K2P2.1 (TREK-1)–activator complexes reveal a cryptic selectivity filter binding site

Crystal structures of an activated two-pore potassium channel reveal a cryptic binding pocket that binds small-molecule activators that restrict the mobility of the selectivity filter and surrounding structure, stabilizing an active ‘leak-mode’ conformation. Binding pocket guards potassium channel gateway The two-pore domain potassium channels (K 2P ) are dimeric channels of the voltage-gated ion channel superfamily and are responsive to temperature, mechanical stimuli and small molecules. Despite their physiological importance in pain and temperature perception as well as mood regulation, their pharmacology (how small molecules modulate their activity) is not well understood. In this work, crystal structures of an activated K 2P channel are reported, revealing a cryptic binding pocket behind the selectivity filter of the chanel. The small-molecule activators bind to this pocket and restrict the mobility of the selectivity filter and the surrounding structure. This stabilizes a 'leak-mode' conformation, activating the channel and allowing current to leak through. Polymodal thermo- and mechanosensitive two-pore domain potassium (K 2P ) channels of the TREK 1 subfamily generate ‘leak’ currents that regulate neuronal excitability, respond to lipids, temperature and mechanical stretch, and influence pain, temperature perception and anaesthetic responses 1 , 2 , 3 . These dimeric voltage-gated ion channel (VGIC) superfamily members have a unique topology comprising two pore-forming regions per subunit 4 , 5 , 6 . In contrast to other potassium channels, K 2P channels use a selectivity filter ‘C-type’ gate 7 , 8 , 9 , 10 as the principal gating site. Despite recent advances 3 , 11 , 12 , poor pharmacological profiles of K 2P channels limit mechanistic and biological studies. Here we describe a class of small-molecule TREK activators that directly stimulate the C-type gate by acting as molecular wedges that restrict interdomain interface movement behind the selectivity filter. Structures of K 2P 2.1 (also known as TREK-1) alone and with two selective K 2P 2.1 (TREK-1) and K 2P 10.1 (TREK-2) activators—an N -aryl-sulfonamide, ML335, and a thiophene-carboxamide, ML402—define a cryptic binding pocket unlike other ion channel small-molecule binding sites and, together with functional studies, identify a cation–π interaction that controls selectivity. Together, our data reveal a druggable K 2P site that stabilizes the C-type gate ‘leak mode’ and provide direct evidenc