Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. They are involved in a vast number of pathophysiological processes in the brain including drug addiction, pain, learning and memory. nAChRs have also been implicated in various Central Nervous System (CNS) disorders such as Alzheimer’s disease, Parkinson’s diseases and schizophrenia. To date, 12 subunits have been cloned from mammalian brain, and include α2-α10 and β2-β4. Although the native subunits-combinations are yet to be fully elucidated, the most dominant subunit combinations are the α7 and α4β2 subtypes. The α4β2 nAChR is known to exist in two functional isoforms with different ACh-activation properties, namely the (α4)2(β2)3 and (α4)3(β2)2 isoforms that differ by the presence of an additional agonist binding site located at the α4-α4 interface on (α4)3(β2)2 receptors.
Methyllycaconitine (MLA) is a natural plant toxin that is a potent competitive antagonist for the α7 and α4β2 receptors. MLA is 1000-fold more selective for α7 than at α4β2 receptors. However, by identifying ligands that are selective for specific nAChR subtypes based on MLA scaffolds may have significant therapeutic potential and contribute to the understanding of the physiological roles of these subtypes in vivo.
In Chapter 3, we investigated MLA analogues in order to identify selective compounds for nAChRs. A series of simple analogues of MLA (BA01-BA12) were evaluated for their ability to discriminate between human α7 and the two stoichiometries of α4β2 nAChRs (α4)3(β2)2, and (α4)2(β2)3) receptors expressed in Xenopus oocytes using two electrode voltage clamp methods. All 12 analogues assessed had a greater selectivity for (α4)3(β2)2 over α7. At 10 μM, (BA07-BA12) analogues successfully discriminated between (α4)3(β2)2 and α7. Since inhibition of BA09, BA11 and BA12 at (α4)3(β2)2 receptors were more compared to their inhibition at (α4)2(β2)3 receptors, further refinement of these chemical structures is required to achieve greater selectivity between the two (α4)3(β2)2, and (α4)2(β2)3) stoichiometries. The Xenopus oocytes used as expression system due to relative absence of endogenous channels which might complicate analysis of electrophysiological measurements.
Chapter 4, describes how AE Succinimide, a simplified MLA analogue, stabilises a closed state of the (α4)3(β2)2 receptor at a distinct site on the channel pore. By utilizing substituted-cysteine accessibility and cross-linking experiments, we showed, using the (α4)3(β2)2 stoichiometry, that upon binding, AE Succinimide induces a conformational change that caused two adjacent mutated α4 subunits at the 13’ to a cysteine react in an act to stabilise a closed state. The cross-reaction was reversed by using the reducing agent, dithiothreitol (DTT). This study demonstrates that AE Succinimide is a pore blocker of nAChRs that not only act by sterically hindering ion permeability but acts by inducing a conformational change to stabilize a closed state.
In Chapter 5, we investigated certain mutations of the (α4)3(β2)2 receptor that causes autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). 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. This chapter describes how the function of the channel can be changed by a disease-causing mutation located in the transmembrane region. Specifically, how mutations in the β2 subunit (β2V308A) that cause ADNFLE alter the function of distinct stoichiometric forms of α4β2 nACh receptors.
Overall, the thesis significantly contributes to the wealth of knowledge about α4β2 receptor stoichiometry. The finding of this thesis has contributed to the knowledge surrounding the structure activity relationship of series of MLA analogues as well as using different approaches to investigate the binding site of AE Succinimide at the two α4β2 receptor stoichiometries and finally to understand and characterize how mutations in the β2 subunit (β2V308A) that cause epilepsy, alter the function of distinct stoichiometric forms of α4β2 nACh receptors. Consequently, this knowledge can serve as a guide in designing selective and potent agents that may be used for therapeutic purposes.