Activation of the Nicotinic Acetylcholine Receptor Involves a Switch in Conformation of the α Subunits

Abstract

The nicotinic acetylcholine (ACh) receptor belongs to a superfamily of synaptic ion channels that open in response to the binding of chemical transmitters. Their mechanism of activation is not known in detail, but a time-resolved electron microscopic study of the muscle-type ACh receptor had suggested that a local disturbance in the ligand-binding region and consequent rotations in the ligand-binding α subunits, connecting to the transmembrane portion, are involved. A more precise interpretation of this structural change is given here, based on comparison of the extracellular domain of the ACh receptor with an ACh-binding protein (AChBP) to which a putative agonist is bound. We find that, to a good approximation, there are two alternative extended conformations of the ACh receptor subunits, one characteristic of either α subunit before activation, and the other characteristic of all three non-α subunits and the protomer of AChBP. Substitution in the three-dimensional maps of α by non-α subunits mimics the changes seen on activation, suggesting that the structures of the α subunits are modified initially by their interactions with neighbouring subunits and switch to the non-α form when ACh binds. This structural change, which entails 15–16° rotations of the inner pore-facing parts of the α subunits, most likely acts as the trigger that opens the gate in the membrane-spanning pore.

Introduction

The nicotinic acetylcholine (ACh) receptor is a member of the Cys-loop superfamily of transmitter-gated ion channels which includes neuronal ACh receptors, GABA_A receptors, 5HT₃ receptors and glycine receptors. It is found in high concentrations at the nerve–muscle synapse, where it mediates fast chemical transmission of electrical signals by opening a narrow pore across the postsynaptic membrane of the muscle cell in response to ACh released from the nerve terminal into the synaptic cleft. Although the ACh receptor is by far the best characterized member of the superfamily in terms of its biochemical and physiological properties,1., 2., 3. the detailed structural mechanism underlying activation of the ion channel is not yet understood.

The ACh receptor is a large (∼290 kDa) glycoprotein comprised of five ∼160 Å long rod-shaped subunits, two (the αs) having identical amino acid sequences, and the three others (β, γ and δ) having similar sequences (36–42% pairwise identity with the αs⁴). The subunits have in common a large N-terminal extracellular domain, four predicted transmembrane regions and an extended cytoplasmic loop. These are arranged around a pseudo 5-fold axis, delineating a cation-selective pathway across the membrane pore 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 (α_γ and α_δ) at sites that are at, or close to, the interfaces made with neighbouring γ and δ subunits.5., 6., 7. These sites are shaped by three separate regions of the α polypeptide chain,2., 3. and include the so-called C-loop, which contains several key residues implicated in ACh binding.

Postsynaptic membranes isolated from the (muscle-derived) electric organ of the Torpedo ray have been a major source of material for structural studies of the receptor. The membranes are readily converted into tubular crystals,8., 9. which have receptors on their surfaces arranged almost as they are in vivo.¹⁰ Electron crystallographic studies of the tubular crystals have provided basic information about three-dimensional structure of the receptor and about how it functions as an ion channel.11., 12., 13., 14. In a previous electron crystallographic investigation of the activation mechanism, a rapid spray-freezing technique was used to represent the synaptic release of ACh and to trap the receptor in the open-channel form.¹⁴ An analysis of the activated structure at 9 Å resolution indicated that the binding of ACh initiates two interconnected events in the extracellular domain. One is a local disturbance in the region of the binding sites, about halfway between the membrane and the synaptic end of the receptor, and the other a larger-scale conformational change, which communicates to the transmembrane portion. The latter appeared to involve small axial rotations predominantly in the ligand-binding α subunits.

A more precise description of the extended conformational change is reported here, based on a correlation between the structure of the ACh receptor at 4.6 Å resolution, obtained from electron images of Torpedo membranes recorded at liquid helium temperatures,¹³ and the atomic model of AChBP complexed with the putative agonist HEPES.¹⁵ AChBP is a soluble, homo-pentameric protein and therefore lacks part of the construction and the physiological properties one would associate with an ion channel. Nevertheless, this protein has the same overall architecture as the extracellular portion of the receptor (Figure 1), and the AChBP protomer has 20–23% sequence identity with the N-terminal ∼200 amino acid residues of the receptor subunits. The structural homology between the two assemblies therefore enables a reliable interpretation of the receptor densities over much of the extracellular domain.

The core of the AChBP protomer is organized around two sets of β-stands, forming “Greek key” motifs, which are linked together through the Cys-loop disulphide bond and folded into a curled β-sandwich.¹⁵ This core was divided into separate “inner” and “outer” parts of the sandwich, which were fitted independently to the densities in the 4.6 Å, resting-state structure. We found that the two parts in all three non-α subunits (β, γ and δ) were oriented approximately as in AChBP, whereas the two parts in the α subunits had a different alignment. Although this distinction between the non-α and α subunits applied to the receptor in the resting state, no such distinction applied in the density map obtained from the activated receptor. The structures of the α subunits therefore appear to be modified initially by their interactions with neighbouring subunits and to switch to the non-α configuration when ACh binds. This conformational change in the extracellular portion of the receptor, parallels other changes observed previously in the transmembrane portion, suggesting it may act as a trigger to open the gate in the membrane-spanning pore.