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The halothane-inhibited K 2P leak potassium channel K 2P 13.1 (THIK-1) 1-3 is found in diverse cells 1,4 including neurons 1,5 and microglia 6-8 where it affects surveillance6, synaptic pruning7, phagocytosis7, and inflammasome-mediated interleukin-1β release 6,8,9 . As with many K 2P s 1,5,10-14 and other voltage-gated ion channel (VGIC) superfamily members 3,15,16 , polyunsaturated fatty acid (PUFA) lipids modulate K 2P 13.1 (THIK-1) 1,5,14,17 via a poorly understood mechanism. Here, we present cryo-electronmicroscopy (cryo-EM) structures of human K 2P 13.1 (THIK-1) and mutants in lipid nanodiscs and detergent. These reveal that, unlike other K 2P s 13,18-24 , K 2P 13.1 (THIK-1) has a two-chamber aqueous inner cavity obstructed by a M4 transmembrane helix tyrosine (Tyr273, the flow restrictor). This hydrophilic barrier can be opened by an activatory mutation, S136P 25 , at natural break in the M2 transmembrane helix and by intrinsic channel dynamics. The structures also reveal a buried lipid in the P1/M4 intersubunit interface at a location, the PUFA site, that coincides with the TREK subfamily K 2P modulator pocket for small molecule agonists 18,26,27 . This overlap, together with the effects of mutation on K 2P 13.1 (THIK-1) PUFA responses, indicates that the PUFA site lipids are K 2P 13.1 (THIK-1) cofactors. Comparison with the PUFA-responsive VGIC Kv7.1 (KCNQ1) 28-31 reveals a shared role for the equivalent pore domain intersubunit interface in lipid modulation, providing a framework for dissecting the effects of PUFAs on the VGIC superfamily. Our findings reveal the unique architecture underlying K 2P 13.1 (THIK-1) function, highlight the importance of the P1/M4 interface in control of K 2P s by both natural and synthetic agents, and should aid development of THIK subfamily modulators for diseases such as neuroinflammation 6,32 and autism 6 ., Competing Interests: Competing interests The authors declare no competing interests.