Two-pore domain (K2P) potassium channels are important regulators of cellular electrical excitability and are capable of integrating a wide range of signalling pathways involving phosphorylation, various lipids, membrane voltage, temperature and intracellular/extracellular pH. Over the last decade, the physiological and pathophysiological importance of K2P channels has become increasingly clear and they now represent important potential therapeutic targets in cardiovascular and neuronal disease states. However, in contrast to this rapidly increasing insight into their physiological role, the structural basis and molecular mechanisms of gating are just starting to emerge. We identified quaternary ammonium (QA) ions as high-affinity pore blockers of all tested K2P channels and used them as tools to probe the inner pore structure and dissect the gating mechanisms in K2P channels. In this context several homology models of the TREK-1 channels pore were generated and scored according to the functional data of the blocker pore interaction. Further, we employed cysteine scanning mutagenesis and state dependent cysteine modification to gain insight into the structural changes that occur in the TREK-1 channels pore during gating. Finally, we will report about the intimate coupling of gating and permeation in K2P channels by exploring the effect of different ions and voltage protocols. From these studies a picture of the gating mechanisms in K2P channel is derived.