The a4~2 nicotinic acetylcholine receptor (nAChR) assembles in two alternate forms, (a4p2ha4 and (a4~2hP2, which display stoichiometry-specific agonist sensitivity. Being heteromeric pentameric ligand-gated ion channels (pLGIC), a4p2 receptors are activated by binding of agonist to sites located at the a4( + )/P2( -) interfaces. These interfaces are present in both stoichiometries they are unlikely to contain structural differences conferring specific properties to (a4~2)2a4 and (a4p2)2p2 receptors. In contrast, the auxiliary subunit can be either a4 or P2, leading to stoichiometry-specific ~2( + )/P2( -) and a4( + )/a4( -) interfaces. Using fully concatenated (a4p2ha4 nAChRs in conjunction with structural modelling, chimeric receptors and functional mutagenesis, this study identified an additional agonist site at the a4( + )/a4( -) interface that accounts for the stoichiometry-specific agonist sensitivity of the (a4p2ha4 receptor. The additional agonist site occupies a region that also contains a potentiating 2n2+ binding site. However, unlike 2n2+, 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 a4(+)/a4(-) 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 a4(+)/a4(-) 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 a4( + )/P2( -) interfaces. Further studies showed that the additional agonist site is less sensitive to desensitisation than the sites at the a4( + )/~2( -) 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 a4(+)/a4(-) differs from that of the sites at the a4(+)/p2(-) interface. The findings may have important consequences for drug discovery programs and the manner by which a4p2 receptor signalling in the brain can be modified in brain pathologies. XIU
Mazzaferro, Simone
Faculty of Health and Life SciencesDepartment of Biological and Medical Sciences
Year: 2013
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