K potassium channels generate leak currents that stabilize the resting membrane potential of excitable cells. Various K 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 K2.1(TREK-1), the founding member of the thermo- and mechanosensitive TREK subfamily; however, its mechanism of action and effects on other K 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 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 function. BL-1249 displays selectivity among the TREK subfamily, activating K2.1(TREK-1) and K10.1(TREK-2) ∼10-fold more potently than K4.1(TRAAK). Investigation of mutants and K2.1(TREK-1)/K4.1(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 channels and provide crucial information for the development of better and more selective K modulators of the TREK subfamily.
The trinuclear ruthenium amine Ruthenium Red (RuR) inhibits diverse ion channels including K2P potassium channels, TRPs, the mitochondrial calcium uniporter, CALHMs, ryanodine receptors, and Piezos. Despite this extraordinary array, there is very limited information for how RuR engages its targets. Here, using X-ray crystallographic and electrophysiological studies of an RuR-sensitive K2P, K2P2.1 (TREK-1) I110D, we show that RuR acts by binding an acidic residue pair comprising the 'Keystone inhibitor site' under the K2P CAP domain archway above the channel pore. We further establish that Ru360, a dinuclear ruthenium amine not known to affect K2Ps, inhibits RuR-sensitive K2Ps using the same mechanism. Structural knowledge enabled a generalizable RuR 'super-responder' design strategy for creating K2Ps having nanomolar sensitivity. Together, the data define a 'finger in the dam' inhibition mechanism acting at a novel K2P inhibitor binding site. These findings highlight the polysite nature of K2P pharmacology and provide a new framework for K2P inhibitor development..
For many voltage-gated
ion channels (VGICs), creation of a properly functioning ion channel
requires the formation of specific protein–protein interactions
between the transmembrane pore-forming subunits and cystoplasmic accessory
subunits. Despite the importance of such protein–protein interactions
in VGIC function and assembly, their potential as sites for VGIC modulator
development has been largely overlooked. Here, we develop meta-xylyl (m-xylyl) stapled peptides that
target a prototypic VGIC high affinity protein–protein interaction,
the interaction between the voltage-gated calcium channel (CaV) pore-forming subunit α-interaction domain (AID) and
cytoplasmic β-subunit (CaVβ). We show using
circular dichroism spectroscopy, X-ray crystallography, and isothermal
titration calorimetry that the m-xylyl staples enhance
AID helix formation are structurally compatible with native-like AID:CaVβ interactions and reduce the entropic penalty associated
with AID binding to CaVβ. Importantly, electrophysiological
studies reveal that stapled AID peptides act as effective inhibitors
of the CaVα1:CaVβ interaction
that modulate CaV function in an CaVβ
isoform-selective manner. Together, our studies provide a proof-of-concept
demonstration of the use of protein–protein interaction inhibitors
to control VGIC function and point to strategies for improved AID-based
CaV modulator design.
The trinuclear ruthenium amine Ruthenium Red (RuR) inhibits diverse ion channels including K2P potassium channels, TRPs, the mitochondrial calcium uniporter, CALHMs, ryanodine receptors, and Piezos. Despite this extraordinary array, there is very limited information for how RuR engages its targets. Here, using X-ray crystallographic and electrophysiological studies of an RuR-sensitive K2P, K2P2.1 (TREK-1) I110D, we show that RuR acts by binding an acidic residue pair comprising the 'Keystone inhibitor site' under the K2P CAP domain archway above the channel pore. We further establish that Ru360, a dinuclear ruthenium amine not known to affect K2Ps, inhibits RuR-sensitive K2Ps using the same mechanism. Structural knowledge enabled a generalizable RuR 'super-responder' design strategy for creating K2Ps having nanomolar sensitivity. Together, the data define a 'finger in the dam' inhibition mechanism acting at a novel K2P inhibitor binding site.These findings highlight the polysite nature of K2P pharmacology and provide a new framework for K2P inhibitor development.
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