Biology subjects

Cheng, W. W.

Publications and source records attributed to Cheng, W. W..

6 recordsLinked to original sources

bioRxiv2025

Structural mechanism of lipid modulation of pentameric ligand-gated ion channel activity

Pentameric ligand-gated ion channels (pLGICs) are sensitive to the lipid environment. However, the structural mechanism of how specific lipids support the agonist response of any pLGIC is poorly understood. Using the model pLGIC, ELIC (Erwinia ligand-gated ion channel), we find that phosphatidylethanolamine (PE) or cardiolipin (CL) are sufficient to support activation of a non-desensitizing mutant called ELIC5. Cryo-EM structures of unliganded and agonist-bound ELIC5 in the absence of PE or CL show increased structural heterogeneity and destabilization of the resting and open-channel states. Importantly, the unliganded structure of ELIC5 in a phosphatidylcholine (PC)-only environment shows variability that resembles agonist-induced changes. The structures also reveal a CL binding site at an outer leaflet M3-M4 site. Together with functional measurements in asymmetric liposomes and coarse-grained molecular dynamics simulations, the data indicate that CL supports ELIC activity by binding to this M3-M4 site thereby stabilizing an agonist-responsive resting state of the channel.

biophysics↗

bioRxiv2025

Cryo-EM structures of a pentameric ligand-gated ion channel in liposomes

Detergents and lipid nanodiscs affect the cryo-EM structures of pentameric ligand-gated ion channels (pLGICs) including ELIC. To determine the structure of a pLGIC in a membrane environment that supports ion channel function, we performed single particle cryo-EM of ELIC in liposomes. ELIC activation and desensitization were confirmed in liposomes with a stopped-flow thallium flux assay. Using WT ELIC and a non-desensitizing mutant (ELIC5), we captured resting, activated and desensitized structures at high resolution. In the desensitized structure, the ion conduction pore has a constriction at the 9 leucine of the pore-lining M2 helix, indicating that 9 is the desensitization gate in ELIC. The agonist-bound structures of ELIC in liposomes are distinct from those in nanodiscs. In general, the transmembrane domain is more loosely packed in liposomes compared to nanodiscs. It has been suggested that large nanodiscs are superior for supporting membrane protein function. However, ELIC localizes to the rim of large circularized nanodiscs, and structures of ELIC in large nanodiscs deviate from the liposome structures more than those in small nanodiscs. Using liposomes for cryo-EM structure determination of a pLGIC increases our confidence that the structures are snapshots of functional states.

biophysics↗

bioRxiv2022

Structural mechanism of leaflet-specific phospholipid modulation of a pentameric ligand-gated ion channel

Pentameric ligand-gated ion channels (pLGICs) mediate synaptic transmission and are sensitive to their lipid environment. The mechanism of phospholipid modulation of any pLGIC is not well understood. We demonstrate that the model pLGIC, ELIC (Erwinia ligand-gated ion channel), is positively modulated by the anionic phospholipid, phosphatidylglycerol, from the outer leaflet of the membrane. To elucidate the mechanism of phosphatidylglycerol modulation, we determine a structure of ELIC in an open conformation. The structure shows a bound phospholipid in an outer leaflet site, and conformational changes in the phospholipid binding site unique to the open state. In combination with streamlined alchemical free energy perturbation calculations and functional measurements in asymmetric liposomes, the data support a mechanism by which an anionic phospholipid stabilizes the open state of a pLGIC by specific, state-dependent binding to this site.

biophysics↗

bioRxiv2021

Polyunsaturated fatty acids inhibit a pentameric ligand-gated ion channel through one of two binding sites

Polyunsaturated fatty acids (PUFAs) inhibit pentameric ligand-gated ion channels (pLGICs) but the mechanism of inhibition is not well understood. The PUFA, docosahexaenoic acid (DHA), inhibits agonist responses of the pLGIC, ELIC, more effectively than palmitic acid, similar to the effects observed in the GABAA receptor and nicotinic acetylcholine receptor. Using photo-affinity labeling and coarse-grained molecular dynamics simulations, we identified two fatty acid binding sites in the outer transmembrane domain (TMD) of ELIC. Fatty acid binding to the photolabeled sites is selective for DHA over palmitic acid, and specific for an agonist-bound state. Hexadecyl-methanethiosulfonate modification of one of the two fatty acid binding sites in the outer TMD recapitulates the inhibitory effect of PUFAs in ELIC. The results demonstrate that DHA selectively binds to multiple sites in the outer TMD of ELIC, but that state-dependent binding to a single intrasubunit site mediates DHA inhibition of ELIC.

biochemistry↗

bioRxiv2020

Site-specific effects of neurosteroids on GABAA receptor activation and desensitization

This study examines how site-specific binding to the three identified neurosteroid binding sites in the 1{beta}3 GABAA receptor (GABAAR) contributes to neurosteroid allosteric modulation. We found that the potentiating neurosteroid, allopregnanolone, but not its inhibitory 3{beta}-epimer epi-allopregnanolone, binds to the canonical {beta}3(+)-1(-) intersubunit site that mediates receptor activation by neurosteroids. In contrast, both allopregnanolone and epi-allopregnanolone bind to intrasubunit sites in the {beta}3 subunit, promoting receptor desensitization and the 1 subunit promoting ligand-specific effects. Two neurosteroid analogues with diazirine moieties replacing the 3-hydroxyl (KK148 and KK150) bind to all three sites, but do not potentiate GABAAR currents. KK148 is a desensitizing agent, whereas KK150 is devoid of allosteric activity. These compounds provide potential chemical scaffolds for site-specific and general neurosteroid antagonists. Collectively, these data show that differential occupancy and efficacy at three discrete neurosteroid binding sites determine whether a neurosteroid has potentiating, inhibitory, or competitive antagonist activity on GABAARs.

biophysics↗

bioRxiv2019

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

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, the L240A mutant known to decrease 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.

biophysics↗