Selective small molecule activators of TREK-2 channels stimulate DRG c-fiber nociceptor K2P currents and limit calcium influx.
AUTHORS
- PMID: 27805811[PubMed].
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 activators 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 DRG c-fiber Ca(2+) influx. Interestingly, some of the selective TREK-2 activators such as 11-deoxy Prostaglandin F2α were found to inhibit the TREK-1 K2P channel. 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 2(nd) pore domain containing extracellular loop and is predicted to play an important role in modulating the activity of TREK channels. 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.
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 activators 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 DRG c-fiber Ca(2+) influx. Interestingly, some of the selective TREK-2 activators such as 11-deoxy Prostaglandin F2α were found to inhibit the TREK-1 K2P channel. 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 2(nd) pore domain containing extracellular loop and is predicted to play an important role in modulating the activity of TREK channels. 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.