A Soluble Fluorescent Binding Assay Reveals PIP₂ Antagonism of TREK-1 Channels

Lipid regulation of ion channels by low-abundance signaling lipids phosphatidylinositol 4,5-bisphosphate (PIP₂) and phosphatidic acid (PA) has emerged as a central cellular mechanism for controlling ion channels and the excitability of nerves. A lack of robust assays suitable for facile detection of a lipid bound to a channel has hampered the probing of the lipid binding sites and measuring the pharmacology of putative lipid agonists for ion channels. Here, we show a fluorescent PIP₂ competition assay for detergent-purified potassium channels, including TWIK-1-related K⁺-channel (TREK-1). Anionic lipids PA and phosphatidylglycerol (PG) bind dose dependently (9.1 and 96 μM, respectively) and agonize the channel. Our assay shows PIP₂ binds with high affinity (0.87 μM) but surprisingly can directly antagonize TREK-1 in liposomes. We propose a model for TREK-1 lipid regulation where PIP₂ can compete with PA and PG agonism based on the affinity of the lipid for a site within the channel.