The Role of Cholesterol in the Activation of Nicotinic Acetylcholine Receptors

Baenziger, John E.; Domville, Jaimee A.; Therien, J. P. Daniel

doi:10.1016/bs.ctm.2017.05.002

Cited by 31 publications

(33 citation statements)

References 177 publications

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“…POPC is commonly used for simulations of mammalian proteins because it is considered representative of mammalian lipid membranes; however, both polyunsaturated fatty acid (PUFA) lipids such as SDPC and cholesterol have been found to make up a significantly high percentage of the lipids found in 5-HT 3A -containing membranes (25). While to date there is a lack of evidence demonstrating the direct effects of these lipids on 5-HT 3A , there is evidence demonstrating that these lipids interact directly with and regulate the function of other pLGICs such as the nAChR, the Gloeobacter violaceus ligand-gated ion channel (GLIC), and the γ-aminobutyric acid receptor (GABA), which are largely homologous in structure to 5-HT 3A (25)(26)(27)(28)(29)(35)(36)(37)(38). This indicates a need to study the effects of these lipids on 5-HT 3A to determine whether they play a role in regulating its function similar to other pLGICs.…”

Section: Resultsmentioning

confidence: 99%

“…Experimentally, cholesterol has been included in lipid membranes to study the structure of 5-HT 3A (16,47), but few conclusions have been reported besides the distribution of 5-HT 3A in the membrane changing from evenly distributed to tightly packed clusters as a result of the removal of cholesterol (47). However, because the structure of 5-HT 3A is largely homologous to other pLGICs including the nAChR, GLIC, and GABA receptors, whose amplitude of channel conductance and rate of desensitization are regulated by cholesterol and PUFA lipids (25)(26)(27)(28)(29)(35)(36)(37)(38), it is likely that cholesterol and PUFA lipids play a role in regulating the structure and function of 5-HT 3A . Notably, MD simulations have previously demonstrated a binding site for cholesterol on GABA (35), which demonstrated that cholesterol interacts with the M2 helix of the TMD, strongly suggesting the regulation of channel conductance.…”

Section: Discussionmentioning

confidence: 99%

“…The importance of lipid membrane type cannot be understated. It has been shown experimentally that cholesterol and polyunsaturated lipids interact directly with pLGICs such as nAChR (25)(26)(27)(28)(29), which is largely homologous in structure to 5-HT 3A , to strongly regulate receptor structure and conductance. Lastly, we included several levels of simulation validation to ensure our model accurately represents experiment, including sufficient lipid membrane equilibration to ensure realistic lipid packing and careful param-eterization of 5-HT for the CHARMM general force field (CGenFF) (30) using free energy perturbation (FEP) simulations to determine its solvation free energy, ensuring the accurate representation of 5-HT binding to 5-HT 3A .…”

Section: Significancementioning

confidence: 99%

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Microsecond-timescale simulations suggest 5-HT–mediated preactivation of the 5-HT_3Aserotonin receptor

Guros

Balijepalli

Klauda

2019

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

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Aided by efforts to improve their speed and efficiency, molecular dynamics (MD) simulations provide an increasingly powerful tool to study the structure–function relationship of pentameric ligand-gated ion channels (pLGICs). However, accurate reporting of the channel state and observation of allosteric regulation by agonist binding with MD remains difficult due to the timescales necessary to equilibrate pLGICs from their artificial and crystalized conformation to a more native, membrane-bound conformation in silico. Here, we perform multiple all-atom MD simulations of the homomeric 5-hydroxytryptamine 3A (5-HT3A) serotonin receptor for 15 to 20 μs to demonstrate that such timescales are critical to observe the equilibration of a pLGIC from its crystalized conformation to a membrane-bound conformation. These timescales, which are an order of magnitude longer than any previous simulation of 5-HT3A, allow us to observe the dynamic binding and unbinding of 5-hydroxytryptamine (5-HT) (i.e., serotonin) to the binding pocket located on the extracellular domain (ECD) and allosteric regulation of the transmembrane domain (TMD) from synergistic 5-HT binding. While these timescales are not long enough to observe complete activation of 5-HT3A, the allosteric regulation of ion gating elements by 5-HT binding is indicative of a preactive state, which provides insight into molecular mechanisms that regulate channel activation from a resting state. This mechanistic insight, enabled by microsecond-timescale MD simulations, will allow a careful examination of the regulation of pLGICs at a molecular level, expanding our understanding of their function and elucidating key structural motifs that can be targeted for therapeutic regulation.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

See 1 more Smart Citation

Microsecond-timescale simulations suggest 5-HT–mediated preactivation of the 5-HT_3Aserotonin receptor

Guros

Balijepalli

Klauda

2019

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

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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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“…Reconstitution of nicotinic receptors into defined lipid preparations has demonstrated that cholesterol is important to nicotinic receptor function 22 . We tested cholesterol derivatives in thermal stabilization assays 23 and found the water-soluble cholesterol analog cholesteryl-hemisuccinate (CHS) potently stabilizes the receptor; thus, we included it throughout purification of the receptor.…”

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confidence: 99%

Structural principles of distinct assemblies of the human α4β2 nicotinic receptor

Walsh

Roh

Gharpure

et al. 2018

Nature

198

191

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SummaryFast chemical communication in the nervous system is mediated by neurotransmitter-gated ion channels. The prototypical member of this class of cell surface receptors is the cation-selective nicotinic acetylcholine receptor. As with most ligand-gated ion channels, nicotinic receptors assemble as oligomers of subunits, usually as hetero-oligomers and often with variable stoichiometries1. This intrinsic heterogeneity in protein composition provides the fine tunability in channel properties essential to brain function but frustrates structural and biophysical characterization. The α4β2 subtype of the nicotinic acetylcholine receptor is the most abundant isoform in the human brain and is the principal target in nicotine addiction. This pentameric ligand-gated ion channel assembles in two stoichiometries of α and β subunits, 2α:3β and 3α:2β. Both assemblies are functional, have distinct biophysical properties, and a misbalance in the ratio of assemblies is tied to both nicotine addiction2,3 and congenital epilepsy4,5. Here we leverage cryo-electron microscopy (cryo-EM) to obtain structures of both receptor assemblies from a single sample. Antibody fragments specific to β2 were used to ‘break’ symmetry during particle alignment and obtain high resolution reconstructions of both stoichiometries, in complex with nicotine. The results reveal principles of subunit assembly and the structural basis of the distinctive biophysical and pharmacological properties of the two different stoichiometries of this receptor.

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The Role of Cholesterol in the Activation of Nicotinic Acetylcholine Receptors

Cited by 31 publications

References 177 publications

Microsecond-timescale simulations suggest 5-HT–mediated preactivation of the 5-HT_3Aserotonin receptor

Microsecond-timescale simulations suggest 5-HT–mediated preactivation of the 5-HT_3Aserotonin receptor

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

Structural principles of distinct assemblies of the human α4β2 nicotinic receptor

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The Role of Cholesterol in the Activation of Nicotinic Acetylcholine Receptors

Cited by 31 publications

References 177 publications

Microsecond-timescale simulations suggest 5-HT–mediated preactivation of the 5-HT3Aserotonin receptor

Microsecond-timescale simulations suggest 5-HT–mediated preactivation of the 5-HT3Aserotonin receptor

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

Structural principles of distinct assemblies of the human α4β2 nicotinic receptor

Contact Info

Product

Resources

About

Microsecond-timescale simulations suggest 5-HT–mediated preactivation of the 5-HT_3Aserotonin receptor

Microsecond-timescale simulations suggest 5-HT–mediated preactivation of the 5-HT_3Aserotonin receptor