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Nicotinic acetylcholine receptor at 4.6 Å resolution: transverse tunnels in the channel1

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Abstract

The nicotinic acetylcholine (ACh) receptor is the neurotransmitter-gated ion channel responsible for the rapid propagation of electrical signals between cells at the nerve/muscle synapse. We report here the 4.6 Å structure of this channel in the closed conformation, determined by electron microscopy of tubular crystals of Torpedo postsynaptic membranes embedded in amorphous ice. The analysis was conducted on images recorded at 4 K with a 300 kV field emission source, by combining data from four helical families of tubes (−16,6; −18,6; −15,7; −17,5), and applying three-dimensional corrections for lattice distortions. The study extends earlier work on the same specimen at 9 Å resolution.

Several features having functional implications now appear with better definition. The gate of the channel forms a narrow bridge, consisting of no more than one or two rings of side-chains, across the middle portion of the membrane-spanning pore. Tunnels, framed by twisted β-sheet strands, are resolved in the extracellular wall of the channel connecting the water-filled vestibule to the putative ACh-binding pockets. A set of narrow openings through which ions can flow are resolved between α-helical segments forming part of the cytoplasmic wall of the channel. It is suggested that the extracellular tunnels are access routes to the binding pockets for ACh, and that the cytoplasmic openings serve as filters to exclude anions and other impermeant species from the vicinity of the pore. Both transverse pathways are likely to be important in achieving a rapid postsynaptic response.

Introduction

Nicotinic ACh receptors are ion channels found in high concentrations in the postsynaptic membranes of muscle cells, where they mediate the chemical transmission of electrical signals from invading motor neurons. ACh released at the synaptic junctions stimulates the channels to open, and the flow of cations through them rapidly depolarises the postsynaptic membrane of the muscle cell, initiating an electrical signal that causes the muscle to contract. Although the ACh receptor is by far the best characterised neurotransmitter-gated ion channel in terms of its biochemical, physiological and pharmacological properties (for recent reviews, see Lester 1992, Karlin and Akabas 1995, Changeux and Edelstein 1998), the detailed structural mechanisms underlying the rapid depolarisation are not yet understood.

The ACh receptor is a large (∼290 kDa) glycoprotein complex. It is made from five membrane-spanning subunits: two (the αs) having identical amino acid sequences, and three others (β, γ and δ) having sequences homologous to the αs. The subunits have in common a large extracellular amino-terminal domain, four predicted transmembrane regions (M1-M4), and a substantial M3-M4 cytoplasmic loop. These are organised around a pseudo 5-fold axis that delineates a cation-selective pathway across the membrane when the channel is open, but a robust barrier to the ions when it is closed. Opening of the channel occurs upon binding of ACh to both α subunits at sites influenced most especially by the neighbouring γ and δ subunits. The site in αγ (the α subunit next to the γ subunit) is distinguished biochemically from the site in αδ (the α subunit next to the δ subunit) by its higher affinity for the competitive antagonist, d-tubocurarine (Neubig & Cohen, 1979).αγ and αδ are separated by one subunit. Electron crystallographic studies, using subunit-specific labels, determined the intervening subunit to be β (Kubalek et al., 1987); other lines of evidence favour γ as the subunit in this location (Karlin, 1993).

Postsynaptic membranes isolated from the (muscle-derived) electric organ of the Torpedo ray are readily converted into tubular vesicles containing receptors arranged regularly on their surfaces, almost as they are in vivo(Heuser & Salpeter, 1979). These tubular crystals, or “tubes”, have overall helical symmetry, making them an ideal kind of specimen for three-dimensional structure determination by electron microscopy. An initial electron microscopic study of tubes frozen to retain their helical symmetry (Toyoshima & Unwin, 1988) showed that the ring of five receptor subunits creates a narrow pore across the membrane, flanked on either side by ∼20 Å wide vestibules. The vestibule on the extracellular side (outside of the tube) is ∼65 Å long. The vestibule on the cytoplasmic side is shorter and has additional density underlying it. This density was not visible in tubes imaged in high pH solution and hence was attributed to the clustering protein, rapsyn, which is normally present in approximately 1:1 stoichiometry with the receptor in Torpedo postsynaptic membranes Sealock 1982, LaRochelle and Froehner 1986, but is released by alkaline pH (Neubig et al., 1979).

A more recent study of the tubes revealed elements of secondary structure in the transmembrane portion of the receptor and also in a region ∼30 Å away from the membrane, where the two α subunits contain cavities: the putative ACh-binding pockets (Unwin, 1993). The structures shaping the two cavities are similar but not identical, indicating that the α subunits have different conformations before ACh binds (Unwin, 1996). Additionally, a time-resolved analysis of the tubes showed that the receptor undergoes a concerted conformational change, involving disturbances around both cavities, after brief reaction with ACh to induce the open-channel form (Unwin, 1995). The gate near the middle of the membrane opens up as a result of this transition, and the pore becomes narrowest close to the cytoplasmic membrane surface.

The resolution in these recent structural investigations (∼9 Å) was limited by the modest performance of the electron microscopes used and by distortions that caused the receptors to be displaced significantly from their correct lattice positions over distances beyond about 800 Å. Here, we have been able to record images of exceptional quality by using a 300 kV field emission electron microscope incorporating a top-entry liquid-helium-cooled stage (Fujiyoshi et al., 1991). We have also applied a computational method to correct in three dimensions for the lattice distortions (Beroukhim & Unwin, 1997), and analysed images from four helical families of tubes. The technical improvements, and the collection of more extensive data, have allowed the structure determination to be extended to 4.6 Å resolution.

