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

Publication date: 8 August 2017
Source:Cell Reports, Volume 20, Issue 6
Author(s): Cerrone Cabanos, Miao Wang, Xianlin Han, Scott B. Hansen
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.

Graphical abstract

Teaser

Cabanos et al. detect anionic signaling lipids bound to potassium channels using a fluorescent PIP2 binding assay. PIP2 binds with high affinity, but surprisingly the lipid can antagonize TREK-1 channels by competing with the agonists phosphatidic acid and phosphatidylglycerol.


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