Abstract
K2P potassium channels generate leak currents that stabilize the resting membrane potential of excitable cells. Various K2P 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 K2P2.1(TREK-1), the founding member of the thermo- and mechanosensitive TREK subfamily; however, its mechanism of action and effects on other K2P channels are not well-defined. Here, we demonstrate that BL-1249 extracellular application activates all TREK subfamily members but has no effect on other K2P 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 K2P function. BL-1249 displays selectivity among the TREK subfamily, activating K2P2.1(TREK-1) and K2P10.1(TREK-2) ∼10-fold more potently than K2P4.1(TRAAK). Investigation of mutants and K2P2.1(TREK-1)/K2P4.1(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 K2P channels and provide crucial information for the development of better and more selective K2P modulators of the TREK subfamily.
Highlights
K2P (KCNK) potassium channels are members of the voltagegated ion channel (VGIC) superfamily, make “background” or “leak” potassium channels that are responsible for the maintenance of cellular resting potential, and play an important role in regulating cellular excitability.[1−3] There are 15 K2P subtypes that form six functionally distinct subfamilies
Our studies show that this compound is a selective agonist of the TREK subfamily when applied extracellularly, having preferential action on K2P2.1(TREK-1) and K2P10.1(TREK-2) over K2P4.1(TRAAK) and establish that its mechanism of action relies on gating at the selectivity filter C-type gate
To test the ability of the chimeras to report on channel determinants for compound action, we examined the responses of the chimeras to two previously characterized activators, ML335, a compound that selectively activates K2P2.1(TREK-1) but not K2P4.1(TRAAK),[6] and ML67-33, an activator showing no clear preference for either channel.[26]
Summary
K2P (KCNK) potassium channels are members of the voltagegated ion channel (VGIC) superfamily, make “background” or “leak” potassium channels that are responsible for the maintenance of cellular resting potential, and play an important role in regulating cellular excitability.[1−3] There are 15 K2P subtypes that form six functionally distinct subfamilies. In contrast to these results, we found no major changes with respect to the responses of the various chimeras to ML67-33 (Figure S1D−F,H) These findings are consistent with the inability of ML67-33 to discriminate between K2P2.1(TREK-1) and K2P4.1(TRAAK).[26] Together, these studies show that this chimera set can identify selectivity determinants for activator compounds within the TREK subfamily. Proved very detrimental to activity and yielded a compound that had only a small amount of stimulatory effect against K2P2.1(TREK-1) (EC50 > 200 μM) and showed a similar profile against K2P4.1(TRAAK) revealing the importance of the bicyclic tetralin ring for BL-1249 function (Figure 6B,E). Our studies demonstrate that both the acidic and tetralin moieties are important contributors to the stimulatory action of BL-1249 against K2P2.1(TREK-1) and indicate that the mobility of the two aryl rings relative to each other is key to its selective effects on K2P2.1(TREK-1) over K2P 4.1(TRAAK). AData derived from at least two independent experiments with each data point averaged from at least three oocytes. bEC50 estimated imposing an upper boundary of 15 (fold activation, I/I0). cEC50 estimated imposing an upper boundary of 20 (fold activation, I/I0)
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