Heterozygous KCNH2 variant phenotyping using Flp-In HEK293 and high-throughput automated patch clamp electrophysiology

Figures

• 

  Figure 1:. Design of a heterozygous KCNH2 expression vector. Nsil was introduced into the KCNH2 cDNA before the IRES, while BstXl, BstEll, and Sbfl were silenced in the KCNH2 cDNA after the IRES. This allows the fragmentation of the KCNH2 gene before the IRES into 5 sections to facilitate subcloning of variants.

• 

  Figure 2:. Overview of PatchControl software. Example of −120 mV tail currents for unmodified (columns 1–6) and modified (columns 7–11) WT hERG channels, as well as, a negative control (column 12). In this example, out of a total of 384 wells, 86% of wells have seal resistance >500 MΩ (green), 4% have a seal resistance in the range 300– 500 MΩ and 10% of wells have seal resistance <300 MΩ (grey).

• 

  Figure 3:. Examples of leak current correction. (A) Uncorrected and (B) corrected current traces. Only corrected current traces that have median current value that are less than ±40 pA from the baseline for the region between 60 and 80 ms in each protocol are accepted for further analysis. Panels (i)–(iii) pass this QC criterion whereas panel (iv) would fail.

• 

  Figure 4:. Current density measurements. (A) Examples of −120 mV tail current traces for (i) unmodified WT, (ii) modified WT, (iii) R56Q:WT, (iv) T618S:WT, and (v) F805C:WT. Voltage protocol is shown as inset on panel (ii) and the dashed lines indicate region of the protocol corresponding to the current traces shown. The peak current is indicated by asterisk. (B) Summary of current densities for all constructs shown as violin plot with median indicated by the red horizontal bar with upper and lower quartiles shown as black bars.

• 

  Figure 5:. Activation phenotyping. (A) Example of −120 mV tail current traces recorded for (i) unmodified and (ii) modified WT KCNH2, (iii) R56Q:WT and (iv) T618S:WT following 1 s of depolarization from the holding potential to voltages in the range −50 to +70 mV. Dashed box on the voltage protocol highlights the portion of the protocol depicted in the current traces. Example of Boltzmann fit to the normalized peak current indicated by asterisk in (i) to show how the V0.5 of activation is calculated. (B) Summary of V0.5 values for steady-state activation derived from fitting a Boltzmann function to the peak tail current amplitudes (see Methods for details). The median values are indicated by the red horizontal bar with upper and lower quartiles shown as black bars. N/A indicates when the n number is <10% of recordings that pass the QC.

• 

  Figure 6:. Deactivation phenotyping. (A) Example −50 mV tail current traces (corresponding to dashed line within the voltage protocol) for (i) unmodified and (ii) modified WT KCNH2, (iii) R56Q:WT, (iv) T618S:WT, and (v) F805C:WT. The double exponential fitting of the deactivation decay is shown as red in (i) with arrows indicating the fast and slow components. (B) Summary of weighted time constant for channel deactivation (see Materials and methods for details). The median values are indicated by the red horizontal bar with upper and lower quartiles shown as black bars. It was only possible to analyse those constructs where the peak tail current was >100 pA, hence, T618S:WT is excluded and F805C:WT has limited numbers. (C) Summary of normalized current amplitude ratio where the current amplitude at 500 ms after the peak current (* in panel i) is divided by the peak current (†). N/A indicates when the n number is <10% of recordings that pass the QC.

• 

  Figure 7:. Inactivation phenotyping. (A) Normalized current traces for the onset of inactivation. The dashed box within the voltage protocol highlights the region shown in the current traces. (i) unmodified and (ii) modified WT KCNH2, (iii) R56Q:WT, (iv) T618S:WT, and (v) F805C:WT. The single exponential fitting of current decay from 500 pA was shown as red in (i). (B) Summary of tau constants for the onset of inactivation at 0 mV. The median values are indicated by the red horizontal bar with upper and lower quartiles shown as black bars. (C) Example tail currents recorded at −50 mV (outward currents) and −120 mV (inward current) for modified WT KCNH2 (i), and T618S:WT (ii). (D) Summary of the normalized current amplitude ratio (i.e. */† in C) for all constructs. The smaller value for the T618S:WT constructs is indicative of enhanced inactivation of this variant. The median values are indicated by the red horizontal bar with upper and lower quartiles shown as black bars.

• 

  Figure 8:. Typical timeline for KCNH2 phenotyping assay. Generation of heterozygous KCNH2 vectors are typically done in a batch of 100. We outsource this process to GenScript Inc (Pistcataway, NJ, USA), which has a turnaround of ∼30 days. Generation of heterozygous KCNH2 Flp-In HEK293 cell lines will take around 25 days. Cell culture and the SyncroPatch assay takes 17 days which is done in batches of 10 variants staggered at weekly intervals. Data analysis and curation takes ∼7 days per batch of variants. In total, it will take less than 3 months from generating the variant vector to the final data analysis.