Transbilayer asymmetry and sphingomyelin composition modulate the preferential membrane partitioning of the nicotinic acetylcholine receptor in Lo domains

Perillo, Vanesa Liliana; Peñalva, Daniel Alejandro; Vitale, Alejandro José; Barrantes, Francisco J.; Antollini, Silvia S.

doi:10.1016/j.abb.2015.12.003

Cited by 33 publications

(29 citation statements)

References 64 publications

(84 reference statements)

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“…For example, following the latter approach Vitrac et al prepared sonicated small unilamellar vesicles (SUVs) with the 12transmembrane protein LacY from E. coli and examined the effect of asymmetric charge distribution on the topology of the protein [4,52]. In a different study, asymmetric SUVs with the nicotinic acetylcholine receptor (AChR) were prepared by using MβCD-loaded lipid complexes to exchange the lipids on the outer leaflet of symmetric SUVs containing AChR, with sphingomyelin [3]. While these examples illustrate how a variety of TM proteins can be incorporated into asymmetric model membranes of different geometries, the effect of the protein (and protein incorporation) on the lipid compositions of the two bilayer leaflets can vary and therefore has to be examined explicitly.…”

Section: Discussionmentioning

confidence: 99%

“…One prominent example is the transverse lipid distribution in cell membranes: whereas a self-assembled lipid bilayer has the same lipid composition in its two leaflets (i.e., it is symmetric), the leaflet compositions in the plasma membranes of eukaryotic cells differ (i.e., the bilayer is asymmetric), and this difference is actively maintained by the cell. Not surprisingly, the biophysical mechanisms underlying membrane asymmetry are the subject of intense studies [1][2][3][4][5] which are rapidly increasing in number as a result of recent advances that have enabled the preparation and biophysical characterization of asymmetric lipid-only model membranes in vitro [6][7][8]. Because such model membranes are not at chemical equilibrium and their asymmetry is not actively maintained, the time window for examining their properties is limited by the gradual redistribution of the lipids between the two leaflets until a symmetric lipid composition is achieved.…”

Section: Introductionmentioning

confidence: 99%

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Gramicidin increases lipid flip-flop in symmetric and asymmetric lipid vesicles

Doktorova

Heberle

Marquardt

et al. 2018

Preprint

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Unlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid distribution. Non-enzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a nonspecific manner and by various hypothesized mechanisms. Using deuterated phospholipids, we examined the acceleration of flip-flop by gramicidin channels which have well-defined structures and known function, features that make them ideal candidates for probing the protein-membrane interactions underlying lipid flip-flop. To study compositionally and isotopically asymmetric proteoliposomes containing gramicidin, we expanded a recently developed protocol for the preparation and characterization of lipid-only asymmetric vesicles. Channel incorporation, conformation, and function were examined with small-angle X-ray scattering, circular dichroism and a stopped-flow spectrofluorometric assay, respectively. As a measure of lipid scrambling we used differential scanning calorimetry to monitor the effect of gramicidin on the melting transition temperatures of the two bilayer leaflets. The two calorimetric peaks of the individual leaflets merged into a single peak over time suggestive of scrambling activity, and the effect of the channel on the transbilayer lipid distribution in both symmetric POPC and asymmetric POPC/DMPC vesicles was quantified from proton NMR measurements. Our results show that gramicidin increases lipid flip-flop in a complex, concentration-dependent manner. To determine the molecular mechanism of the process we used molecular dynamics simulations and further computational analysis of the trajectories to estimate the amount of membrane deformation in the samples. Together, the experimental and computational approaches were found to constitute an effective means for studying the effects of transmembrane proteins on lipid distribution in both symmetric and asymmetric model membranes.All rights reserved. No reuse allowed without permission.was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Gramicidin increases lipid flip-flop in symmetric and asymmetric lipid vesicles

Doktorova

Heberle

Marquardt

et al. 2018

Preprint

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“…Three generations of investigation into the mechanism have followed, with the first generation of studies aiming to differentiate between the role of bulk, annular, and non-annular cholesterol. The second generation (16,18,(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54) of studies probed membrane-mediated effects on organization of multiple nAChRs, while the third generation (55)(56)(57)(58) has applied x-ray crystallography and high-resolution cryo electron microscopy to directly observe lipid binding modes.…”

Section: Introductionmentioning

confidence: 99%

Boundary lipids of the nicotinic acetylcholine receptor: Spontaneous partitioning via coarse-grained molecular dynamics simulation

Sharp¹,

Salari²,

Brannigan³

2019

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Reconstituted nicotinic acetylcholine receptors (nAChRs) exhibit significant gain-of-function upon addition of cholesterol to reconstitution mixtures, and cholesterol affects organization of nAChRs within domain-forming membranes, but whether nAChR partitions to cholesterol-rich liquid-ordered ("raft" or l o ) domains or cholesterol-poor liquiddisordered (l do ) domains is unknown. We use coarse-grained molecular dynamics simulations to observe spontaneous interactions of cholesterol, saturated lipids, and polyunsaturated (PUFA) lipids with nAChRs. In binary Dipalmitoylphosphatidylcholine:Cholesterol (DPPC:CHOL) mixtures, both CHOL and DPPC acyl chains were observed spontaneously entering deep "non-annular" cavities in the nAChR TMD, particularly at the subunit interface and the β subunit center, facilitated by the low amino acid density in the cryo-EM structure of nAChR in a native membrane. Cholesterol was highly enriched in the annulus around the TMD, but this effect extended over (at most) 5-10Å. In domain-forming ternary mixtures containing PUFAs, the presence of a single receptor did not significantly affect the likelihood of domain formation. nAChR partitioned to any cholesterol-poor l do domain that was present, regardless of whether the l do or l o domain lipids had PC or PE headgroups. Enrichment of PUFAs among boundary lipids was positively correlated with their propensity for demixing from cholesterol-rich phases. Long n − 3 chains (tested here with Docosahexaenoic Acid, DHA) were highly enriched in annular and non-annular embedded sites, partially displacing cholesterol and completely displacing DPPC, and occupying sites even deeper within the bundle. Shorter n − 6 chains were far less effective at displacing cholesterol from non-annular sites.

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“…This allowed preparation of artificial vesicles that mimic natural membranes by having both natural lipid composition and asymmetry. The approach is beginning to see applications by other laboratories (19)(20)(21)(22).…”

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confidence: 99%

Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids

Kim

Huang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

107

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Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70-80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids.lipid exchange | plasma membrane outer leaflet | lipid asymmetry | methyl-alpha-cyclodextrin | mass spectrometry

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Transbilayer asymmetry and sphingomyelin composition modulate the preferential membrane partitioning of the nicotinic acetylcholine receptor in Lo domains

Cited by 33 publications

References 64 publications

Gramicidin increases lipid flip-flop in symmetric and asymmetric lipid vesicles

Gramicidin increases lipid flip-flop in symmetric and asymmetric lipid vesicles

Boundary lipids of the nicotinic acetylcholine receptor: Spontaneous partitioning via coarse-grained molecular dynamics simulation

Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids

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