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Title: The emerging role of the α4(+)α4(-) interface as a determinant of functional signatures of α4ß2 nicotinic acetylcholine receptors
Author: Mazzaferro, Simone
Awarding Body: Oxford Brookes University
Current Institution: Oxford Brookes University
Date of Award: 2013
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The α4β2 nicotinic acetylcholine receptor (nAChR) assembles in two alternate forms, (α4β2)₂α4 and (α4β2)₂β2, which display stoichiometry-specific agonist sensitivity. Being heteromeric pentameric ligand-gated ion channels (pLGIC), α4β2 receptors are activated by binding of agonist to sites located at the α4(+)/β2(-) interfaces. These interfaces are present in both stoichiometries they are unlikely to contain structural differences conferring specific properties to (α4β2)₂α4 and (α4β2)₂β2 receptors. In contrast, the auxiliary subunit can be either α4 or β2, leading to stoichiometry-specific β2(+)/β2(-) and α4(+)/α4(-) interfaces. Using fully concatenated (α4β2)₂α4 nAChRs in conjunction with structural modelling, chimeric receptors and functional mutagenesis, this study identified an additional agonist site at the α4(+)/α4(-) interface that accounts for the stoichiometry-specific agonist sensitivity of the (α4β2)₂α4 receptor. The additional agonist site occupies a region that also contains a potentiating Zn²⁺ binding site. However, unlike Zn²⁺, the agonist binding influences agonist responses by directly contributing to channel gating. By engineering a receptor with a C226S mutation to provide a free cysteine in loop C in the + side of the α4(+)/α4(-) interface, this study found that the acetylcholine (ACh) responses of the additional agonist site are modified following modification of the substituted cysteine with sulfhydryl reagents. These findings suggested that agonist occupation of the site at the α4(+)/α4(-) interface leads to channel gating through a coupling mechanism involving a conformational switch in loop C. The sulfhydryl reagents had similar effects on substituted cysteines in the α4(+)/β2(-) interfaces. Further studies showed that the additional agonist site is less sensitive to desensitisation than the sites at the α4(+)/β2(-) interfaces, suggesting that the agonist sites are functionally non-equivalent. Non-functional equivalency was also indicated by the finding that the agonist selectivity of the site at the α4(+)/α4(-) differs from that of the sites at the α4(+)/β2(-) interface. The findings may have important consequences for drug discovery programs and the manner by which α4β2 receptor signalling in the brain can be modified in brain pathologies.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available