Biophysical characterization of lynx‐nicotinic receptor interactions using atomic force microscopy

Nicotinic acetylcholine receptors (nAChRs) are broadly expressed in the central and peripheral nervous systems, playing essential roles in cholinergic neurotransmission. The lynx family proteins, a subset of the Ly6/uPAR superfamily expressed in multiple brain regions, have been shown to bind to nAChRs and modulate their function via allosteric regulation. The binding interactions between lynx and nAChRs, however, have not been systematically quantified and compared. In this work, we characterized the interactions between lynx1 or lynx2 and α3β4‐ or α7‐nAChRs using single‐molecule atomic force microscopy (AFM). The AFM technique allows the quantification of the off‐rate of lynx‐nAChR binding and of the energetic barrier width between the bound state and transition state, providing a biophysical means to compare the selectivity of lynx proteins for nAChR subtypes. Results indicate that lynx1 has a marginal preference for α7‐ over α3β4‐nAChRs. Strikingly, lynx2 exhibits a two order of magnitude stronger affinity for α3β4‐ compared to α7‐nAChRs. Together, the AFM assay serves as a valuable tool for the biophysical characterization of lynx‐nAChR binding affinities. Revealing the differential affinities of lynx proteins for nAChR subtypes will help elucidate how lynx regulates nAChR‐dependent functions in the brain, including nicotine addiction and other critical pathways.