Stopped-Flow Fluorometric Ion Flux Assay for Ligand-Gated Ion Channel Studies

Posson, David J.; Rusinova, Radda; Andersen, Olaf S.; Nimigean, Crina M.

doi:10.1007/978-1-4939-7362-0_17

Cited by 18 publications

(13 citation statements)

References 26 publications

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“…To examine ELIC activity in the absence of POPG, a fluorescence-based stopped-flow flux assay was performed (Posson et al, 2018). ELIC was reconstituted into either POPC-alone or 2:1:1 POPC:POPE:POPG liposomes encapsulating the fluorophore ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid).…”

Section: Resultsmentioning

confidence: 99%

Direct binding of phosphatidylglycerol at specific sites modulates desensitization of a ligand-gated ion channel

Tong

Petroff

Hsu

et al. 2019

eLife

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Pentameric ligand-gated ion channels (pLGICs) are essential determinants of synaptic transmission, and are modulated by specific lipids including anionic phospholipids. The exact modulatory effect of anionic phospholipids in pLGICs and the mechanism of this effect are not well understood. Using native mass spectrometry, coarse-grained molecular dynamics simulations and functional assays, we show that the anionic phospholipid, 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG), preferentially binds to and stabilizes the pLGIC, Erwinia ligand-gated ion channel (ELIC), and decreases ELIC desensitization. Mutations of five arginines located in the interfacial regions of the transmembrane domain (TMD) reduce POPG binding, and a subset of these mutations increase ELIC desensitization. In contrast, a mutation that decreases ELIC desensitization, increases POPG binding. The results support a mechanism by which POPG stabilizes the open state of ELIC relative to the desensitized state by direct binding at specific sites.

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Section: Resultsmentioning

confidence: 99%

Direct binding of phosphatidylglycerol at specific sites modulates desensitization of a ligand-gated ion channel

Tong

Petroff

Hsu

et al. 2019

eLife

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“…The mechanism responsible for that difference is not yet understood. We investigated the kinetics of SthK channel activation by examining nonequilibrium properties of SthK using a fluorescence-based, stopped-flow flux assay with millisecond time resolution ( Rusinova et al, 2014 ; Posson et al, 2018 ).…”

Section: Resultsmentioning

confidence: 99%

“…Kinetics of SthK response to cyclic nucleotides were monitored with a sequential-mixing, stopped-flow fluorescence-ensemble assay ( Rusinova et al, 2014 ; Posson et al, 2018 ) based on the Tl + -induced quenching of a liposome-encapsulated fluorescent dye (8-aminonaphthalene-1,3,6-trisulfonic acid [ANTS], disodium salt; Life Technologies). For this assay, the gel filtration was performed in 10 mM HEPES, 140 mM KNO 3 , and 0.5 mM DDM, pH 7.4.…”

Section: Methodsmentioning

confidence: 99%

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Ligand binding and activation properties of the purified bacterial cyclic nucleotide–gated channel SthK

Schmidpeter

Gao

Uphadyay

et al. 2018

Journal of General Physiology

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“…To examine ELIC activity in the absence of POPG, a fluorescence-based stopped-flow flux assay was performed (37). ELIC was reconstituted into either POPC-alone or 2:1:1 POPC:POPE:POPG liposomes encapsulating the fluorophore ANTS (8-Aminonaphthalene-1,3,6-Trisulfonic acid).…”

Section: Resultsmentioning

confidence: 99%

Direct Binding of Phosphatidylglycerol at Specific Sites Modulates Desensitization of a Pentameric Ligand-Gated Ion Channel

Tong

Petroff

Hsu

et al. 2019

Preprint

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27Pentameric ligand-gated ion channels (pLGICs) are essential determinants of synaptic 28 transmission, and are modulated by specific lipids including anionic phospholipids. The exact 29 modulatory effect of anionic phospholipids in pLGICs and the mechanism of this effect are not 30 well understood. Using native mass spectrometry, coarse-grained molecular dynamics 31 simulations and functional assays, we show that the anionic phospholipid, 1-palmitoyl-2-oleoyl-32 phosphatidylglycerol (POPG), preferentially binds to and stabilizes the pLGIC, Erwinia ligand-33 gated ion channel (ELIC), and decreases ELIC desensitization. Mutations of five arginines located 34 in the interfacial regions of the transmembrane domain (TMD) reduce POPG binding, and a 35 subset of these mutations increase ELIC desensitization. In contrast, the L240A mutant known to 36 decrease ELIC desensitization, increases POPG binding. The results support a mechanism by 37 which POPG stabilizes the open state of ELIC relative to the desensitized state by direct binding 38 at specific sites. 39 40 42 transmission, and the targets of many allosteric modulators including general anesthetics and 43 anti-epileptics (1). These ion channels are embedded in a heterogeneous and dynamic lipid 44 environment (2), and the presence of specific lipids fine-tunes the function of pLGICs and may 45 play a role in regulating neuronal excitability and drug sensitivity (3-5). One nearly ubiquitous 46 example is that of anionic phospholipids, which are known to modulate pentameric ligand-gated 47 ion channels (pLGICs) such as the nicotinic acetylcholine receptor (nAchR) (6), as well as inward 48 rectifying potassium channels (7), K(2P) channels (8), voltage-gated potassium channels (9, 10), 49 and cyclic nucleotide-gated channels (11). In pLGICs, anionic phospholipids have been shown to 50 shift the conformational equilibrium of the channel from an uncoupled or desensitized state to a 51 resting state, in which agonist binding is effectively coupled to channel activation (12)(13)(14). Studies of lipid modulation of ion channel function including modulation of pLGICs have53 focused on two central questions: 1) what is the exact effect of the lipid on channel function and 54 structure, and 2) is the effect attributable to direct binding of the lipid at specific sites? Torpedo 55 nAchR channel activity measured from flux assays (6, 15, 16) and agonist-induced conformational 56 changes (13, 17) depend on anionic phospholipids. However, only a few studies have employed 57 fast solution changes to measure current responses of pLGICs in model membranes (18), which 58 is necessary to distinguish the effect of lipids on channel gating, specifically transitions between 59 resting, open and desensitized states. With regard to lipid binding, early studies using electron 60 paramagnetic resonance (EPR) of spin-labeled lipids or lipid-induced modification of fluorescent 61 probes revealed an immobilized layer of lipids surrounding nAchRs that is enriched for certain 62 phospholipids (...

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Stopped-Flow Fluorometric Ion Flux Assay for Ligand-Gated Ion Channel Studies

Cited by 18 publications

References 26 publications

Direct binding of phosphatidylglycerol at specific sites modulates desensitization of a ligand-gated ion channel

Direct binding of phosphatidylglycerol at specific sites modulates desensitization of a ligand-gated ion channel

Ligand binding and activation properties of the purified bacterial cyclic nucleotide–gated channel SthK

Direct Binding of Phosphatidylglycerol at Specific Sites Modulates Desensitization of a Pentameric Ligand-Gated Ion Channel

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