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

Cabanos, Cerrone; Wang, Miao; Han, Xianlin; Hansen, Scott B.

doi:10.1016/j.celrep.2017.07.034

Cited by 70 publications

(114 citation statements)

References 40 publications

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“…2H) at 100 mV with 270 nM Ca 2+ . This falls within the range reported for PI(4,5)P2 binding to other ion channels (0.12 µM to 4.6 µM, (47)(48)(49)). At -100 mV, inward currents were more sensitive to diC8-PI(4,5)P2 with EC50 < 0.1 µM ( Fig.…”

Section: The Effect Of Pi(45)p2 Is Dependent On Voltage and Ca 2+supporting

confidence: 88%

A Network of Phosphatidylinositol 4,5-bisphosphate Binding Sites Regulate Gating of the Ca²⁺-activated Cl⁻ Channel ANO1 (TMEM16A)

Jiang

Cui

et al. 2019

Preprint

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ANO1 (TMEM16A) is a Ca 2+ -activated Clchannel that regulates diverse cellular functions including fluid secretion, neuronal excitability, and smooth muscle contraction. ANO1 is activated by elevation of cytosolic Ca 2+ and modulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Here we describe a closely concerted experimental and computational study, including electrophysiology, mutagenesis, functional assays, and extended sampling of lipid-protein interactions with molecular dynamics (MD) to characterize PI(4,5)P2 binding modes and sites on ANO1. ANO1 currents in excised inside-out patches activated by 270 nM Ca 2+ at +100 mV are increased by exogenous PI(4,5)P2with an EC50 = 1.24 µM. The effect of PI(4,5)P2 is dependent on membrane voltage and Ca 2+ and is explained by a stabilization of the ANO1 Ca 2+ -bound open state. Unbiased atomistic MD simulations with 1.4% PI(4,5)P2 in a phosphatidylcholine bilayer identified 8 binding sites with significant probability of binding PI(4,5)P2. Three of these sites captured 85% of all ANO1 -PI(4,5)P2interactions. Mutagenesis of basic amino acids near the membrane-cytosol interface found three regions of ANO1 critical for PI(4,5)P2 regulation that correspond to the same three sites identified by MD. PI(4,5)P2 is stabilized by hydrogen bonding between amino acid sidechains and phosphate/hydroxyl groups on PI(4,5)P2. Binding of PI(4,5)P2 alters the position of the cytoplasmic extension of TM6, which plays a crucial role in ANO1 channel gating, and increases the accessibility of the inner vestibule to Clions. We propose a model consisting of a network of three PI(4,5)P2 binding sites at the cytoplasmic face of the membrane allosterically regulating ANO1 channel gating.of the authors and does not necessarily represent the official views of the National Institutes of Health. We also acknowledge computing resources provided by Blue Waters at National Center for Supercomputing Applications, and Extreme Science and Engineering Discovery Environment (grant MCA06N060 to ET).

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Section: The Effect Of Pi(45)p2 Is Dependent On Voltage and Ca 2+supporting

confidence: 88%

A Network of Phosphatidylinositol 4,5-bisphosphate Binding Sites Regulate Gating of the Ca²⁺-activated Cl⁻ Channel ANO1 (TMEM16A)

Jiang

Cui

et al. 2019

Preprint

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“…2c) failed to respond and suggests that PLD2 localization is a significant contributor to TREK-1 activation. Since the PLD-TREK-1 complex was shown above to translocate to PIP2 domains and PIP2 is a TREK-1 antagonist 44 , it is likely that without co-localization the local concentration of PA does not reach sufficient levels to compete with PIP2 binding 11 . It may be possible if PLD2 dependent PA production could reach activating concentration if the membrane and or cytoskeleton were sufficiently disrupted.…”

Section: Discussionmentioning

confidence: 99%

“…2, 3e). Since PIP2 directly antagonizes TREK-1 44,45 , the localization of TREK-1 to PIP2 before and after shear (Fig 3c,e) explains the strong inhibition of TREK-1 by xPLD2 ( Fig. 2b-c).…”

Section: The Trek-1 Force Transduction Pathwaymentioning

confidence: 92%

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Mechanical activation of TWIK-related potassium channel by nanoscopic movement and rapid second messenger signaling

