α6 nAChR subunit residues that confer α-conotoxin BuIA selectivity

Nicotinic acetylcholine receptors (nAChRs) containing alpha 6 and/or alpha 4 subunits modulate the release of dopamine. However, few compounds can effectively discriminate between ligand-binding sites that contain alpha 6 vs. alpha 4 nAChR subunits. Using a chimeric (alpha 6/alpha 4) subunit, we showed that alpha-conotoxin BuIA binds the extracellular rat alpha 6 beta 2 vs. alpha 4 beta 2 interface with similar to 60,000-fold selectivity. Chimeras containing residues from the alpha 6 subunit were inserted into the homologous position of the alpha 4 subunit to identify critical sequence segments. The region between residues 184 and 207 in the alpha 6 subunit accounted for the potency difference. Chimeras within this region followed by point mutations were constructed for further definition. alpha 6 Lys185, Thr187, and Ile188 form a triad of key residues that influence BuIA binding; when these 3 alpha 6 residues were inserted into the alpha 4 subunit, there was an similar to 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 alpha 6 and beta 2 subunits. The results indicate that the triad of alpha 6 residues lies outside the C loop and is distantly located from bound BuIA (> 10 angstrom). 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 alpha 6 and alpha 3 subunits. Together, the findings provide insight into differences between the alpha 6 and other alpha subunits that may be exploited by alpha-conotoxins to achieve binding selectivity.-Kim, H.-W., McIntosh, J. M. alpha 6 nAChR subunit residues that confer alpha-conotoxin BuIA selectivity. FASEB J. 26, 4102-4110 (2012). www.fasebj.org