Abstract
TRAAK (KCNK4, K2P4.1) is a two-pore domain potassium channel (K2P) and like other K2Ps is responsible for carrying background (or leak) potassium current. K2P channels contribute to the maintenance of cellular resting membrane potential and therefore their activation leads to a reduction in cellular excitability. There are 15 members of the K2P superfamily, which are further divided into six subfamilies. TRAAK is a member of the TREK subfamily. Multiple lines of evidence have suggested a role for TREK-1 and TREK-2 in pain signalling. Similarly, studies with TRAAK KO rodent models have demonstrated a reduced threshold for painful stimuli and human genetic studies have shown an association between TRAAK and post-surgical neuropathic pain. Taken together this data suggests that TRAAK could be an attractive target for novel pain therapeutics.
In common with other K2P’s, a lack of pharmacological tools has hindered the investigation of TRAAK’s contribution to pathophysiology. To address this, we sought to establish a thallium flux assay suitable for the identification of TRAAK channel activators. A BacMam transduction system was developed to express TRAAK in U-2 OS cells. Initially, a set of known K2P modulators was screened against TRAAK to identify tool compounds and confirm pharmacology at TRAAK. A pilot screen was then completed using a library of FDA approved and drug like molecules. Hit compounds were subsequently screened against related TREK channels and then other K2P’s to probe selectivity. A library of 11,000 compounds, representing LifeArc’s larger collection, was then screened and several novel TRAAK activators were identified. The screen delivered excellent screening statistics (Mean Z’ = 0.68 and Mean Signal:Background = 17.8) and a ‘hit’ rate of 0.3% which is in line with previous screens for K2P channel activators. The ‘hit’ compounds were analysed using automated patch clamp electrophysiology (SyncroPatch) to confirm activity at TRAAK.
We have successfully developed a novel cell-based assay for TRAAK, which is suitable for the identification of channel activators. We have identified several tool activators, with initial data suggesting that these compounds selectively activate TRAAK compared to TREK-1 and TREK-2. These are some of the first selective activators of TRAAK to be reported and they will help us to gain a greater understanding of the physiological roles of TRAAK in pain. These molecules will also enable us to provide further validation for TRAAK as a potential therapeutic target.
