Reconstitution of homomeric GluA2(flop) receptors in supported lipid membranes: functional and structural properties

Abstract

AMPA receptors (AMPARs) are glutamate-gated ion channels ubiquitous in the vertebrate central nervous system, where they mediate fast excitatory neurotransmission and act as molecular determinants of memory formation and learning. Together with detailed analyses of individual AMPAR domains, structural studies of full-length AMPARs by electron microscopy and x-ray crystallography have provided important insights into channel assembly and function. However, the correlation between the structure and functional states of the channel remains ambiguous particularly because these functional states can be assessed only with the receptor bound within an intact lipid bilayer. To provide a basis for investigating AMPAR structure in a membrane environment, we developed an optimized reconstitution protocol using a receptor whose structure has previously been characterized by electron microscopy. Single-channel recordings of reconstituted homomeric GluA2(flop) receptors recapitulate key electrophysiological parameters of the channels expressed in native cellular membranes. Atomic force microscopy studies of the reconstituted samples provide high-resolution images of membrane-embedded full-length AMPARs at densities comparable to those in postsynaptic membranes. The data demonstrate the effect of protein density on conformational flexibility and dimensions of the receptors and provide the first structural characterization of functional membrane-embedded AMPARs, thus laying the foundation for correlated structure-function analyses of the predominant mediators of excitatory synaptic signals in the brain.

FIGURE 1.

Modular architecture of AMPARs. A, schematic representation of an AMPAR monomer comprising an ATD, a LBD, a transmembrane region (TM) consisting of three membrane helices (M1, M3, and M4) and a re-entrant M2, and a carboxyl-terminal domain (CTD). The amino terminus is positioned extracellularly and the carboxyl terminus intracellularly. The pink asterisk represents an agonist molecule bound in the LBD cleft. B, crystal structure of the nearly full-length GluA2 homotetramer. Individual monomers are color-coded (blue, green, red, and yellow), and domain layers are boxed to emphasize the modular structure (Protein Data Bank code 3kg2 (11)).

FIGURE 2.

Effect of different reconstitution parameters on density of reconstituted GluA2 receptors. Black bars are reconstituted samples, and white bars are negative controls (reconstitutions without protein). The reconstitutions were performed with brain lipid extract and CHAPS detergent unless stated otherwise. All data are shown as means ± S.E. A, effect of different detergents: CHAPS (10 mm), C₁₂E₈ (9.8 mm), DDM (2.8 and 8.4 mm), and DM (0.3 and 20 mm). The highest protein density of 78.4 ± 10.8 proteins μm⁻² (n = 33) was achieved with CHAPS. B, two different concentrations of DDM were tested. At 2.8 mm DDM, liposomes were saturated with the detergent when the protein was added, and at 8.4 mm DDM, the liposomes were solubilized as described (22). Solubilizing and saturating concentrations were also tested for DM (data not shown). C, the receptors were reconstituted in different lipid mixtures: brain total lipid extract, fusion lipids (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine/cholesterol at a 10:5:5:4 molar ratio), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG). D, the reconstitutions were performed at room temperature (RT) and +4 °C. E, the mixture of lipids, proteins, and detergent was mixed with Bio-Beads to remove the detergent. The length of the Bio-Beads treatment was varied between 1 h and overnight. F, different lipid/protein mass ratios were tested (13 and 667). Lower lipid/protein mass ratio resulted in higher protein density but also in higher aggregation of proteins.

FIGURE 3.

Fluorescence microscopy illustrating protein reconstitution in liposomes. Receptors with GFP fused upstream from the ATD were reconstituted into 18:1 Liss Rhod PE-labeled liposomes, which was confirmed by co-localization of both emission wavelengths. A, negative control showing only liposomes with 0.01% (w/w) 18:1 Liss Rhod PE. B, non-reconstituted protein only. C and D, co-localization of fluorescently labeled membrane (red) and protein (green) in reconstituted samples resulted in yellow spots (enlarged in the insets). All images were acquired at magnification ×100; insets are ×10 zooms of the corresponding image. Red circles mark areas used for background subtraction.

FIGURE 4.

Activity of reconstituted GluA2 receptors. A, single-channel current measured at +80 mV in 5 mm l-Glu and 250 μm CTZ, indicating conductance levels of 9 and 12 pS. Closed and open levels are marked as c, o1, and o2, respectively. The signal was absent in the presence of NBQX (+80 mV; middle trace) and in the negative control (liposomes without receptors, +100 mV; lower trace). B, dwell time histogram of open events with a two-term exponential fit yielding time constants τ_o1 = 0.9 ± 0.1 ms and τ_o2 = 5.6 ± 0.1 ms. N, number of bin counts. C, dwell time histogram of closed events with a three-term exponential fit yielding time constants τ_c1 = 0.6 ± 0.1 ms, τ_c2 = 5.3 ± 0.1 ms, and τ_c3 = 44.7 ± 0.3 ms. D–F, single-channel conductance was determined by Gaussian fit to amplitude histograms. Conductances of 5 pS (D), 9 and 12 pS (E), and 20 pS (F), characteristic of AMPARs, were measured at +100, +80, and +40 mV, respectively.

FIGURE 5.

Multichannel recording of reconstituted GluA2 receptors. A, multiple opening levels indicate concurrent gating of more than one channel in the bilayer. The holding voltage was +40 mV. Closed and open levels are marked as c, o1, and o2, respectively. B, when multiple openings were recorded, a higher conductance of 47.5 pS was observed, together with its multiple at 97.5 pS.

FIGURE 6.

AFM images of reconstituted GluA2 homomers showing isolated and clustered proteins. The lipid bilayer is the flat light brown area. Defects in the lipid bilayer expose imaging surface (mica; dark brown area). Bright specks are proteins reconstituted in the lipid bilayer. Big white clumps in C and D are unresolved aggregates of proteins and/or lipids. A, negative (protein-less) control of reconstituted samples. Note the absence of the proteins (i.e. bright specks) in the flat lipid bilayer. Scale bar = 500 nm. B, isolated proteins surrounded by only the membrane (average density of 27 proteins μm⁻²). Three isolated proteins are indicated by black arrowheads. Scale bar = 500 nm. C and D, small clusters (circled) containing up to eight receptors (average density of 223 particles μm⁻²). Examples of isolated proteins are indicated by black arrowheads. Scale bars = 500 nm (C) and 200 nm (D). Samples in C and D were incubated overnight at +4 °C.

FIGURE 7.

A, AFM image of isolated receptors reconstituted in the lipid bilayer. Scale bar = 500 nm. B, heights obtained from the line scan. Yellow and green arrows indicate protein features covered by the scan line. C, distribution of extracellular protrusions corresponding to a height of 11.4 ± 3.1 nm (n = 179). D, high-resolution AFM images of isolated receptors revealing an internal structure that is mostly tetrameric. Subunit contours for each receptor are shown below the corresponding scan. Scale bars = 20 nm.

FIGURE 8.

AFM scan of clustered GluA2 receptors. A, small linear cluster containing three receptors (circled) in contact. Scale bar = 20 nm. B, three-dimensional reconstruction of the cluster revealing the presence of a central pore in each receptor. C, the height profile of the black dotted line in A. D, the inset shows the uppermost receptor from the cluster in A, and the pore dimensions are visible from the height profile of the white line in the inset (6.5 nm across and 2 nm deep).