Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.786279
Title: The role of subunit interfaces in the function of (α4β2)2β2 nAChRs
Author: New, Karina L.
ISNI:       0000 0004 7971 7468
Awarding Body: Oxford Brookes University
Current Institution: Oxford Brookes University
Date of Award: 2018
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Abstract:
Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels formed from homologous subunits, of which there are many different subtypes. The ability to combine different types of subunits into an individual pentamer enables a wide diversity of functional properties to meet a range of physiological needs. In the brain, the vast majority of high-sensitivity 3H-nicotine binding sites are due to nAChRs containing α4 and β2 subunits. These subunits assemble into pentamers with alternate stoichiometries (α4β2)2β2 and (α4β2)2α4. These two receptors differ in sensitivity to ACh, unitary current amplitude, selectivity for different agonists, antagonists an, and potentiation by ions or drugs. The alternate stoichiometries are present in neurones and although they tend to co-express, there are regions in the brain such as the striatum, where only one stoichiometry is present. Recent studies of the (α4β2)2α4 nAChR have shown that this receptor type functions with three agonist sites, two of these are on the α4/β2 interfaces of the receptor and are thus classical nAChR agonist sites, whereas the other site is on the α4/α4 interface, the signature interface of this receptor type. Pharmacological studies have shown convincingly that the site at the α4/α4 interface accounts for the unique pharmacology of the (α4β2)2α4 nAChRs. In the case of the (α4β2)2β2, there is a signature β2(+)/β2(-) interface that homology models suggest may house an agonist site. The β2(+)/β2(-) interface forms between the fifth subunit of the receptor and another β2 subunit that also contributes, through its complementary face, to an agonist site. In common with the (α4β2)2α4 nAChR, the α4/β2 interfaces of (α4β2)2β2 house each an agonist site. To test the possibility of an operational agonsit site at the β2(+)/β2(-) interface, and to answer the question of whether the unique functional behaviour of the (α4β2)2β2 can be ascribed to an additional agonist site, the work presented here used targeted single point-mutations, functional analysis and the substituted cysteine scanning approach. By using these approaches, it was found that the β2(+)/β2(-) interface does not house an agonist site; however, it was found that this interface is an important site for inter-subunit communication and that this communication encodes agonist efficacy elements. The inter-subunit communication occurs between residues of the E loop of the fifth subunit of the receptor and conserved aromatic residues in loop B of the complementary subunit of one of the agonist sites found on α/β interfaces. By alanine substituting agonist sites on the α4/β2 interfaces and determining the consequences of the mutations on the pattern of covalent reaction between a methanethiosulphonate compound and one of the β2 subunits contributing to the β2(+)/β2(-) interface, it was found that the agonist sites communicate with the β2(+)/β2(-) interface via the interactions between the E loop residues and conserved aromatic residues. Further studies with a compound that enabled direct measurements of changes in agonist efficacy relative to that ACh established that agonist efficacy is dependent on primarily on binding of the agonist to the agonist sites on the α4/β2 interfaces and then on the E loop-conserved aromatic residues interactions. The results are discussed in the context of recent cryo-electron microscopy structures of the muscle nAChR that show, for the first time, that the fifth subunit of nAChRs may play a key role in gating of the ion channel.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.786279  DOI:
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