Structural requirements of membrane phospholipids for M-type potassium channel activation and binding.
M-channels are voltage-gated potassium channels that regulate cell excitability. They are heterotetrameric assemblies of Kv7.2 and Kv7.3 subunits. Their opening requires the presence of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)). However, the specificity of PI(4,5)P(2) as a binding and activating ligand is unknown. Here, we tested the ability of different phosphoinositides and lipid phosphates to activate or bind to M-channel proteins. Activation of functional channels was measured in membrane patches isolated from cells coexpressing Kv7.2 and Kv7.3 subunits. Channels were activated to similar extents (maximum open probability of ∼0.8 at 0 mV) by 0.1-300 μM dioctanoyl homologs of the three endogenous phosphoinositides, PI(4)P, PI(4,5)P(2), and PI(3,4,5)P(3), with sensitivity increasing with increasing numbers of phosphates. Non-acylated inositol phosphates had no effect up to 100 μM. Channels were also activated with increasing efficacy by 1-300 μM concentrations of the monoacyl monophosphates fingolimod phosphate, sphingosine 1-phosphate, and lysophosphatidic acid but not by phosphate-free fingolimod or sphingosine or by phosphate-masked phosphatidylcholine or phosphatidylglycerol. An overlay assay confirmed that a fusion protein containing the full-length C terminus of Kv7.2 could bind to a broad range of phosphoinositides and phospholipids. A mutated Kv7.2 C-terminal construct with reduced sensitivity to PI(4,5)P showed significantly less binding to most polyphosphoinositides. We concluded that M-channels bind to, and are activated by, a wide range of lipid phosphates, with a minimum requirement for an acyl chain and a phosphate headgroup. In this, they more closely resemble inwardly rectifying Kir6.2 potassium channels than the more PI(4,5)P(2)-specific Kir2 channels. Notwithstanding, the data also support the view that the main endogenous activator of M-channels is PI(4,5)P(2).
Department of Neuroscience, Physiology, and Pharmacology, University College London, London WC1E 6BT and.
Vsevolod
V
Telezhkin
Department of Neuroscience, Physiology, and Pharmacology, University College London, London WC1E 6BT and.
Joanne M
JM
Reilly
William Harvey Heart Centre, Barts and The London School of Medicine and Dentistry, London EC1M 6BQ, United Kingdom.
Alison M
AM
Thomas
William Harvey Heart Centre, Barts and The London School of Medicine and Dentistry, London EC1M 6BQ, United Kingdom.
Andrew
A
Tinker
Department of Neuroscience, Physiology, and Pharmacology, University College London, London WC1E 6BT and. Electronic address: d.a.brown@ucl.ac.uk.
David A
DA
Brown
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D051658:KCNQ2 Potassium Channel / Q000235:genetics / Q000378:metabolism*; D051659:KCNQ3 Potassium Channel / Q000235:genetics / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000235:genetics / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000235:genetics / Q000378:metabolism*; D010743:Phospholipids / Q000235:genetics / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000378:metabolism; D000099161:Kcnj11 Channel
D051658:KCNQ2 Potassium Channel; D051659:KCNQ3 Potassium Channel; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010743:Phospholipids; D024661:Potassium Channels, Inwardly Rectifying; D000099161:Kcnj11 Channel; C060974:phosphatidylinositol 3,4,5-triphosphate