The nicotinic acetylcholine (ACha
) receptor (nAChR) is the prototypical agonist-gated ion channel responsible for rapid excitatory neurotransmission.1
The nAChR agonists include many toxicants, potential therapeutic agents acting peripherally or centrally, and the important neonicotinoid insecticides.2–5
Initial attempts to understand agonist-receptor interactions involved site-directed or chimeric mutagenesis, estimating the role of specific region(s) or amino acid(s) on ligand occupation and/or the pharmacological response. The structural biology approach of high-resolution X-ray crystallography reveals orientations of functional amino acids at atomic resolution in the ligand-bound state and conformational rearrangements of the protein upon ligand interaction. Alternatively, incorporation of an unnatural amino acid or photoaffinity labeling defines the receptor recognition properties in a physiologically-relevant, aqueous solution environment.6,7
Binding site interactions of nicotinoid and neonicotinoid agonists have been characterized by comparative structural and chemical biology approaches8–13
using mollusk ACh binding protein (AChBP), which is a suitable structural surrogate of the extracellular ligand-binding domain of the nAChR.9,14
5-Azido-6-chloropyridin-3-yl nicotinic photoaffinity probes have played an important role in studying agonist-nAChR interactions.7
In principle, these probes bind reversibly to the specific site and then the reactive nitrene intermediate, generated by photoirradiation, reacts covalently with the target protein (). Upon photoaffinity labeling of Aplysia californica
AChBP, the azidochloropyridinyl photoprobes reacted at one location which was at the interface between loop C (Y195) on the principal or (+)-face subunit and loop E (M116) on the partnering or (−)-face subunit (),10,11
establishing a bound ligand position and conformation consistent with those observed in crystal structures.8,9,13
Interestingly, a nicotinic photoaffinity ligand of this type exclusively labeled the α4 subunit of the chick α4β2 nAChR subtype,10
in which Y225 in loop C on the α4 subunit and F137 in loop E on the β2 subunit are in homologous residue positions, spatially equivalent to Y195 and M116, respectively, of AChBP. These observations may be rationalized by the difference in the reactivity between amino acids and the photoactivated probe molecule. Therefore, the present investigation designs a methionine- and tyrosine-scanning approach on the loops C and E domains involving 17 AChBP mutants regarding photoreactivity of the 5-azido-6-chloropyridin-3-yl nicotinic photoprobes,10,15–17
ultimately mapping the specific site undergoing photoderivatization and the precise position and conformation of the bound ligand.
Two Chemotypes of Nicotinic Agonists and Their 5-Azido-Substituted Photoaffinity Probes (top) and Scheme for Photoaffinity Labeling (bottom)a.
Figure 1 AzEPI-docked structure of Aplysia AChBP, indicating the positions for the mutants studied. Relevant amino acids in yellow are from the (+)-face or principal subunit while in aquamarine are from the (−)-face or partnering subunit. This docking (more ...)