gated potassium channels have drawn particular attention due to their role in disorders such as Long QT Syndrome (e.g., LQT1: KCNQ1; LQT2: KCNH2; LQT5: KCNE1; LQT7/Andersen-Tawil Syndrome: KCNJ2).¹ Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney (HEK293) cells are widely used models for functional studies of candidate channelopathy-associated variants. CHO cells are often favored due to their minimal endogenous ion channel expression, ease of culture, and robust heterologous protein expression. HEK cells, benefiting from their human origin and high transfection efficiency, serve as a powerful alternative. Recent advances have produced “landing-pad” HEK cells that support stable integration of cDNA constructs, enabling high-throughput studies across multiple ion channel disorders.² However, a limitation in HEK cell-based studies of potassium channel variants is a prominent endogenous voltage-dependent potassium current, the molecular identity of which has remained elusive.³

To address this, we used a combination of RNA sequencing, pharmacological inhibition, and CRISPR-Cas9 genome editing to identify the gene responsible for the endogenous current in both wild-type and “landing-pad” HEK cells (Figure A). We first evaluated baseline potassium currents in CHO and HEK “landing-pad” cells using whole-cell patch-clamp electrophysiology (Figures B–D). Cells were voltage-clamped using 1-second depolarizing pulses from a holding potential of −80 mV to a test potential of +60 mV to elicit background potassium currents. HEK cells exhibited outward voltage-gated potassium currents, whereas CHO cells showed minimal background currents (n = 5, p < 0.01). To identify candidate genes underlying the endogenous HEK cell current, we performed RNA sequencing. Among potassium channel genes, KCNQ2 was the most abundantly expressed transcript. KCNQ2 is primarily expressed in neurons, and loss-of-function mutations are associated with developmental and epileptic encephalopathy (OMIM: 613720).

Figure.

Chemical and genetic knockout strategies identify KCNQ2 as the primary gene underlying endogenous voltage-gated potassium current in HEK cells.

A) Schematic overview of the experimental strategy, integrating transcriptomics, pharmacology, and CRISPR-based gene editing.

B) Representative outward current traces from CHO cells, demonstrating minimal endogenous voltage-gated potassium current. Inset: voltage-clamp protocol used for panels B, C, D and H.

C) Voltage-dependent endogenous potassium currents recorded from HEK293 cells under the same protocol.

D) Current-voltage plot of the HEK cell potassium currents (n = 5 per group). Error bars indicate standard error of the mean (SEM).

E) Time course of current suppression during bath application of ML-252 (1 µM), a selective KCNQ2 inhibitor. Currents were evoked every 10 seconds; traces from the 1st, 50th, and 90th pulses are shown. Inset: voltage protocol used.

F) Sanger sequencing traces of KCNQ2 Exon 4 from wild-type (WT) HEK cells and the homozygous 5 bp deletion clone (HEK-5hom) generated via CRISPR-Cas9.

G) Current–voltage relationship in WT vs. KCNQ2 knockout (HEK-5hom) cells, showing marked reduction in voltage-gated potassium current (n = 5 per group). Error bars indicate standard error of the mean (SEM).

H) Representative traces of WT I_Ks (KCNQ1 + KCNE1) alongside KCNQ1 variant p.Ala302Val and WT I_Kr (KCNH2) alongside KCNH2 variant p.Arg752Gln. Respective WT and variant are shown at similar scales. Four cells studied per condition – voltage protocol similar to B and C, with 2 seconds per segments.

Figure created in part with BioRender.

We next experimentally tested whether KCNQ2 contributes to the HEK cell background current by performing cellular electrophysiology with ML-252, a selective KCNQ2 inhibitor (1 µM, Cayman Chemical). This molecule was previously shown to have high selectivity for KCNQ2 during its medicinal chemistry development.⁴ ML-252 treatment resulted in a substantial current reduction (Figures E). After 15 minutes, the current density was significantly reduced in treated cells compared to untreated controls (n = 5, p < 0.01, paired two-tailed t-test). To confirm the role of KCNQ2 channel, we generated a KCNQ2 knockout HEK cell line using CRISPR-Cas9, following previously described methods.⁵ A plasmid encoding Cas9 and a guide RNA targeting Exon 4 of KCNQ2—a conserved exon present in all Ensembl isoforms—was transfected into HEK cells. We isolated a clone with a homozygous 2 bp deletion (Figure F), predicted to cause a frameshift and result in transcript degradation via nonsense-mediated decay. Whole-cell patch-clamp experiments revealed that the KCNQ2 knockout line (HEK-2hom) exhibited broad reductions in endogenous potassium current across tested voltages compared to the WT line (Figure G). The current density at +40 mV was 43.8 ± 6.0 pA/pF in unedited cells, vs 8.9 ± 3.1 pA/pF in edited cells, strongly supporting KCNQ2 as the primary contributor to the endogenous voltage-gated potassium current in HEK cells. These results were consistent in both wild-type and “landing-pad” HEK cell backgrounds. To highlight the value of this new cell line, we studied two LQTS-associated variants in KCNQ1 and KCNH2. We observed that the KO cell line could be readily transfected with plasmids expressing the potassium currents I_Ks (KCNQ1 + KCNE1) and I_Kr (KCNH2; Panel H). The Pathogenic/Likely Pathogenic variants KCNQ1 p.Ala302Val (ClinVar: 36439) and KCNH2 p.Arg752Glu (ClinVar: 14435) showed absence of normal currents. Frozen cells were readily thawed, passaged, and transfected with these plasmids.

While our experiments support the technical application of KCNQ2 knockout lines to ablate undesirable background currents in multiple experiments, we cannot exclude the possibility of unintended transcriptional dysregulation among other channel transcripts.

In summary, we describe a combined pharmacological and genetic strategy to resolve a limitation in HEK293-based functional studies of potassium ion channel variants. Our results establish KCNQ2 as the major source of endogenous voltage-gated potassium current in HEK cells. We will provide both WT and “landing-pad” HEK cell lines with KCNQ2 knockout as a resource to the community upon request. We anticipate that these cells will be helpful tools for future functional assays.

Data Availability

All data supporting this study are available from the corresponding author upon reasonable request.