Protein and Chemical Determinants of BL-1249 Action and Selectivity for K_2P Channels

K_2P potassium channels generate leak currents that stabilize the resting membrane potential of excitable cells. Various K_2P channels are implicated in pain, ischemia, depression, migraine, and anesthetic responses, making this family an attractive target for small molecule modulator development efforts. BL-1249, a compound from the fenamate class of nonsteroidal anti-inflammatory drugs is known to activate K_2P2.1(TREK-1), the founding member of the thermo- and mechanosensitive TREK subfamily; however, its mechanism of action and effects on other K_2P channels are not well-defined. Here, we demonstrate that BL-1249 extracellular application activates all TREK subfamily members but has no effect on other K_2P subfamilies. Patch clamp experiments demonstrate that, similar to the diverse range of other chemical and physical TREK subfamily gating cues, BL-1249 stimulates the selectivity filter "C-type" gate that controls K_2P function. BL-1249 displays selectivity among the TREK subfamily, activating K_2P2.1(TREK-1) and K_2P10.1(TREK-2) ∼10-fold more potently than K_2P4.1(TRAAK). Investigation of mutants and K_2P2.1(TREK-1)/K_2P4.1(TRAAK) chimeras highlight the key roles of the C-terminal tail in BL-1249 action and identify the M2/M3 transmembrane helix interface as a key site of BL-1249 selectivity. Synthesis and characterization of a set of BL-1249 analogs demonstrates that both the tetrazole and opposing tetralin moieties are critical for function, whereas the conformational mobility between the two ring systems impacts selectivity. Together, our findings underscore the landscape of modes by which small molecules can affect K_2P channels and provide crucial information for the development of better and more selective K_2P modulators of the TREK subfamily.