TRESK: Sensory Physiology and Pain Identification of Channel Activators and Inhibitors

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TRESK (TWIK-RElated Spinal cord K+) belongs to the K2P family of potassium channels, known for their role in regulating the excitability of specific subtypes of sensory neurons, suggesting that TRESK may have a role in pain sensitivity. 

In this study, we examine the activity of TRESK expression in cells to identify both activators and inhibitors of the TRESK channel. We used a selected subset of 20,000 compounds screened in the FLuorescence Imaging Plate (FLIPR) reader. Furthermore TREK, a closely associated potassium channel, was screened as a selectivity assay. TREK is expressed in the heart and we needed to identify compounds that had TRESK and not TREK activity.

TRESK: Sensory Physiology and Pain – Identification of Channel Activators and Inhibitors

Potassium channels are the largest family of ion channels, with about 80 genes encoding for alpha subunits. Four large families of K+ channels have been identified in mammals according to their structural and functional characteristics: voltage-gated K+ (Kv) channels, Ca2+-activated K+ (KCa) channels, inwardly rectifying K+ (Kir) channels, and two-pore domain background K+ (K2P) channels. TRESK belongs to the K2P family.

Among the different K+ channel families, the family of K2P K+ channels is the last one identified and described, which, to date, has 15 members, grouped into six subfamilies (TWIK, TREK, TASK, TALK, THIK and TRESK) based on sequence and functional similarities. TRESK (K2P18, KCNK18) is the only K2P regulated by intracellular Ca2+ concentration through calcineurin-mediated dephosphorylation. 

In contrast to other K+ channel families that have one pore-forming domain for each subunit, K2P channels have two pore domains and four transmembrane domains. K2P channel activity produces constitutive leak currents that are mostly independent of the membrane potential. Their main role in most cell types is attributed to the regulation of membrane potential, as they constitute the leak of potassium through the plasma membrane (accordingly they are commonly referred to as leak or background potassium channels). They then equilibrate with the function of the Na+/K+ pump and help to set the resting membrane potential. 

For this reason, they influence neuronal excitability over a wide range of membrane potentials, especially between resting and the action potential threshold. Thus, K2P channels maintain sustained K+ conductance to establish the resting membrane potential in neurons, where they can also shape the duration, frequency, and amplitude of the action potential.


To identify activators and inhibitors of the TRESK channel that are specific for the TRESK channel by screening 20,000 compounds as a High Throughput Screen (HTS). This was followed by retest, dose-response and selectivity screening against TREK. These compounds were then further studied in electrophysiology assays and examination of in vivo activity.

TRESK screening cascade

Figure 1. Screening cascade designed to identify compounds with human TRESK channel stimulatory activity. Initially developed as a 384 well TRESK kinetic assay using the FluxOR kit, screen 20,000 compounds, selectivity test hits against wide type U2OS cells followed by selectivity against TREK, dose-response, and further SAR work.

What We Did...

We utilized the FLIPR instruments in our laboratory to study the activity of the TRESK channel using a Thallium Flux kit called FluxOR from Molecular Probes. The FluxOR Potassium Ion Channel Assay is a solution used for the screening of potassium ion channel and transporter activities.

The assay takes advantage of the well described permeability of potassium channels to thallium (Tl+) ions. When thallium is added to the extracellular solution with a stimulus to open channels, thallium flows down its concentration gradient into the cells, and channel or transporter activity is detected with a proprietary indicator fluorogenic dye that increases in cytosolic fluorescence. In this way, the fluorescence reported becomes an indicator of any ion channel activity or transport process that allows thallium into cells.

By monitoring changes in luminescent signal following treatment with autophagy inducers or inhibitors, we were able to accurately assess alterations in autophagic flux. Notably, the NanoBiT assay demonstrated consistency across both 96 and 384 well formats, with pharmacological standards effectively inducing and inhibiting autophagy in a dose-dependent manner.

