Nicotinic acetylcholine receptor–lipid interactions: Mechanistic insight and biological function

Baenziger, John E.; Hénault, Camille M.; Therien, J. P. Daniel; Sun, J.

doi:10.1016/j.bbamem.2015.03.010

Cited by 67 publications

(60 citation statements)

References 149 publications

(204 reference statements)

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“…(72) Our results here suggest that this framing (lipid-mediated vs. protein-mediated) is misleading; general anesthetics don't act via lipids, they act like lipids: amphipathic molecules that displace water from the protein surface and (in pLGICs) transmembrane cavities. This interpretation is consistent with the established sensitivity of the pLGIC family to lipids (73)(74)(75)(76)(77)(78) and implies that lipid-specificity and general anesthetic specificity are deeply related questions.…”

Section: Resultssupporting

confidence: 86%

Competitive dewetting underlies site-specific binding of general anesthetics to GABA(A) receptors

Murlidaran

Hénin

Brannigan

2019

Preprint

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GABA(A) receptors are pentameric ligand-gated ion channels playing a critical role in the modulation of neuronal excitability. These inhibitory receptors, gated by -aminobutyric acid (GABA), can be potentiated and even directly activated by intravenous and inhalational anesthetics. Intersubunit cavities in the transmembrane domain have been consistently identified as putative binding sites by numerous experiment and simulation results. Synaptic GABA(A) receptors are predominantly found in a 2↵:2 :1 stoichiometry, with four unique inter-subunit interfaces. Experimental and computational results have suggested a perplexing specificity, given that cavity-lining residues are highly conserved, and the functional effects of general anesthetics are only weakly sensitive to most mutations of cavity residues. Here we use Molecular Dynamics simulations and thermodynamically rigorous alchemical free energy perturbation (AFEP) techniques to calculate affinities of the intravenous anesthetic propofol and the inhaled anesthetic sevoflurane to all intersubunit sites in a heteromeric GABA(A) receptor. We find that the best predictor of general anesthetic affinity for the intersubunit cavity sites is water displacement: combinations of anesthetic and binding site that displace more water molecules have higher affinities than those that displace fewer. The amount of water displacement is, in turn, a function of size of the general anesthetic, successful competition of the general anesthetic with water for the few hydrogen bonding partners in the site, and inaccessibility of the site to lipid acyl chains. The latter explains the surprisingly low affinity of GAs for the ↵ intersubunit site, which is missing a bulky methionine residue at the cavity entrance and can be occupied by acyl chains in the unbound state. Simulations also identify sevoflurane binding sites in the subunit centers and in the pore, but predict that these are lower affinity than the intersubunit sites. SignificanceAfter over a century of research, it is established that general anesthetics interact directly with hydrophobic cavities in proteins. We still do not know why not all small hydrophobic molecules can act as general anesthetics, or why not all hydrophobic cavities bind these molecules. General anesthetics can even select among homologous sites on one critical target, the GABA(A) heteropentamer, although the origins of selectivity are unknown. Here we used rigorous free energy calculations to find that binding affinity correlates with the number of released water molecules, which in turn depends upon the lipid content of the cavity without bound anesthetic. Results suggest a mechanism that reconciles lipid-centered and protein-centered theories, and which can directly inform design of new anesthetics.

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Section: Resultssupporting

confidence: 86%

Competitive dewetting underlies site-specific binding of general anesthetics to GABA(A) receptors

Murlidaran

Hénin

Brannigan

2019

Preprint

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“…Likewise, the L240A mutant, which reduces desensitization, increases POPG binding. The results support the hypothesis that anionic phospholipids stabilize the open state of pLGICs by direct binding to sites in the TMD adjacent to the lipid-facing transmembrane helix 4 (TM4) (3).…”

Section: Introductionsupporting

confidence: 81%

“…Pentameric ligand-gated ion channels (pLGICs) are essential determinants of synaptic transmission, and the targets of many allosteric modulators including general anesthetics and anti-epileptics (1). These ion channels are embedded in a heterogeneous and dynamic lipid environment (2), and the presence of specific lipids fine-tunes the function of pLGICs and may play a role in regulating neuronal excitability and drug sensitivity (3–5). One nearly ubiquitous example is that of anionic phospholipids, which are known to modulate pentameric ligand-gated ion channels (pLGICs) such as the nicotinic acetylcholine receptor (nAchR) (6), as well as inward rectifying potassium channels (7), K(2P) channels (8), voltage-gated potassium channels (9, 10), and cyclic nucleotide-gated channels (11).…”

Section: Introductionmentioning

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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“…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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Nicotinic acetylcholine receptor–lipid interactions: Mechanistic insight and biological function

Cited by 67 publications

References 149 publications

Competitive dewetting underlies site-specific binding of general anesthetics to GABA(A) receptors

Competitive dewetting underlies site-specific binding of general anesthetics to GABA(A) receptors

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

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

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