AE Succinimide, an Analogue of Methyllycaconitine, When Bound Generates a Nonconducting Conformation of the α4β2 Nicotinic Acetylcholine Receptor

Abstract

Nicotinic acetylcholine (nACh) receptors are pentameric ligand-gated ion channels that mediate fast synaptic transmission. The α4β2 nACh receptor is highly expressed in the brain and exists in two functional stoichiometries: the (α4) 2 (β2) 3 and (α4) 3 (β2) 2 that differ by an ACh-binding site at the α4−α4 interface of (α4) 3 (β2) 2 receptors. Methyllycaconitine (MLA) is an nACh receptor antagonist, and while potent at both α7 and α4β2 nACh receptors, it has a higher selectivity for the α7 nACh receptor. The anthranilate-succinimide ester side-chain is important for its activity and selectivity. Here we identify a simplified MLA analogue that contains only the A and E ring skeleton of MLA, AE succinimide, that binds close to the channel lumen to display insurmountable inhibition at α4β2 nACh receptors. Although inhibition by AE succinimide was found to be voltagedependent indicating a possible pore channel blocker, substituted-cysteine accessibility experiments indicated it did not bind between 2′−16′ region of the channel pore. Instead, we found that upon binding and in the presence of ACh, there is a conformational change to the channel membrane that was identified when the compound was assessed against (α4 V13′C)β2 nACh receptors. It was found that in the 3:2 stoichiometry the two adjacent α4 subunits containing 13′ cysteine mutations formed a disulfide bond and occluded ion conductance. This was reversed by treatment with the reducing agent, dithiothreitol. Thus, AE succinimide has a different mechanism of inhibition to both MLA and other AE analogues, such as AE bicyclic alcohol, in that upon binding to an as yet unidentified site, AE succinimide in the presence of ACh induces a conformational change to the channel that generates a ligand-bound closed state.

Structural Studies of Norditerpenoid Alkaloids: Conformation Analysis in Crystal and in Solution States

“…A synthetic A/E-[3.3.1]azabicyclic ketone HCl salt (19,Figure 5c) adopts a true-boat/true-chair conformation in its crystal lattice, but it adopts true-chair/true-chair conformation in D 2 O as its ketal hydrate (20) where C9 is now sp 3 hybridised rather than the sp 2 ketone. We conclude that it is the nature of a protonated [3.3.1]azabicycle to adopt a boat/chair conformation in the crystal lattice, which does not require any interaction such as an intramolecular bond or intramolecular repulsion like that between 1-OMe/12-H e in NDAs.…”

“…However, their affinities at nAChRs never reach that of MLA (7). [19,20] We considered that the conformation, especially the solution conformation, of the A/E-rings in the NDAs is different from that of the simple synthetic analogues. The skeletal structure, especially of the A/E-[3.3.1]azabicyclic rings, in the crystal lattice and in solution states are worthy of detailed investigation.…”

“…Some of these analogues substituted with the key 2 S ‐(methylsuccinimido)benzoate moiety have been biologically evaluated. However, their affinities at nAChRs never reach that of MLA ( 7 ) [19,20] . We considered that the conformation, especially the solution conformation, of the A/E‐rings in the NDAs is different from that of the simple synthetic analogues.…”