Structural basis for differential inhibition of THIK1 by chemically related inhibitors

TWIK-related halothane-inhibited potassium channel 1 (THIK1) is a two-pore domain potassium (K2P) channel highly expressed in microglia that contributes to microglial homeostasis and neuroinflammatory responses. Pharmacological inhibition of THIK1 suppresses tonic K ⁺ currents and attenuates NLRP3-dependent IL-1β release, highlighting THIK1 as a potential therapeutic target for neuroinflammatory disorders. However, the structural basis of THIK1 inhibition remains poorly understood. Here we report cryo-electron microscopy (cryo-EM) structures of full-length human THIK1 in complex with C101248, a commercially available preclinical compound, and CVN293, an inhibitor currently advancing toward Phase II clinical trials. The two inhibitors occupy a similar site within the inner vestibule formed by TM2 and TM4 beneath the selectivity filter, implicating this region in THIK1 inhibition. Consistent with their closely related chemical scaffolds and shared binding site, both inhibitors remodel the Y273 inner gate; however, they differ in their effects on the C-terminal gate. Electrophysiological analyses further demonstrate that the C-terminal gate contributes differently to inhibition by the two compounds. Together, our findings provide structural and functional insights into how chemically related inhibitors can exert different effects on THIK1 despite engaging a common binding site.