Structure and gating mechanism of the α7 nicotinic acetylcholine receptor

Abstract

The α7 nicotinic acetylcholine receptor plays critical roles in the central nervous system and in the cholinergic inflammatory pathway. This ligand-gated ion channel assembles as a homopentamer, is exceptionally permeable to Ca²⁺, and desensitizes faster than any other Cys-loop receptor. The α7 receptor has served as a prototype for the Cys-loop superfamily yet has proven refractory to structural analysis. We present cryo-EM structures of the human α7 nicotinic receptor in a lipidic environment in resting, activated, and desensitized states, illuminating the principal steps in the gating cycle. The structures also reveal elements that contribute to its function, including a C-terminal latch that is permissive for channel opening, and an anionic ring in the extracellular vestibule that contributes to its high conductance and calcium permeability. Comparisons among the α7 structures provide a foundation for mapping the gating cycle and reveal divergence in gating mechanisms in the Cys-loop receptor superfamily.

Keywords: Cys-loop receptor; acetylcholine receptor; cryo-EM; ion channel; ligand-gated ion channel; nicotinic receptor; α7.

Figure 1:. α7 nicotinic receptor structures and function.

A, Whole-cell patch-clamp electrophysiology of WT α7 (black) vs. the EM construct (red). B, Single-channel electrophysiology, comparing responses of acetylcholine with and without PNU-120596. C, D, and E, cryo-EM maps of the α7 receptor (teal) bound to α-bungarotoxin (green), epibatidine + PNU-120596 (red), and epibatidine alone (purple). Top panels show the side view of maps normal to the plane of the cell membrane. Bottom panels show the top view looking down the central axis of the pore. The nanodisc shell is represented as a semitransparent surface.

Figure 2:. Neurotransmitter-binding pocket.

A, α-Bungarotoxin-bound (resting) α7 model on left; perspective magnified on right is indicated. α-Bgt is shown in green, receptor in teal. Key interacting residues from the toxin and the receptor are shown. B, Epibatidine + PNU-bound (activated) α7 model on left; perspective magnified on right is indicated. Epibatidine is shown in gold with coordinating residues shown. (+) indicates the principal subunit of the interface and (−) indicates the complementary subunit.

Figure 3:. Coupling region interactions and conformational differences.

A, Alignments of amino acids from α subunits in the nicotinic receptor family for coupling regions. B-D, The coupling region in resting (B), activated (C), and desensitized (D) conformations. Dashed lines indicate electrostatic interactions with distances in Angstrom (Å).

Figure 4:. Conformational transitions in α7 transmembrane and intracellular domains.

A, Global superposition of the three α7 structures. α-Helices are represented as cylinders. Teal, resting. Red, activated. Purple, Desensitized. Boxed areas indicate regions in panels B-C. B, Experimental density maps aligned by their ECD showing tilting of M1, M3 and M4 helices from a single subunit from each structure. The Mx density has been removed for clarity. C, the TMD helix bundle as viewed from the pore. The M2-M4 salt bridge between K238 and D445 is indicated. K45 capping of the M2 helix (dashed lines) is shown in the activated (red) and desensitized (purple) states.

Figure 5:. Ion permeation pathway.

A-C, permeation pathways for the three conformational states represented by solid surface colored by hydrophobicity. Constriction points are indicated with distances in A. D-F, Two M2 helices are shown for each of the conformational states with side chains shown for pore-lining residues, numbering on the left. Diameters (A) are indicated with dashed lines.

Figure 6:. Structural and functional interrogation of ECD vestibule constriction.

A, Top view of α7 in an activated conformation, highlighting residues involved in ECD constriction. B, amino acids related to the ECD constriction shown from the three receptor conformations. Residues and distances (A) are shown for one subunit interface. C, Side view showing three subunits from the perspective within the ECD vestibule, highlighting the same residues as in A and B. D, Orientation from C, focusing on interactions at one subunit interface and their differences as a function of conformational state. E, F, Current-Voltage (IV) relationships for two concentrations of CaCl₂ are shown. G, Whole cell recordings in the absence and presence of calcium. Asterisk (*) indicates tail current seen from calcium-induced channel block. H, Single channel current-voltage relationships for WT and E97A. Lines are least squares fits with slope conductance in units of pS indicated.

Figure 7:. Global conformational changes during the nicotinic receptor gating cycle.

A-C, Two-subunit interfaces of the resting, activated and desensitized states are shown with black rods drawn down the axis of the center of mass of the ECD or TMD of the principal subunit. Arrows indicate motions undergone from resting to activated (B) and activated to desensitized (C) states. D-F, top down view of the TMD pore of resting (D), activated (E) and desensitized (F) states.