Atomic force microscopy to characterize binding properties of α7-containing nicotinic acetylcholine receptors on neurokinin-1 receptor-expressing medullary respiratory neurons.

In the present study, we used atomic force microscopy (AFM) to examine the ligand-binding properties of α7-containing nicotinic acetylcholine receptors (nAChRs) expressed on neurons from the ventral respiratory group. We also determined the effect of acute and prolonged exposure to nicotine on the binding probability of nAChRs. Neurons from neonatal (postnatal day 5-10) and juvenile rats (3-4 weeks old) were cultured. Internalization of Alexa Fluor 488-conjugated substance P was used to identify respiratory neurons that expressed the neurokinin-1 receptor (NK1-R), a recognized marker of ventral respiratory group neurons. To assess functional changes in nAChRs, AFM probes conjugated with anti-α7 subunit nAChR antibody were used to interact cyclically with the surface of the soma of NK1-R-positive neurons. Measurements were made of the frequency of antibody adhesion to the α7 receptor subunit and of the detachment forces between the membrane-attached receptor and the AFM probe tip. Addition of α-bungarotoxin (a specific antagonist of α7 subunit-containing nAChRs) to the cell bath produced a 69% reduction in binding to the α7 subunit (P < 0.05, n = 10), supporting specificity of binding. Acute exposure to nicotine (1 μM added to culture media) produced an 80% reduction in nAChR antibody binding to the α7 subunit (P < 0.05, n = 9). Prolonged incubation (72 h) of the cell culture in nicotine significantly reduced α7 binding in a concentration-dependent manner. Collectively, these findings demonstrate that AFM is a sensitive tool for assessment of functional changes in nAChRs expressed on the surface of living NK1-R-expressing medullary neurons. Moreover, these data demonstrate that nicotine exposure decreases the binding probability of α7 subunit-containing nAChRs.