Assay Principles:

The FluxORTM reagent is loaded into cells as a membrane-permeable AM ester. Once inside the cell, a small amount of thallium is added to the cells with a stimulus solution that opens potassium-permeant ion channels using a mild depolarization or agonist addition.

Thallium then passes into cells through open potassium channels according to a strong inward driving force. Upon binding cytosolic thallium, the de-esterified dye exhibits a strong increase in fluorescence intensity at its peak emission of 525 nm. Baseline and stimulated fluorescence are monitored in real time to give a dynamic, functional readout of thallium redistribution across the membrane with no interference from quencher dyes. In this case we use the FLIPR to measure the movement of thallium into the cells.

TRESK fig 2 combined use

Figure 2. Left: Thallium redistribution in the FluxOR™ assay. Basal fluorescence from cells loaded with FluxOR dye is low, as shown in the “resting” panel, until potassium channels are stimulated. When thallium is added to the assay with the stimulus, the thallium flows down its concentration gradient into the cells, activating the dye as shown in the “stimulated” panel. Right: The assay protocol designed to investigate the stimulatory or inhibitory activity of the compounds.

Establishing Controls to be used for High Throughput Screening Purposes

Control wells

  • Control 1 (yellow) = Basal Activity (vehicle)
  • Control 2 (blue) – Maximal inhibition (EC100TPA)
  • Control 3 (red) = EC100 (Cloxyquin)

Data Normalisation

  • Rate of Thallium Flux at 75 seconds calculated
  • Rate data were expressed relative to Controls 1 & 2
  • Vehicle = 100%, TPA = 0%, Cloxyquin ~200%

Hit Identification

  • Hits were identified using ≥3SD (~130% response) cut off
  • Hits were selected using an arbitrary >150% response cut off
Compounds with (putative) interference e.g. elevated basal fluorescence, were identified and removed.

Figure 3. 384 well plate layout for high throughput screening purposes. As the screen was biased towards identifying inhibitors of the TRESK channel, 28 wells were used as the inhibited control and 4 wells used as controls to demonstrate the channel could be activated by Cloxyquin. Both compounds were used at an EC100 concentration in columns 1 and 2 of the 384 well plate. Columns 23 and 24 were used as the control activation level.

High Throughput Screen

66 x 384 well plates were screened in the FLIPR instrument as outlined in Figure. 2.

For the cell plate: U2OS cells were transfected in T175 flasks for 24-hours using a Bacmam construct containing the TRESK channel. Cells were removed from the flask using TryplE and dispensed at 20,000 cells per well in 50 µL of media. After a 24-hour incubation, media was removed and replaced with 15 µL of assay buffer.

For compound plates: 1 mM compound at 100% DMSO was laid out in columns 3 through 22 of the source compound plate, and 2 µL dispensed and diluted in an intermediate daughter plate to 40 µM in diluent buffer to 4% DMSO, then 5 µL of this was added to the cell plate and incubated for 30 min at room temperature. Control standard pharmacology compounds were also added at this point.

The cell plate was placed into the FLIPR and, whilst reading (1 second update for 260 seconds), 2 mM thallium was added in 5 µL of buffer using the FLIPR pipetting head.

  • 66 library plates screened
  • 21,120 compounds screened (28,416 wells)
  • Single-shot @10 µM (1% DMSO)
  • S:B = 3.1 (±0.9)
    Range 5.2 – 2.0
  • 3xSD (statistical) cut off = 133% (±12)
    Range 158%-115%

Figure 4. Control Data: Left: Data for 66 x 384 well plates expressed as the rate of flux per second for controls TPA, Cloxyquin and DMSO across each of the 66 plates. Right: The same data expressed as a percent response using the average DMSO response for each plate expressed as the 100% response.

