?6 nAChR subunit residues that confer ?-conotoxin BuIA selectivity.

ABSTRACT: Nicotinic acetylcholine receptors (nAChRs) containing ?6 and/or ?4 subunits modulate the release of dopamine. However, few compounds can effectively discriminate between ligand-binding sites that contain ?6 vs. ?4 nAChR subunits. Using a chimeric (?6/?4) subunit, we showed that ?-conotoxin BuIA binds the extracellular rat ?6?2 vs. ?4?2 interface with ?60,000-fold selectivity. Chimeras containing residues from the ?6 subunit were inserted into the homologous position of the ?4 subunit to identify critical sequence segments. The region between residues 184 and 207 in the ?6 subunit accounted for the potency difference. Chimeras within this region followed by point mutations were constructed for further definition. ?6 Lys185, Thr187, and Ile188 form a triad of key residues that influence BuIA binding; when these 3 ?6 residues were inserted into the ?4 subunit, there was an ?2000-fold increase in toxin potency. We used a crystal structure of BuIA bound to the acetylcholine-binding protein together with the structure of the Torepedo marmorata nAChR to build a homology model of BuIA bound to the interface between ?6 and ?2 subunits. The results indicate that the triad of ?6 residues lies outside the C loop and is distantly located from bound BuIA (>10 Å). This suggests that alterations in potency are not caused by the direct interaction between the triad and BuIA. Instead, alterations in C-loop 3-dimensional structure and/or flexibility may account for differential potency. Thr198 and Tyr205 also contributed to BuIA potency. In addition, Thr198 caused BuIA potency differences between the closely related ?6 and ?3 subunits. Together, the findings provide insight into differences between the ?6 and other ? subunits that may be exploited by ?-conotoxins to achieve binding selectivity.

SUBMITTER: Kim HW 

PROVIDER: S-EPMC3448775 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature