Structure and function at the lipid-protein interface of a pentameric ligand-gated ion channel

Abstract

Although it has long been proposed that membrane proteins may contain tightly bound lipids, their identity, the structure of their binding sites, and their functional and structural relevance have remained elusive. To some extent, this is because tightly bound lipids are often located at the periphery of proteins, where the quality of density maps is usually poorer, and because they may be outcompeted by detergent molecules used during standard purification procedures. As a step toward characterizing natively bound lipids in the superfamily of pentameric ligand-gated ion channels (pLGICs), we applied single-particle cryogenic electron microscopy to fragments of native membrane obtained in the complete absence of detergent-solubilization steps. Because of the heterogeneous lipid composition of membranes in the secretory pathway of eukaryotic cells, we chose to study a bacterial pLGIC (ELIC) expressed in Escherichia coli's inner membrane. We obtained a three-dimensional reconstruction of unliganded ELIC (2.5-Å resolution) that shows clear evidence for two types of tightly bound lipid at the protein-bulk-membrane interface. One of them was consistent with a "regular" diacylated phospholipid, in the cytoplasmic leaflet, whereas the other one was consistent with the tetra-acylated structure of cardiolipin, in the periplasmic leaflet. Upon reconstitution in E. coli polar-lipid bilayers, ELIC retained the functional properties characteristic of members of this superfamily, and thus, the fitted atomic model is expected to represent the (long-debated) unliganded-closed, "resting" conformation of this ion channel. Notably, the addition of cardiolipin to phosphatidylcholine membranes restored the ion-channel activity that is largely lost in phosphatidylcholine-only bilayers.

Keywords: Cys-loop receptors; cardiolipin; cryo-EM; nicotinic receptors; styrene–maleic acid nanodiscs.

Fig. 1.

ELIC function in E. coli polar-lipid membranes. Outward currents recorded from inside-out patches of membrane excised from E. coli polar-lipid liposomes reconstituted with wild-type ELIC. (A) Response of a representative excised patch of membrane to a 1-min pulse of 1 mM propylammonium. Openings are upward deflections. The distribution of ions across the membrane was asymmetrical (∼150 mM/5 mM K⁺, inside/outside). The command potential was zero, and the +2.7-mV liquid-junction potential between the pipette and bath solutions was offset. As a result, the membrane potential was close to zero, thus approximating the conditions used for cryo-EM image acquisition. The black dashed lines denote the zero-current baseline. (B–E) Details of A at expanded time scales.

Fig. 2.

Cryo-EM structure of unliganded ELIC in SMA nanodiscs. Global superposition of two atomic models of unliganded ELIC: reconstituted in SMA nanodiscs and detergent solubilized [PDB ID: 2VL0 (36)]. The two models were superposed “globally” in such a way as to minimize the Cα–Cα distance between identically numbered residues in the stretch ranging from Pro-11 (the first residue in both models) to Ile-317 (the last residue modeled in detergent-solubilized ELIC) of all five subunits and omitting Gly-164 (which was not included in the 2VL0 model). (A–C) Different views of the ELIC–SMA model displayed in ribbon representation and colored according to Cα–Cα distance from the 2VL0 model. The color code is the same for all three panels. (D and E) View (parallel to the plane of the membrane) of the extracellular and transmembrane domains, respectively. For clarity, only one subunit is displayed in ribbon representation; all others are displayed as lines. The color code is the same for both panels. (F) Cα–Cα distances as a function of residue number. Distances ≥2 Å are denoted with black symbols. Including all superposed residues, the mean and RMSD values of the Cα–Cα distance were 0.5 and 1.2 Å, respectively. The molecular images were prepared with Visual Molecular Dynamics (37).

Fig. 3.

Protein–membrane interface of unliganded ELIC. (A) Unfiltered sum of experimental half maps sharpened locally. The density features modeled as cardiolipin (cyan) and phosphatidylglycerol (yellow) are highlighted. (B) Atomic models of firmly bound phospholipids and corresponding densities (display level = 0.7). (C) Atomic model of the protein–membrane interface. The phospholipids are displayed in stick representation, and the protein, in ribbon. The two front subunits are colored in different shades of magenta to highlight the binding sites of cardiolipin and phosphatidylglycerol in our unliganded ELIC–SMA model. At every subunit–subunit interface, the individual subunits are referred to as “principal” (“+”) or “complementary” (“–”). (D) A lower-magnification view of the model shown in (C) including the entire protein. The molecular images were prepared with Visual Molecular Dynamics (37) and Chimera X (42).

Fig. 4.

ELIC function in cardiolipin-free and cardiolipin-containing POPC bilayers. Outward currents recorded from inside-out patches of membrane excised from liposomes of the indicated composition reconstituted with wild-type ELIC. The distribution of ions across the membrane was asymmetrical (∼150 mM/5 mM K⁺, inside/outside). The command potential was zero, and the +2.7-mV liquid-junction potential between the pipette and bath solutions was offset. As a result, the membrane potential was close to zero, thus approximating the conditions used for cryo-EM image acquisition. (A) Three representative responses to the application of 1 mM propylammonium for each type of bilayer. Openings are upward deflections. In each case, ion-channel activity is shown for 1 min. (B) A 6-s stretch of ELIC activity during the application of 1 mM propylammonium to a patch of cardiolipin-containing POPC bilayer. The overall ion-channel activity of this recording was lower than that in the traces shown in (A), and thus, single-channel openings, with the highly characteristic subconductance behavior of ELIC, could be clearly observed. A black dashed line denotes the zero-current baseline. As expected from a negatively charged lipid, the addition of cardiolipin to POPC bilayers increased the single-channel conductance of this cation-selective channel.

Fig. 5.

Cardiolipin-binding site in unliganded ELIC. (A–C) Atomic model of cardiolipin and protein atoms within 5 Å of the phosphate–glycerol–phosphate polar head group. The corresponding densities are also displayed (display level = 0.7). Ser-202, Leu-205, and Trp-206 are in transmembrane α-helix M1; Thr-259, in the M2–M3 linker; Gln-264, in M3; and Arg-318, in M4. Phospholipid and amino-acid atoms are shown in stick representation with carbons colored cyan and purple, respectively. The molecular images were prepared with Chimera X (42). (D and E) Outward currents recorded in the whole-cell configuration from HEK-293 cells transiently transfected with wild-type ELIC or one of the indicated alanine mutants in response to short and long applications of saturating (10 mM) propylammonium. For wild-type ELIC, the mean (black solid line) ± 1 SD (gray error bars) of responses recorded from eight (short pulses) or nine (long pulses) different whole-cell experiments are displayed. For the mutants, the displayed traces are currents recorded from representative individual cells. The distribution of ions across the membrane was asymmetrical (∼150 mM/5 mM K⁺, inside/outside). The command potential was zero, and the −1.6-mV liquid-junction potential between the pipette and bath solutions was offset. As a result, the membrane potential was close to zero, thus approximating the conditions used for cryo-EM image acquisition. The black dashed lines denote the zero-current baseline. The mutants S202A and L205A + W206A, both in M1, did not elicit measurable currents despite displaying cell-surface expression levels that were comparable to those of the other three mutants.