Understanding structure-function relationships of the human neuronal acetylcholine receptor: insights from the first crystal structures of neuronal subunits

Abstract

Nicotinic ACh receptors (nAChRs) are the best studied members of the superfamily of pentameric ligand-gated ion channels (pLGICs). Neuronal nAChRs regulate neuronal excitability and neurotransmitter release in the nervous system and form either homo- or hetero-pentameric complexes with various combinations of the 11 neuronal nAChR subunits (α2-7, α9, α10 and β2-4) known to exist in humans. In addition to their wide distribution in the nervous system, neuronal nAChRs have been also found in immune cells and many peripheral tissues. These nAChRs are important drug targets for neurological and neuropsychiatric diseases (e.g. Alzheimer's, schizophrenia) and substance addiction (e.g. nicotine), as well as in a variety of diseases such as chronic pain, auditory disorders and some cancers. To decipher the functional mechanisms of human nAChRs and develop efficient and specific therapeutic drugs, elucidation of their high-resolution structures is needed. Recent studies, including the X-ray crystal structures of the near-intact α4β2 nAChR and of the ligand-binding domains of the α9 and α2 subunits, have advanced our knowledge on the detailed structure of the ligand-binding sites formed between the same and different subunits and revealed many other functionally important interactions. The aim of this review is to highlight some of the structural and functional findings of these studies and to compare them with recent breakthrough findings on other pLGIC members and earlier data from their homologous ACh-binding proteins.

Linked articles: This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc.

Figure 1

Overall architecture of the α4β2 nAChR and α2‐ECD. (A) View of α4β2 parallel to the plasma membrane (PDB ID: 5KXI) (Morales‐Perez et al., 2016). α subunits are shown in green and β in cyan, while nicotine is in orange spheres. Solid lines indicate the approximate limits of the membrane. (Β) View of α4β2 along the channel axis. Colour coding as in (A). (C) Side‐view of α2‐ECD (PDB ID: 5FJV) (Kouvatsos et al., 2016). Each of the α2 subunit is coloured differently and epibatidine is shown in orange spheres. (D) The protomer of the human α4 subunit participating in α4β2 nAChR (PDB ID: 5KXI). The critical domains, characteristic of pLGICs, are shown. The ECD and the intracellular helix (MX) are coloured in green, while each TM helix is in different colour. The coordinates of all the structures depicted were retrieved from Protein Data Bank (http://www.wwpdb.org), and PyMol (http://www.pymol.org) was used to generate the figures.

Figure 2

Close views of wild‐type or chimeric nAChR ligand‐binding sites. (A) The α7‐AChBP bound to epibatidine (PDB ID: 3SQ9) (Li et al., 2011). (B) Engineered AChBP towards α4/α4 bound to NS3920 (PDB ID: 4UM3) (Shahsavar et al., 2015). (C) The α2‐ECD bound to epibatidine (PDB ID: 5FJV) (Kouvatsos et al., 2016). (D) The α4β2 nAChR bound to nicotine (PDB ID: 5KXI) (Morales‐Perez et al., 2016). The principal sides are shown in green, the complementary in cyan and the agonists in magenta. Interactions are shown in black dashed lines. The coordinates of all the structures depicted were retrieved from Protein Data Bank (http://www.wwpdb.org), and PyMol (http://www.pymol.org) was used to generate the figures.

Figure 3

Rearrangements upon agonist binding. (A) The epibatidine‐bound α2 subunit showing the interaction of loop‐C Tyr²¹⁹ with the β7‐strand Lys¹⁷⁴, probably weakening the interaction between the residues of β7 and β10 strands. The (+) side is shown in green, the agonist in magenta and the (−) subunit in cyan. (B) Similarly for the α4 subunit bound to nicotine. Colours as in (A). (C) The β2‐subunit Asp198 on β10‐strand acquires a rotamer never observed before in α subunits. It is further stabilized by interactions with two positively charged residues of β7 strand. The β2 subunit is shown in cyan. α4 and β2 subunits were retrieved from PDB ID: 5KXI (Morales‐Perez et al., 2016) and α2 subunit from PDB ID: 5FJV (Kouvatsos et al., 2016). The coordinates of all the structures depicted were retrieved from Protein Data Bank (http://www.wwpdb.org), and PyMol (http://www.pymol.org) was used to generate the figures.

