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.
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.
Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center , Nashville, Tennessee 37232, United States.
Prasanna K
PK
Dadi
Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center , Nashville, Tennessee 37232, United States.
Nicholas C
NC
Vierra
Institute of Chemical Biology, Vanderbilt University Medical Center , Nashville, Tennessee 37232, United States.
Emily
E
Days
Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center , Nashville, Tennessee 37232, United States.
Matthew T
MT
Dickerson
Department of Biochemistry, Vanderbilt University Medical Center , Nashville, Tennessee 37232, United States.
Paige N
PN
Vinson
Department of Pharmacology, Vanderbilt University Medical Center , Nashville, Tennessee 37232, United States.
C David
CD
Weaver
Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center , Nashville, Tennessee 37232, United States.
David A
DA
Jacobson
D000200:Action Potentials / Q000187:drug effects / Q000502:physiology*; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015232:Dinoprostone / Q000031:analogs & derivatives / Q000494:pharmacology; D004558:Electric Stimulation; D005473:Fluoxetine / Q000494:pharmacology; D005727:Ganglia, Spinal / Q000166:cytology*; D057809:HEK293 Cells; D006801:Humans; D037102:Lectins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation / Q000235:genetics; D009619:Nociceptors / Q000187:drug effects* / Q000378:metabolism*; D026902:Potassium Channel Blockers / Q000494:pharmacology; D024683:Potassium Channels, Tandem Pore Domain / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013752:Tetracycline / Q000494:pharmacology
C481059:11-deoxy,16,16-dimethyl prostaglandin E(2); D000906:Antibodies; C503445:Kcnk10 protein, mouse; D037102:Lectins; D026902:Potassium Channel Blockers; D024683:Potassium Channels, Tandem Pore Domain; D011500:Protein Synthesis Inhibitors; C103742:potassium channel protein TREK-1; D005473:Fluoxetine; D013752:Tetracycline; D015232:Dinoprostone; D002118:Calcium
DRG neuron; TREK-1; TREK-2; pain; thallium flux; two-pore-domain potassium channel