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

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 voltage dependent 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 stateThis research was supported by a Project (APP1069417) from the Australian National Health and Medical Research Council (M.D.M. and M.C.) and by a Discovery Project (DP0986469) from the Australian Research Council (M.D.M. and M.C.). T.Q., G.Q., J.I.H. were supported by Australian Postgraduate Award. D.I. was supported by International Postgraduate Research Scholarship, and T.Q., G.X.Q., J.I.H., and D.I. were also supported by the John A. Lamberton scholarshi

Revisiting autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) mutations in the nicotinic acetylcholine receptor reveal an increase in efficacy regardless of stochiometry

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a genetic form of epilepsy that is caused by mutations in several genes, including genes encoding for the α4 and β2 subunits of the nicotinic acetylcholine (nACh) receptor. Pentameric α4β2 nACh receptors are the most abundant nicotinic receptor in the mammalian brain and form two stoichiometries, the (α4)3(β2)2 and (α4)2(β2)3 receptors that differ in their physiological and pharmacological properties. The purpose of this study was to investigate how ADNFLE mutations β2V287M, β2V287L or α4T293I manifest themselves in different receptor stoichiometries. We expressed wild-type and mutant receptors in Xenopus oocytes and measured the response to ACh and other agonists at both receptor stoichiometries. For all three mutations, the efficacy of ACh at (α4)2(β2)3 receptors was increased. At (α4)3(β2)2 receptors, the efficacy of activation was increased both when two molecules of agonist, either ACh or the siteselective agonist sazetidine-A, were bound at the α4-β2 interfaces, and when a third ACh molecule was bound at the α4-α4 site. Regardless of stoichiometry, the mutations increased the current elicited by low concentrations of ACh. Further, the smoking cessation agents, nicotine, varenicline and cytisine increased activation of mutant (α4)3(β2)2 receptors, while only nicotine increased activation of mutant (α4)2(β2)3 receptors. Chronic exposure of all agonists reduced ACh-activation levels at low and high ACh concentrations. From this, we concluded that mutations that cause ADNFLE manifest themselves in a change in efficacy regardless of the stoichiometry of the receptor

Covalent trapping of methyllycaconitine at the α4-α4 interface of the α4β2 nicotinic acetylcholine receptor: antagonist binding site and mode of receptor inhibition revealed

Background: Methyllycaconitine is an antagonist at subtypes of the nicotinic acetylcholine receptor. Results: A reactive methyllycaconitine probe was covalently trapped by a cysteine introduced on the complementary face of the α4 subunit and only in th