Characterization of alternate forms of the α4�2 nicotinic acetylcholine receptor

• Main Author: Moroni, Mirko
• Published: Oxford Brookes University 2008
• Subjects: 615.1
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.491310

Ligand-gated ion channels (LOIC) mediate fast synaptic transmission in the central nervous system and at the neuro-muscular junction. In addition, LOIC can also influence neuronal signalling by influencing the release of neurotransmitters from presynaptic ternlinals. Neuronal nicotinic acetylcholine receptors (nAChRs) are wellknown examples of LOIC that modulate neurotransmitter release. These receptors are located mostly presynaptically in neurones, and due to their high calcium pernleability and fast activation upon ligand binding, they represent a key component of the physiological mechanisms that regulate neurotransmitter release and therefore neuronal signalling. This thesis is concerned with the properties of the a4f32 nAChR, the most abundant nAChR in the brain. The a4f32 nAChR is involved in a variety of physiological tasks, such as nociception, memory and vision, but it is also associated with pathological states such as Alzheimer's disease, Parkinson's disease, schizophrenia and autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Furthermore, due to its major contribution to the central effects of nicotine, the a4f32 nAChR is one of the most studied brain nAChR. The a4f32 nAChR is made up of five subunits, but its stoichiometry remained uncertain until recent insights. In this study, by using site-directed mutagenesis and the voltage-clamp technique, the stoichiometric arrangements of this receptor have been determined by heterologous expression in Xenopus oocytes. It can be concluded that two different arrangements in Xenopus oocytes two homogenous populations are produced: (a4))(f32h and (a4)2(f32)). The pharmacology of the two receptor subtypes has been characterised and subtype-selective drugs were used to investigate the effect of chronic nicotine exposure on both stoichiometric arrangements. The data presented here support the idea of nicotine acting as a chaperone in the endoplasmic reticulum, driving the receptor assembly of the a4 and f32 subunits towards the (a4)2(f32h stoichiometry. This thesis also examined the sensitivity of both a4f32 receptor stoichiometries to modulation by 2n2+. 2n2+ is the second most abundant ion present in the brain and its physiological relevance in modulating fast synaptic transmission has been widely documented. Here it is shown that the stoichiometry of the a4J32 receptor determines how the receptor responds to 2n2+: 2n2+ only inhibits (a4h(f32)) receptors, whilst it potentiates or inhibits, depending upon its concentration, the function of (a4))(f32)2 receptors. Mutational studies in combination with receptor modelling showed that the structural deternlinants responsible for 2n2 + potentiation and 2n2 + inhibition reside at the a4-a4 and at the f32-a4 subunit interfaces, respectively. Finally, to circumvent any uncertainty on the stoichiometry of a4J32 expressed in Xenopus oocytes or other heterologous systems, the expression of the two a4f32 receptor stoichiometries was constrained by means of subunit concatenation. In Xenopus oocytes concatenated receptors have produced functional expression levels comparable to those obtained by non-linked subunits. The data presented here suggest that subunit concatenation does not alter the receptor properties, thus making pentameric concatenated constructs a powerful tool for addressing the fine study of the structure and function of receptors.