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

ABSTRACT: 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 ?M, respectively) and agonize the channel. Our assay shows PIP2 binds with high affinity (0.87 ?M) 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.

SUBMITTER: Cabanos C

PROVIDER: S-EPMC5586213 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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A Soluble Fluorescent Binding Assay Reveals PIP<sub>2</sub> Antagonism of TREK-1 Channels.

Cabanos Cerrone C Wang Miao M Han Xianlin X Hansen Scott B SB

Cell reports 20170801 6

Lipid regulation of ion channels by low-abundance signaling lipids phosphatidylinositol 4,5-bisphosphate (PIP<sub>2</sub>) 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 PIP<s ...[more]

PMID: 28793254

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