Selective Small Molecule Activators of TREK-2 Channels Stimulate Dorsal Root Ganglion c-Fiber Nociceptor Two-Pore-Domain Potassium Channel Currents and Limit Calcium Influx

Abstract

The two-pore-domain potassium (K2P) channel TREK-2 serves to modulate plasma membrane potential in dorsal root ganglia c-fiber nociceptors, which tunes electrical excitability and nociception. Thus, TREK-2 channels are considered a potential therapeutic target for treating pain; however, there are currently no selective pharmacological tools for TREK-2 channels. Here we report the identification of the first TREK-2 selective activators using a high-throughput fluorescence-based thallium (Tl+) flux screen (HTS). An initial pilot screen with a bioactive lipid library identified 11-deoxy prostaglandin F2α as a potent activator of TREK-2 channels (EC50 ≈ 0.294 µM), which was utilized to optimize the TREK-2 Tl+ flux assay (Z′ = 0.752). A HTS was then performed with 76 575 structurally diverse small molecules. Many small molecules that selectively activate TREK-2 were discovered. As these molecules were able to activate single TREK-2 channels in excised membrane patches, they are likely direct TREK-2 activators. Furthermore, TREK-2 activators reduced primary dorsal root ganglion (DRG) c-fiber Ca2+ influx. Interestingly, some of the selective TREK-2 activators such as 11-deoxy prostaglandin F2α were found to inhibit the K2P channel TREK-1. Utilizing chimeric channels containing portions of TREK-1 and TREK-2, the region of the TREK channels that allows for either small molecule activation or inhibition was identified. This region lies within the second pore domain containing extracellular loop and is predicted to play an important role in modulating TREK channel activity. Moreover, the selective TREK-2 activators identified in this HTS provide important tools for assessing human TREK-2 channel function and investigating their therapeutic potential for treating chronic pain.

Characterization of temperature-sensitive leak K+ currents and expression of TRAAK, TREK-1, and TREK2 channels in dorsal root ganglion neurons of rats

“…In heterologous expression system, riluzole produces phasic potentiation followed by inhibition of TREK-1 and TREK-2 channels, and it also produces tonic potentiation of TRAAK channels; norfluoxetine inhibits both TREK-1 and TREK2 channels [ 7 , 18 ]. Effects of PGF2α on leak K + channels have not been reported previously but its derivative 11-deoxy-PGF2α has been shown to inhibit TREK-1 and activate TREK-2 channel [ 5 ]. We show that all three compounds produced inhibitory effects on temperature-sensitive leak K + currents.…”

“…On the other hand, K2P channel activators may decrease nociceptors’ excitability and may be used to alleviate pathological pain. Efforts have been put recently to synthesize selective K2P channel activators for potential therapeutic uses for pain management [ 5 , 19 ].…”

GI‐530159, a novel, selective, mechanosensitive two‐pore‐domain potassium (K2P) channel opener, reduces rat dorsal root ganglion neuron excitability

“…Also recently, Dadi et al . () have shown that PG F2α and a number of other small molecules activate TREK2 channels and stimulate K 2P currents in a proportion of DRG neurons. 2‐Aminoethoxydiphenyl borate has also been suggested to be a selective activator of TREK2 channels (Zhuo et al ., ).…”

TALK-1 channels control β cell endoplasmic reticulum Ca 2+ homeostasis

“…To confirm the specificity of the effect of TALK-1 on Ca 2+ ER , we compared Ca 2+ ER storage in cells stably and inducibly expressing different K2P channels (43) (Fig. 4A, Fig.…”

“…The cells were then washed with buffer supplemented with 1 mM EGTA and incubated in a 5% CO 2 incubator at 37 °C for 8 minutes before imaging. Plates were then loaded into a whole-plate kinetic-imaging Functional Drug Screening System (FDSS 6000, Hamamatsu, Bridgewater, NJ) and imaged at 37 °C as previously described (43). …”