Characterizing different stoichiometric forms of nicotinic acetylcholine receptors (nAChR) using molecular pharmacology and kinetic modelling

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels involved in fast synaptic transmission. They are pentameric structures formed from a combination of different or identical subunits to produce heteromeric or homomeric channels. The heteromeric receptor subtypes form distinct stoichiometric forms, which respond differently to the endogenous agonist, acetylcholine (ACh), and other clinically relevant agonists and antagonists. The current thesis is focused on developing our understanding both of the pharmacology of different stoichiometries, and their mechanism of activation. The heteromeric α9α10 nAChR subtype is well-known for its role in the auditory system, being expressed in cochlear hair cells, and also been involved in immune-modulation. Antagonists of α9α10 nAChRs, like the α-conotoxin Vc1.1, have effects in controlling neuropathic pain. Unlike other nAChR subtypes there is no evidence that functional receptor stoichiometries of α9α10 exist, until work contained in this thesis was published. By using 2-electrode voltage clamp method and maintaining a constant intracellular Ca2+ concentration, we observed a biphasic activation curve for ACh that is dependent on receptor stoichiometry. Vc1.1, but not the other α9α10 antagonists RgIA or atropine, inhibits ACh-evoked currents in a biphasic manner. Characteristics of the ACh and Vc1.1 activation and inhibition curves can be altered by varying the ratio of α9 and α10 mRNA injected into oocytes, changing the curves from biphasic to monophasic when an excess of α10 mRNA is used. These results highlight the difference in the pharmacological profiles of at least two different α9α10 nAChR stoichiometries, possibly (α9)3(α10)2 and (α9)2(α10)3, although other stoichiometries may exist. As a result, we infer that there is an additional binding site for ACh and Vc1.1 at the α9-α9 interface on the hypothesized (α9)3(α10)2 nAChR, in addition to the α10-α9 and or α9-α10 interfaces that are common to both stoichiometries. This study provides further evidence - 7 - that receptor stoichiometry contributes another layer of complexity in understanding Cys-loop receptors. The α4β2 nAChR is the most abundant subtype in the brain and exists in two functional stoichiometries: (α4)3(β2)2 and (α4)2(β2)3. A distinct feature of the (α4)3(β2)2 receptor is the biphasic activation response to ACh, where it is activated with high potency and low efficacy when two α4-β2 binding sites are occupied and with low potency/high efficacy when a third α4-α4 binding site is occupied. Further, exogenous ligands can bind to the third α4-α4 binding site and potentiate the activation of the receptor by ACh that is bound at the two α4-β2 sites. We propose that perturbations of the recently described pre-activation step when a third binding site is occupied are key drivers of these distinct activation properties. To investigate this, we used a combination of simple linear kinetic models and voltage clamp electrophysiology to determine whether transitions into the pre-activated state were increased when three binding sites were occupied. We separated the binding at the two different sites with ligands selective for the α4-β2 site (Sazetidine-A and TC- 2559) and the α4-α4 site (NS9283) and identified that when a third binding site was occupied, changes in the concentration-response curves were best explained by an increase in transitions into a pre-activated state. We proposed that perturbations of transitions into a pre-activated state are essential to explain the activation properties of the (α4)3(β2)2 receptor by acetylcholine and other ligands. This information can be used to develop selective ligands that bind at distinct pharmacological sites to modulate receptor activation with the desired effects. 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 nACh receptor. To determine how ADNFLE mutations alter the function of the different stoichiometries, we injected cRNA encoding for wild-type and mutant receptors containing the - 8 - ADNFLE mutations β2V287M, β2V287L or α4T293I and measured the response to acetylcholine and other agonists at both receptor stoichiometries. For all three mutations, the efficacy of receptor activation by acetylcholine at (α4)2(β2)3 receptors was increased. At (α4)3(β2)2 receptors, the efficacy of activation was increased both when two molecules were bound at the α4-β2 interfaces, and when a third ACh molecule was bound at the α4-α4 site. Regardless of stoichiometry, the ADNFLE mutations resulted in increased current elicited by low concentrations of ACh. The smoking cessation agents, nicotine, varenicline and cytisine increased activation of (α4)3(β2)2 receptors, while only nicotine increased activation of (α4)2(β2)3 receptors. However chronic exposure of nicotine, varenicline or cytisine reduced the ACh activation levels at both low and high ACh concentrations. This study provides a basic understanding on how mutations that cause ADNFLE manifest themselves in a change in efficacy regardless of the stoichiometry they are expressed in. Overall, the thesis significantly adds to our current understanding of receptor stoichiometry in nAChR subtypes. Several approaches are used including understanding how clinically significant ligands such as Vc1.1 act at the α9α10 nAChRs, use of kinetic modelling for mechanistic understanding of how agonists act at the α4-α4 binding site within the α4β2 receptor subtype and finally to understand and characterize some of the native disease mutations that cause epilepsy, in the context of receptor stoichiometry