Investigating the correlation between thallium flux and automated patch-clamp for ion channel activators

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Case Study

Case Study title

Investigating the correlation between thallium flux and automated patch-clamp for ion channel activators

Authors

David McCoull¹, Jonathan M Large¹, Alex Loucif², Raymond Tang², Ian Witton², Lewis Byrom¹, Edward Stevens², Jeff Jerman¹, Paul D Wright¹

1. LifeArc, Accelerator Building, Open Innovation Campus, Stevenage, SG1 2FX, United Kingdom
2. Metrion Biosciences Ltd, Riverside 3; Suite 1, Granta Park, Cambridge, CB21 6AD, United Kingdom

Overview

• Ion channels play a key role in regulating resting membrane potential and cell excitability and are attractive targets for therapeutic intervention. 

• Thallium (Tl⁺) flux assays, which measure the flow of Tl⁺ through potassium channels, offer a high throughput method for the identification of potassium channel activators. However, these assays are a surrogate for channel function and it is important to have an appropriate panel of orthogonal and translational electrophysiology assays in place to confirm activity at the channel of interest. 

• We used a Tl⁺ flux assay to screen a library of 100K compounds and identified 173 ‘hit’ compounds as potential activators of a K₂P potassium channel. These compounds were then screened in an automated patch-clamp (APC) assay to confirm activity. 

• Compounds with the highest activity in Tl⁺ flux were the most likely to confirm in automated patch-clamp, but the correlation between the two systems was not entirely uniform. 

• Aim: to investigate the correlation between thallium flux and automated patch-clamp.

Methods

Figure 1: Schematic showing the use of intracellular thallium-sensitive dye to measure ion conduction through potassium channels as a screening assay to measure the effects of modulator compounds.

Thallium Flux

• FLIPR Potassium Assay Kit (Molecular Devices, USA) used according to manufacturers’ guidelines. 

• Tl⁺ sensitive dye within the cell detects the flow of Tl⁺ through potassium channels. 

• Typically, compounds are pre-incubated with cells for 30 minutes prior to Tl⁺ addition. Alternative pre-incubation periods were studied. 

• 5-10K cells per well. Channel activity is measured as the rate of fluorescence increase following Tl⁺addition.

APC (QPatch)

• Cells in extracellular solution (ECS) move through a microfluidic system and suction pulls a single cell to the patch-clamp hole. 

• After a gigaseal is formed, the membrane is ruptured, allowing whole-cell electrophysiological recordings to be performed. 

• Test compounds are applied to the inlet wells. Continuous recording allows multiple concentrations / compounds to be applied to a single cell. 

• Perforated-cell patch-clamp is a variant of traditional patch clamp, where pores are formed in the membrane, allowing electrical access but maintaining cytoplasmic constituents within the cell.

Figure 2: Schematic illustrating an automated patch clamp ‘chip’ well where cells are flowed over holes in a borosilicate substrate to obtain whole-cell access and enable patch clamp recordings of ion channel activity, and the effects of compounds added to each well (yellow).

Compound testing

• Dose response curves were prepared using either an acoustic (ECHO, Labcyte) or a tip-based (Biomek FX, Beckman Coulter) dispenser. Compounds were run in Tl⁺ flux assays with a 30 minute pre-incubation. Rate (15-28s) denotes the rate of Tl⁺ flux. (Data represent mean ± SD, n=2). 

• A subset of compounds displayed a reduction in the magnitude of the response when they were handled on the acoustic dispenser, demonstrating that the liquid handling method can influence the response in Tl⁺ flux.

Automated and Manual Patch-clamp

• Whole-cell, perforated-cell QPatch and whole-cell manual patch-clamp data for LA-05 and LA-06. (Data represent mean ± SEM, n≥1). 

• Whole-cell and perforated-cell APC methods produce comparable results for both active and inactive compounds. Manual whole-cell patch-clamp (the gold standard patch-clamp technique), confirmed APC potency and efficacy data for active compounds.

Correlation Between Tl⁺ flux and APC

Figure 5: Comparison of K2P hit compound activity obtained by thallium flux HTS assay with hit validation activity from an APC (QPatch) platform.

• 173 compounds identified as ‘hits’ in a Tl⁺ flux screen of 100K compounds were screened in APC (QPatch). 

• Plot shows correlation between Tl⁺ flux and APC data, coloured by activity in APC. 

• Tl⁺ flux data represents fold-stimulation relative to DMSO control. APC data represents percentage of maximal channel activation. 

• Compounds with the highest activity in Tl⁺ flux were the most likely to confirm in APC, but the correlation was not entirely uniform.

Figure 6: Correlation between thallium flux and APC hit compound activity is strongest for the most active compounds (bins 1-2) and for less active compounds pre-incubated in the thallium flux assay (green bars).

• Compounds were screened in Tl⁺ flux, with either a 30 minute (t=30) or no (t=0) pre-incubation. 

• Compounds were then binned based on Tl⁺ flux activity; Bin 1 highest activity to Bin 5 lowest activity. 

• For each bin, the percentage of compounds which were active in APC was then calculated. 

• Data confirmed that compounds with the highest activity in Tl⁺ flux were the most likely to be active in APC. 

• A subset of compounds showed higher activity at t=30 compared to t=0.

Compound Incubation Time

• Compounds were screened in dose response format in the Tl⁺ flux assay with either a 30 minute (t=30), a 15 minute (t=15) or no pre-incubation (t=0). Rate (15-28s) denotes the rate of Tl⁺ flux. (Data represent mean ± SD, n=2). * denotes compounds which were active in APC. 

• A subset of compounds displayed a marked time dependency in the magnitude of their response. The magnitude of the response at t=0 may be indicative of the response in APC for some compounds.

Compound Properties

• Correlation between Tl⁺ flux and APC data, coloured by ACDlogP (left) or CNSMPO (right). ACDlogP is a measure of lipophilicity and CNSMPO a score for predicted CNS penetration. 

• Neither metric could adequately explain the correlation between the two systems, but phys-chem properties of individual compounds may contribute to differential responses in the two systems.

Conclusions

• No single factor tested can adequately explain the correlation between the two systems. 

• Compound incubation time and liquid handling method can influence the response in Tl⁺ flux. 

• Looking at these factors in combination, prior to selecting compounds for electrophysiology, may enable us to prioritise screening outputs.