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...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2017-08-01
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| Series: | Cell Reports |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2211124717310008 |
| _version_ | 1811323513828540416 |
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| author | Cerrone Cabanos Miao Wang Xianlin Han Scott B. Hansen |
| author_facet | Cerrone Cabanos Miao Wang Xianlin Han Scott B. Hansen |
| author_sort | Cerrone Cabanos |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T13:56:29Z |
| format | Article |
| id | doaj.art-3b2d05653228457784817828d5ee5d80 |
| institution | Directory Open Access Journal |
| issn | 2211-1247 |
| language | English |
| last_indexed | 2024-04-13T13:56:29Z |
| publishDate | 2017-08-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Cell Reports |
| spelling | doaj.art-3b2d05653228457784817828d5ee5d802022-12-22T02:44:10ZengElsevierCell Reports2211-12472017-08-012061287129410.1016/j.celrep.2017.07.034A Soluble Fluorescent Binding Assay Reveals PIP2 Antagonism of TREK-1 ChannelsCerrone Cabanos0Miao Wang1Xianlin Han2Scott B. Hansen3Departments of Molecular Medicine and Neuroscience, The Scripps Research Institute, Jupiter, FL 33458, USACenter for Metabolic Origins of Disease, Sanford Burnham Prebys Medical Discovery Institute, 6400 Sanger Road, Orlando, FL 32827, USACenter for Metabolic Origins of Disease, Sanford Burnham Prebys Medical Discovery Institute, 6400 Sanger Road, Orlando, FL 32827, USADepartments of Molecular Medicine and Neuroscience, The Scripps Research Institute, Jupiter, FL 33458, USALipid 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.http://www.sciencedirect.com/science/article/pii/S2211124717310008PIP2TREKTRAAKKirpotassium channelfluorescent assaylipid bindingarachidonic acidsignaling lipidphosphatidic acid |
| spellingShingle | Cerrone Cabanos Miao Wang Xianlin Han Scott B. Hansen A Soluble Fluorescent Binding Assay Reveals PIP2 Antagonism of TREK-1 Channels Cell Reports PIP2 TREK TRAAK Kir potassium channel fluorescent assay lipid binding arachidonic acid signaling lipid phosphatidic acid |
| title | A Soluble Fluorescent Binding Assay Reveals PIP2 Antagonism of TREK-1 Channels |
| title_full | A Soluble Fluorescent Binding Assay Reveals PIP2 Antagonism of TREK-1 Channels |
| title_fullStr | A Soluble Fluorescent Binding Assay Reveals PIP2 Antagonism of TREK-1 Channels |
| title_full_unstemmed | A Soluble Fluorescent Binding Assay Reveals PIP2 Antagonism of TREK-1 Channels |
| title_short | A Soluble Fluorescent Binding Assay Reveals PIP2 Antagonism of TREK-1 Channels |
| title_sort | soluble fluorescent binding assay reveals pip2 antagonism of trek 1 channels |
| topic | PIP2 TREK TRAAK Kir potassium channel fluorescent assay lipid binding arachidonic acid signaling lipid phosphatidic acid |
| url | http://www.sciencedirect.com/science/article/pii/S2211124717310008 |
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