Here, we compare the receptor structures determined from the four helical families of tubes and show that these structures are equivalent. The four three-dimensional density maps could therefore be combined to obtain an average structure, contributed by more than 500,000 molecules. The average structure reveals, with better definition, the gate of the channel and features affecting ACh binding and ion transport. We now find that there are tunnels in the extracellular wall of the channel connecting the vestibule to the putative ACh-binding pockets. Structural parallels with the “gorge” in acetylcholinesterase suggest that these tunnels are access routes for ACh to the binding pockets. Narrow openings are also resolved in the cytoplasmic wall of the receptor, which may serve as filters to exclude anions and other impermeant species from the vicinity of the pore. Direct flow of ions into or from the interior of the cell appears to be obstructed not only by rapsyn, as originally thought, but also by a part of the receptor itself.

Section snippets

Subdivision into helical families

ACh receptor tubes form from ribbons of receptor dimers (Brisson & Unwin, 1984). The ribbons associate side-by-side to make a surface lattice which in two dimensions would have the plane group symmetry, p2. The unit cell vectors of this lattice and the principal 1,0 and 0,1 lines are shown in Figure 1(a). To represent a tube, the same sets of lines may be considered as helical traces over the surface of a cylinder. They thus give rise to a range of helical families depending on the number of

Discussion

The results presented above have led to a description of the high-resolution structure of the closed-channel form of the ACh receptor. Four helical families of tubular crystals were examined: the one most frequently encountered, (−16,6), and three others having the same surface lattice, but different helical parameters. We showed by pairwise correlation that three-dimensional maps of the receptor obtained from each family were essentially the same, when differences in numbers of images were

Tunnels to the ACh-binding pockets

The extracellular domains of both α subunits contain cavities that were proposed in an earlier study to be the ACh-binding pockets (Unwin, 1993). This assignment was based on two lines of evidence. First, the two ACh-binding α subunits were the only subunits found to contain these specialised internal features. Second, the cavities aligned, in projection, with the locations of the binding sites that were determined, using as a marker the competitive antagonist α-bungarotoxin (Kubalek et al.,

Gate of the channel

In the open-channel form of the receptor, the pore would make a continuous water-filled passage from one side of the membrane to the other, narrowing towards the cytoplasmic surface Villarroel et al 1991, Lester 1992. However, in this closed-channel form, a narrow strip of density bridges the pore at a level close to the middle of the membrane (Figure 11). This density, almost 15 Å away from the cytoplasmic membrane surface, must correspond to the gate of the channel, since it is the only

Passages through the cytoplasmic wall

The cytoplasmic wall of the receptor was shown to contain openings wide enough for the ions to pass through, made by the spaces between ∼30 Å long rods that protrude from each of the subunits towards the cell interior (Figure 12(a) and (b)). Interestingly, the spaces are not all equal in size, the widest being those on opposite faces of the two α subunits (arrows, Figure 12(b)) and the next widest those on either side of the intervening (β) subunit. However, the openings are all less than 10 Å

Attachment to rapsyn

The central mass underlying the cytoplasmic vestibule of the receptor was interpreted in an earlier, lower resolution analysis of the tubular crystals to be composed entirely of non-receptor protein (Toyoshima & Unwin, 1988). This interpretation arose because the mass was absent in maps obtained from tubes frozen under conditions (pH 11) known to release the clustering protein, rapsyn (Neubig et al., 1979). Furthermore, tannic-acid-stained material was observed to be redistributed within the

Architecture of the sub-synaptic membrane

Figure 15 is a schematic drawing highlighting some functional aspects of the sub-synaptic membrane suggested by the structural results. Facing the synaptic cleft are the extracellular vestibules of the channels, which are wide enough to provide freely diffusing pathways for both the small inorganic cations and ACh. Opening into these vestibules are the tunnels, which selectively guide the ACh molecules to their binding sites. Near the middle of the membrane are the gate-forming amino acid

Outlook

The work reported here has combined a new method to determine structure from tubular crystals with the use of liquid helium temperatures to minimise radiation damage. This has allowed a resolution approaching atomic dimensions to be attained from only about 500,000 molecules: a limit that is primarily a result of the smallness in number of molecules analysed, according to the Fourier shell correlations (Figure 8). Since the resolution limit is not due to an inherent property of the specimen,

The specimen

Tubular crystals of about 800 Å in diameter were grown from postsynaptic membranes isolated from Torpedo marmorata electric organ in the standard way (Kubalek et al., 1987), using fish killed in the autumn or winter. The preparation involves no high salt or detergent treatment, and retains the native protein and lipid components of the postsynaptic membrane. While tubes could be obtained from most fish, only a small proportion of preparations gave high yields of tubes having the required

Acknowledgements

We thank our colleagues in Japan, the UK and the Scripps Research Institute, California, for their help and encouragement. Rameen Beroukhim participated in pilot studies designed to optimise the recording and analysis of the 4 K images. Ikuyo Arimoto (IIAR) and Yoko Hiroaki (Kyoto University), and staff at LMB, Cambridge, provided valuable assistance in many aspects of the work. The Marine Station, Roscoff, France, generously supplied the Torpedo electric rays. The research was supported in

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