Development of automated patch clamp assays to overcome the burden of variants of uncertain significance in inheritable arrhythmia syndromes

Figures

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  FIGURE 1. The transition from the conventional patch-clamp system to a planar patch-clamp system has enabled simultaneous patching of cells and underlies many of the APC used today. (A) In conventional whole-cell patch-clamp recordings, a pipette filled with electrolyte solution contains an electrode enabling the experimenter to apply various stimuli and measure the cell’s response. It takes considerable skill to attach the pipette to the cell using micromanipulators and a microscope. (B) In planar patch-clamp technology, cells are drawn down and adhered to a micropore using suction, acting similar to a pipette tip. Here, the cell is ruptured enabling stimulation and measurement of electrical activities. Chips used in modern APC platforms can record up to 768 cells in parallel (see Table 1). No micromanipulation is necessary. Figure developed using Biorender.

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  FIGURE 2. Cell-to-cell variability necessitates large numbers of replicate measurements to obtain an accurate estimate of the mean value. (A) Violin plots of KCNH2 WT (i) and negative (ii) control lines show experimental cell-to-cell variability, plate-to-plate variability across 24 plates, and overall dynamic range of the assay revealing clear separation between positive and negative controls (iii). Data are shown as mean ± 95% CI. (B) Square-root transformations enable gaussian-distributed data for analysis as seen with KCNH2 before (i) and after (ii) transformation (N = 1,198), and (C) SCN5A before (i) and after (ii) (N = 609). Transformations are applied to all data, including variants, enabling distinction of any changes in current density (iii). Solid lines denote median. Dashed lines denote quartiles. (A) Adapted from Jiang et al.(2022), with permission from Elsevier.

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  FIGURE 3. Stringent quality control is required to remove bad quality recordings. (A) The SyncroPatch 384 PE offers high throughput APC of mammalian cell lines. A minimum seal resistance of 500 MΩ is adequate to produce a high-quality seal (green wells). Cells with seal resistant <300 MΩ (grey) and between 300 and 500 MΩ (blue) indicate lower quality seals. (B) Current traces illustrating the activation of two WT SCN5A HEK293 cells (−100 mV to +60 mV, Δ5 mV). The top row shows an example of current recorded with poor voltage control whilst the bottom row shows recordings from a cell with good quality voltage control. Due to the rapid kinetics of sodium channel, a “time to peak” measurement can be used to identify poorly clamped cells. In this instance, time to peak is applied at 0 mV (blue) and dotted line represents 1.1 ms post-activation. (C) Respective current-voltage curves revealing steep jump in current in the poorly clamped cell (top). (D) Steady-state activation and Boltzmann function of respective cells reveal the poorly clamp cell (top) have visibly severely impacted V50 (voltage required to activate half of the cells’ channels) and slope, which is typically used for comparison…