Swapped and non-swapped TRAAK states co-exist in membranes at a ratio influenced by temperature

The potassium two-pore domain (K2P) ion channel TRAAK is expressed in the nervous system and regulates the fast action potential in membranes. Like all K2P channels, TRAAK possesses a distinct extracellular cap, which adopts a swapped and a non-swapped conformation^1,2. However, the proportional representation of these two species within native membranes - and the trigger or stimulus associated with this conformational transition - are unknown. Here, we utilise Pulse Dipolar EPR Spectroscopy combined with heterologous single-subunit spin-labelling and monitor the complete conformational ensemble of TRAAK's cap domain in membranes. We demonstrate the coexistence of the swapped and non-swapped states, quantify their populations within the TRAAK ensemble, and show that the swapped conformation dominates, with the ratio being influenced by temperature. Native lipid analysis shows that TRAAK selectively associates with and is activated by signalling lipids to the exclusion of membrane-dominant phosphatidylcholine lipids from its vicinity, forming a distinct microdomain. Our approach can identify the immediate lipid environment, detect and quantify cap state populations in homo-/hetero-K2P channels and link domain swapping to specific triggers.