Figure 4

Membrane‐facing networks. (A) Close view of the interactions between structural elements at the lower part of the ECDs, viewed from the bottom of the ECD. These interactions are present in most of the resolved structures of pLGICs [α9‐ECD in green, PDB ID: 4D01 (Zouridakis et al., 2014); GABA_A receptor in pink, PDB ID: 4COF (Miller and Aricescu, 2014); 5‐HT₃ receptor in yellow, PDB ID: 4PIR (Hassaine et al., 2014); GLIC in orange, PDB ID: 3EAM (Bocquet et al., 2009)]. The invariant arginine at the end of β10 strand or pre‐M1 loop interconnects Cys‐loop, β1–β2 loop and in most cases β8–β9 loop. (B) Side view of the superimposed structures of the glycine receptor determined in closed (PDB ID: 3JAD), open (PDB ID: 3JAE) and desensitized (PDB ID: 3JAF) states (Du et al., 2015), shown in green, magenta or orange respectively. The interaction network in (B) is shown in equatorial orientation, while the aromatic residues that sandwich the charged residues of the network are in axial positions. Representative interactions are shown in black dashed lines. The coordinates of all the structures depicted were retrieved from Protein Data Bank (http://www.wwpdb.org), and PyMol (http://www.pymol.org) was used to generate the figures.

Figure 5

Comparison of subunit interfaces. (A) Superposition of β2/α4 [in cyan or orange, respectively; PDB ID: 5KXI (Morales‐Perez et al., 2016)], α2/α2 [in green or yellow, respectively; PDB ID: 5FJV (Kouvatsos et al., 2016)] and nicotine‐bound AChBP [in purple or magenta, respectively; PDB ID: 1UW6 (Celie et al., 2004)]. The lack of one tyrosine in loop C of the β2 subunit allows the radical rotation of its Tyr¹⁹⁶ to occupy space that in α subunits is occupied by the other tyrosine (e.g. α2‐Tyr²¹⁹). β2‐Tyr¹⁹⁶ along with the β2‐Tyr⁹⁵ from loop A stabilize the β2‐Arg¹⁴⁹ that rams the cavity. This is possible only after β2‐Tyr⁹⁵ recedes towards the complementary subunit, occupying the space where in α subunits the loop‐B tryptophan (e.g. α2‐Trp¹⁷⁸) is found. As a result, β2‐Trp¹⁵¹ presents an extreme rotational movement towards β4–β5 loop. Notably, the α2‐ECD pentameric structure shows that this rotamer of loop‐B tryptophan is also possible in α subunits, but not in AChBPs where this space is occupied by β4–β5 loop. (B) The same as (A) rotated by 90^o. The coordinates of all the structures depicted were retrieved from Protein Data Bank (http://www.wwpdb.org), and PyMol (http://www.pymol.org) was used to generate the figures.

Figure 6

Models of α9/α10 and α10/α9 binding sites. (A–C) Superpositions of the ACh‐bound AChBP crystal structure [AChBP in blue; ACh in orange; PDB ID: 3WIP (Olsen et al., 2014)] with models of the ACh‐bound α9α10 binding sites (α9 and α10 in green; ACh in magenta) (Azam et al., 2015). (A) Side‐view of the α10/α9 interface, showing a similar binding mode for ACh with that in AChBP, although ACh and loop C are shifted upwards. (B) The same as in (A), rotated by 90^o, also showing a lateral shift of loops B and C of α10(+) side. The stable salt bridge from the α9(−) side is also shown. (C) Top‐view of the α9/α10 interface, showing an extreme shift of ACh outwards, causing an equal shift of α9(+) loop C. A second arginine from α10(−) side penetrates the binding cavity, forming an uncommon charged environment. All interactions are shown in black dashed lines. The coordinates of all the structures depicted were retrieved from Protein Data Bank (http://www.wwpdb.org), and PyMol (http://www.pymol.org) was used to generate the figures.