  • Control peaks at 100% and 0% with no obvious skew
  • Samples cluster tightly around 100% (no effect) in keeping with null hypothesis

Clear evidence of skew in negative tail due to the (relatively) high abundance of inhibitors

Based on this data, the controls appeared to be statistically reproducible and there were compounds that were channel activators in this collection of compounds. 

These were catagorized based on activity as shown below (figure 6).

Figure 5. “Hit” Compound Distribution Profiles. Left: Number of hits versus percent response for controls, centred around 0% is the Cloxyquin response and centred around 100% is the TEA response. Right: Numbers of hits versus the percent response as a frequency distribution plot.

TRESK Fig 6 new (2)

Compounds selected based on this cut-off were cherry picked from the master stock tubes. 

They were  reformatted into 384 well plates as side-by-side plate duplicates. 

These were re-screened in the original assay format (see figure. 8).

Figure 6. Distribution of hits from the HTS. A cut off greater than 150% gave a hit rate of 0.5%, broken down below based on isolation into different activity “bins”.

  • Hit confirmation rate ~65%
  • Highly reproducible

Based on this cut off and retest confirmation, compounds were selected for dose-response screening in the FLIPR.

As shown in Figure 8, hits were selected from the master stock tubes, not the daughter 384 well plates.

These were arrayed as 10-point dose-response in duplicate, 16 compounds per plate plus Cloxyquin and TEA as controls.

Figure 7. Retest “Hit” Confirmation. Compounds were re-arrayed from 96 well mother plates into 384 well daughter plates for screening. Controls clustered around 0% (TPA – blocked channel – green dots) and 100% (DMSO – blue dots) plus activation of the channel using Cloxyquin (red dots). The hit confirmation rate was 65%, confirming that the assay was highly reproducible with an R2 of 0.9 (yellow dots).

TRESK fig 8

Figure 8. Compounds for dose-response were again cherry picked from the master stock plates as shown.


Compounds achieving greater than 150% cut off were tested in the TRESK assay as dose response. Most of these showed EC50 activity in the 10µM range comparing favorably with Cloxyquin.

TRESK fig 9

Figure. 9. Example dose-responses of 16 hit compounds run alongside Cloxyquin in the TRESK FluxOR assay.

At this stage we need to distinguish TRESK selectivity from those with activity on the TREK channel. Using the FluxOR reagents, we developed a TREK assay using a commercially available human TREK channel expressed in a HEK293T cell line. 

Again, TEA was used as the channel inhibitor, whilst BL-1249 was used to stimulate the opening of the channel.


Following validation of the TREK assay with these standard control compounds, we screened compounds that demonstrated a dose-response in the TRESK assay against the TREK channel. 

Only 2 of the hits from the TRESK screen crossed over into the TREK assay (figure. 11).


Figure 10. TREK assay control data. Channel opening was blocked by TEA, while channel opening was stimulated with BL-1249.

Amost all hits are ‘TRESK specific’

Following on from the HTS and selectivity screen, the chemistry workflow proceeded as per below.  

Favored structures were used to explode the Structure Activity Relationship (SAR). 

These compounds were evaluated in both the TRESK and TREK FluxOR assays. 

This was in addition to more phenotypic electrophysiology assays and further ion channel selectivity screening and eventually in vivo.

Figure. 11. Correlation of TRESK versus TREK compounds screened at 10 µM in both assays on the same day using the same compound source plates.

Chemistry Workflow

Following on from the HTS and selectivity screen, the chemistry workflow proceeded as per the below. 

TRESK fig 12 chemistry workflow


In conclusion, we have developed a FLIPR based FluxOR assay to examine the activity of the TRESK two-pore potassium channel.

The assay was developed in 384 well format and used to screen over 20,000 compounds, looking to identify both channel inhibitors and channel activators.

Hit compounds were retested and compounds with activator activity greater than 150% were screened as dose-response.

Hit compounds were re-screened for selectivity against the TREK channel, most showed selectivity.

Hit compounds went on for further iteration in more detailed downstream cascade assays.