Gudheti

et al. 2019

Preprint

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The transduction of force into a biological signal is critical to all living organisms. Recently, disruption of ordered lipids has emerged as an 'atypical' force sensor in biological membranes; however, disruption has yet to link with canonical channel mechanosensation. Here we show that forceinduced disruption and lipid mixing activates TWIKrelated K + channel (TREK-1), and that this activation is dependent on phospholipase D2 (PLD2). PLD2 transduces the force into a chemical signal phosphatidic acid (PA) that is then sensed by TREK-1 with a latency of <3 ms. TREK-1 then produces a mechanically induced change in membrane potential. Hence, in a biological membrane, we show the ordered lipid is the force sensor, PLD2 is a chemical transducer, and the 'mechanosensitive' ion channel TREK-1 is a downstream effector of mechanical transduction. Confirming this central role for PA singling in force transduction, genetic deletion of PLD decreases mechanosensitivity and pain thresholds in D. melanogaster.

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“…PLD is known to be sensitive to its localization to lipid domains 5 and recent research has shown that anesthetics have a significant effect on the organization of these domains 2,19,20 . PLD and its activating lipid PA have also been shown to directly control the activity of the mammalian potassium channel TREK-1, a hyperpolarizing channel 7,21 . While it is unknown what channels with which the fly ortholog of PLD are associated, recent experiments have shown that it too is likely helping to regulate a hyperpolarizing channel like TREK-1.…”

Section: Discussionmentioning

confidence: 99%

Measuring anesthetic resistance in Drosophila by VAAPR

Petersen

Clowes

Hansen

2019

Preprint

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Volatile anesthetics are compounds which are commonly used to induce a reversable loss of consciousness (LOC) in animals. The molecular mechanism of how anesthetics induce LOC is largely unknown. However, observations have been made which show that there are genetically-encoded traits which influence the effective concentration of anesthetics in the inducement of LOC. Despite this longterm observation, little progress has been made in identifying genes involved in anesthetic sensitivity. One reason for this is that many techniques to test anesthetic sensitivity are technically challenging and are inhibitory for high-throughput studies. Here we introduce a technique for testing volatiles and aerosols with positional recording (VAAPR), a method which allows for high-throughput testing of the effect of anesthetics and other aerosolized drugs using Drosophila. Using VAAPR we show that the enzyme phospholipase D (PLD) significantly shifts the concentration of diethyl ether, chloroform, and isoflurane needed to induce LOC in Drosophila. We also show that PLD is required for a paradoxical hyperactivity phenotype. We expect that this technique will allow for additional genes to be found which control anesthetic sensitivity as well as other behavioral phenotypes.

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

Cited by 70 publications

References 40 publications

A Network of Phosphatidylinositol 4,5-bisphosphate Binding Sites Regulate Gating of the Ca²⁺-activated Cl⁻ Channel ANO1 (TMEM16A)

A Network of Phosphatidylinositol 4,5-bisphosphate Binding Sites Regulate Gating of the Ca²⁺-activated Cl⁻ Channel ANO1 (TMEM16A)

Mechanical activation of TWIK-related potassium channel by nanoscopic movement and rapid second messenger signaling

Measuring anesthetic resistance in Drosophila by VAAPR

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

Cited by 70 publications

References 40 publications

A Network of Phosphatidylinositol 4,5-bisphosphate Binding Sites Regulate Gating of the Ca2+-activated Cl− Channel ANO1 (TMEM16A)

A Network of Phosphatidylinositol 4,5-bisphosphate Binding Sites Regulate Gating of the Ca2+-activated Cl− Channel ANO1 (TMEM16A)

Mechanical activation of TWIK-related potassium channel by nanoscopic movement and rapid second messenger signaling

Measuring anesthetic resistance in Drosophila by VAAPR

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A Network of Phosphatidylinositol 4,5-bisphosphate Binding Sites Regulate Gating of the Ca²⁺-activated Cl⁻ Channel ANO1 (TMEM16A)

A Network of Phosphatidylinositol 4,5-bisphosphate Binding Sites Regulate Gating of the Ca²⁺-activated Cl⁻ Channel ANO1 (TMEM16A)