BPS2026 – Probing uncharted conformational ensembles of K2P channels in membranes by pulse dipolar EPR spectroscopy

The human potassium two-pore domain (K2P) ion channel TRAAK is a critical regulator of action potentials in the nervous system. While its unique extracellular cap is known to exist in swapped and non-swapped conformations, the native membrane population dynamics and the triggers for this transition are unclear. We here employ pulse dipolar EPR spectroscopy (PDS) combined with purposely developed heterologous single-subunit spin-labeling (HSS-SL) to explore the entire conformational landscape of TRAAK's cap in native membranes. We demonstrate that both the swapped and non-swapped states coexist within the channel's ensemble and quantitatively establish that the swapped state is predominant. We show that the population ratio of these states is temperature-sensitive. Native lipid analysis further reveals that TRAAK selectively forms a specialized microdomain by associating with and activated by specific signaling lipids, while excluding common phosphatidylcholine from its vicinity. Our combined methodology provides a robust platform for monitoring and quantifying conformational ensembles and identifying the triggers associated with such transitions in K2P channels and other membrane proteins.