(A) Western blot showing trafficked (fully glycosylated, ~155 kDa) and non-trafficked (core glycosylated, ~135 kDa) Kv11.1 protein. Twenty-four-hour treatment with E4031 increases trafficking in 2 variants. (B) High-throughput, Tl⁺-flux trafficking screen in 384-well plates using HEK-293 cells expressing trafficking-deficient Kv11.1 variants plated at 15,000 cells per well. Cells were treated for 22 hours with drug prior to Tl⁺-flux experiments. Schematic depiction of individual wells treated with vehicle or therapeutic and resulting fluorescence generated via Tl⁺ ions traversing (Tl⁺-flux) Kv11.1 potassium channels (black arrows). Tl⁺ binding to an intracellular indicator (Thallos AM) generates fluorescence. Fluorescence (F) recordings are normalized to baseline fluorescence (F₀). Pharmacological chaperone treatment increases Kv11.1 variant trafficking and surface expression, resulting in a larger fluorescence signal (red trace) generated by Tl⁺ flux. (C) Median robust z scores were calculated by taking the difference of slope between drug-treated (10 μmol/L) wells and the median slope for the same plate [(ΔF/s)_drug – (ΔF/s)_median] and then dividing by the median absolute deviation (MAD) of that individual plate (see Methods and Supplemental Methods). Dot plots show the results of 1,680 clinically used drugs screened in 2 trafficking-deficient Kv11.1 variants and then grouped by drug class. The median of the robust z score from replicate screens (n = 2–4 wells/drug) is shown with hits designated as median robust z scores ≥3 (dotted red line).