Open-channel structure of a pentameric ligand-gated ion channel reveals a mechanism of leaflet-specific phospholipid modulation

Abstract

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 explore the mechanism of phosphatidylglycerol modulation, we determine a structure of ELIC in an open-channel conformation. The structure shows a bound phospholipid in an outer leaflet site, and structural changes in the phospholipid binding site unique to the open-channel. 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 activated, open-channel state of a pLGIC by specific, state-dependent binding to this site.

Lipid nanodisc scaffold and size alters the structure of a pentameric ligand-gated ion channel

“…The copyright holder for this preprint this version posted November 20, 2022. ; https://doi.org/10.1101/2022.11.20.517256 doi: bioRxiv preprint conformations, respectively, as well as a structure of a non-desensitizing mutant, ELIC5 (P254G/C300S/V261Y/G319F/I320F), which is consistent with an open-channel conformation 19 . These structures will be referred to as apo-MSP1E3D1 ELIC (WT ELIC without agonist in MSP1E3D1), MSP1E3D1 ELIC (WT ELIC with agonist in MSP1E3D1) and ELIC5 (ELIC5 with agonist in MSP1E3D1) (Fig.…”

“…One might therefore expect that an agonist-bound structure of ELIC in nanodiscs using these lipids should show a desensitized conformation. However, cryo-EM structures of agonist-bound ELIC in MSP1E3D1 nanodiscs using 2:1:1 lipids or only POPC show identical structures most consistent with a pre-active conformation 19,21 . We hypothesized that the nanodisc is altering the conformational landscape of ELIC, producing a discrepancy between the functional measurements in liposomes and structures in nanodiscs.…”

“…The ELIC5 structure is apparently open with 2' (Q233) forming the narrowest point of the pore (Fig. 1) 19 .…”

“…POPG (1-palmitoyl-2-oleoyl-phosphatidylglycerol) phospholipids (hereafter called 2:1:1 lipids) shows greater agonist responses than in POPC-only liposomes 19 . The peak open probability of ELIC in response to saturating concentrations of agonist in HEK membranes has been estimated to be >0.96 20 .…”

“…We previously showed that ELIC in liposomes composed of 2:1:1 POPC (1-palmitoyl-2-oleoyl-phosphatidylcholine): POPE (1-palmitoyl-2-oleoyl-phosphatidylethanolamine): POPG (1-palmitoyl-2-oleoyl-phosphatidylglycerol) phospholipids (hereafter called 2:1:1 lipids) shows greater agonist responses than in POPC-only liposomes 19 . The peak open probability of ELIC in response to saturating concentrations of agonist in HEK membranes has been estimated to be >0.96 20 .…”