Polymodal K2P (KCNK) thermo- and mechanosensitive TREK1 potassium channels, generate ‘leak’ currents that regulate neuronal excitability, respond to lipids, temperature, and mechanical stretch, and influence pain, temperature perception, and anesthetic responses1–3. These dimeric voltage-gated ion channel (VGIC) superfamily members have a unique topology comprising two pore forming regions per subunit4–6. Contrasting other potassium channels, K2Ps use a selectivity filter ‘C-type’ gate7–10 as the principal gating site. Despite recent advances3,11,12, K2Ps suffer from a poor pharmacologic profile limiting mechanistic and biological studies. Here, we describe a new small molecule TREK activator class that directly stimulates the C-type gate by acting as molecular wedges that restrict interdomain interface movement behind the selectivity filter. Structures of K2P2.1(TREK-1) alone with two selective K2P2.1(TREK-1) and K2P10.1(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 unveil a previously unknown, druggable K2P site that stabilizes the C-type gate ‘leak mode’ and provide direct evidence for K2P selectivity filter gating.

Voltage-gated sodium channels (NaVs) are central elements of cellular excitation. Notwithstanding advances from recent bacterial NaV (BacNaV) structures, key questions about gating and ion selectivity remain. Here, we present a closed conformation of NaVAe1p, a pore-only BacNaV derived from NaVAe1, a BacNaV from the arsenite oxidizer Alkalilimnicola ehrlichei found in Mono Lake, California, that provides insight into both fundamental properties. The structure reveals a pore domain in which the pore-lining S6 helix connects to a helical cytoplasmic tail. Electrophysiological studies of full-length BacNaVs show that two elements defined by the NaVAe1p structure, an S6 activation gate position and the cytoplasmic tail ‘neck’, are central to BacNaV gating. The structure also reveals the selectivity filter ion entry site, termed the ‘outer ion’ site. Comparison with mammalian voltage-gated calcium channel (CaV) selectivity filters, together with functional studies shows that this site forms a previously unknown determinant of CaV high affinity calcium binding. Our findings underscore commonalities between BacNaVs and eukaryotic voltage-gated channels and provide a framework for understanding gating and ion permeation in this superfamily.

SUMMARY Mechanical and thermal activation of ion channels is central to touch, thermosensation, and pain. The TRAAK/TREK K2P potassium channel subfamily produces background currents that alter neuronal excitability in response to pressure, temperature, signaling lipids, and anesthetics. How such diverse stimuli control channel function is unclear. Here we report structures of K2P4.1 (TRAAK) bearing C-type gate-activating mutations that reveal a tilting and straightening of the M4 inner transmembrane helix and a buckling of the M2 transmembrane helix. These conformational changes move M4 in a direction opposite to that in classical potassium channel activation mechanisms and open a passage lateral to the pore that faces the lipid bilayer inner leaflet. Together, our findings uncover a unique aspect of K2P modulation, indicate a means for how the K2P C-terminal cytoplasmic domain affects the C-type gate which lies ~40Å away, and suggest how lipids and bilayer inner leaflet deformations may gate the channel.

Background: HCN2 and HCN4 respond to cAMP, whereas HCN1 does not. Results: The C-linker plus CNBD of HCN2 and HCN4 show cAMP-induced tetramerization, whereas that of HCN1 contains prebound cAMP and is tetrameric. Conclusion: HCN1 does not respond to the addition of cAMP because its CNBD contains cAMP already. Significance: Tetramerization of the C terminus controls ligand gating in HCN channels.