Protein-lipid interplay at the neuromuscular junction

Abstract

Many new structures of membrane proteins have been determined over the last decade, yet the nature of protein-lipid interplay has received scant attention. The postsynaptic membrane of the neuromuscular junction and Torpedo electrocytes has a regular architecture, opening an opportunity to illuminate how proteins and lipids act together in a native membrane setting. Cryo electron microscopy (Cryo-EM) images show that cholesterol segregates preferentially around the constituent ion channel, the nicotinic acetylcholine receptor, interacting with specific sites in both leaflets of the bilayer. In addition to maintaining the transmembrane α-helical architecture, cholesterol forms microdomains - bridges of rigid sterol groups that link one channel to the next. This article discusses the whole protein-lipid organization of the cholinergic postsynaptic membrane, its physiological implications and how the observed details relate to our current concept of the membrane structure. I suggest that cooperative interactions, facilitated by the regular protein-lipid arrangement, help to spread channel activation into regions distant from the sites of neurotransmitter release, thereby enhancing the postsynaptic response.

Keywords: cholesterol microdomain; cryo-EM; nicotinic acetylcholine receptor; postsynaptic membrane; synaptic transmission.

Fig. 1.

Organization of acetylcholine receptors in postsynaptic membranes from frog neuromuscular junction (freeze-fracture, deep-etch image of in situ synaptic surface, left) and Torpedo electric organ (negative-stain image of extracted membrane, right). Both images show long, close-packed ribbons of receptors extending across the membrane surfaces. The enlargement of an isolated vesicle on the right shows that ribbons are comprised of dimers of receptors in oblique alignment (see also Fig. 2b). While the structural findings reported here have been performed entirely on the more experimentally tractable Torpedo membrane, the close architectural similarities between the two systems, and their shared developmental origin, make it unlikely that their protein–lipid interactions and organization are fundamentally different. The images are from Hirokawa and Heuser [4] (left) and Brisson and Unwin [5] (right). Scale bar, 1000 Å.

Fig. 2.

Structures of the acetylcholine receptor and a tubular vesicle from Torpedo postsynaptic membrane. (a) The receptor is a heteropentamer (stoichiometry: α_δ, α_γ, β, γ, δ), which includes four TM helices (M1–M4) in each subunit, and a transverse sub-membrane helix, MX. The structure shown is that of a recent model (PDB ID 6uwz [9]), modified such that its transmembrane portion fits the densities of the (cholesterol-complexed) receptor in the tubes (Unwin, unpublished). The orange bars identify the levels of the phosphate moieties of the outer and inner phospholipid headgroups, which are 30 Å apart [7]; subunit colours: α_γ, red; α_δ, orange; β, green; γ, cyan; δ, blue. (b) The tubular vesicle consists of curved ribbons of δ subunit-linked receptor dimers packed closely together and embedded in a bilayer matrix, which is composed of the native lipids (one ribbon, grey; one dimer, pink).

Fig. 3.

Cross-sections through a helical reconstruction displaying the postsynaptic membrane in profile. Cholesterol in the outer leaflet gives rise to gaps next to the protein surfaces (red arrows) in the otherwise continuous (light grey) densities associated with the phospholipid headgroups. A similar weakening of density associated with cholesterol occurs in the inner leaflet, above the MX helices (blue arrows).

Fig. 4.

Organization of cholesterol next to acetylcholine receptors, viewed in tangential sections encompassing the phospholipid headgroups. (a) Outer leaflet and (b) inner leaflet: cholesterol-rich patches appear as small gaps (dark grey) among the headgroup densities (smooth light grey areas) next to the TM helices (red bars), and as more extended gaps – microdomains – bridging neighbouring proteins. The pair of receptors comprising the central δ–δ dimer is bridged by microdomains in both leaflets of the bilayer. The pairs of broken lines track a dimer ribbon (seen at lower magnification in Figs. 1 and 2b). The numbering 1–4 identifies TM helices M1–M4. Adapted from [7].

Fig. 5.

Hypothetical arrangements of cholesterol molecules based on the cryo-EM images from Torpedo: (a) wedging between helices M4–M1 and M1–M3 in outer leaflet; (b) supporting helices M3 and M4 in inner leaflet; (c) bridging the δ–δ dimer in the outer leaflet. Subunit colours as in Fig. 2a.

Fig. 6.

Optimal packing of acetylcholine-receptor channels in the vicinity of the active zone, based on image reconstruction from Torpedo tubular vesicles (dimer ribbon in grey; see also Fig. 2b). Channels at the crest of the junctional fold are closest to the active zone, where acetylcholine is released (pink shading). Those located down the side of the infolded membrane (and at more distant regions within the synaptic cleft) are exposed to less neurotransmitter, which is diluted further through its rapid hydrolysis by acetylcholinesterase [37]. Cooperative interactions, implicating the cholesterol-bridged (green dots) δ–δ dimers and α_γ–α_γ contacts, may help to spread channel activation into the side region (arrows) and into distant cleft regions, thereby magnifying the postsynaptic response.