Photoaffinity labeling the agonist binding domain of α4β4 and α4β2 neuronal nicotinic acetylcholine receptors with [125I]epibatidine and 5[125I]A-85380

The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human alpha 4 beta 2 and rat alpha 4 beta 4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [I-125]epibatidine and 5[I-125]A-85380. Our results show that both agonists photo incorporated into the beta 4 subunit with little or no labeling of the 2 and alpha 4 subunits respectively. [I-125]epibatidine labeling in the M subunit was mapped to two overlapping proteolytic fragments that begin at beta 4V102 and contain Loop E (beta 4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that beta 4Q117 in Loop E is the principal site of [I-125]epibatidine labeling. This was accomplished by substituting residues in the beta 2 subunit with the beta 4 homologs and finding [I-125] epibatidine labeling in beta 4 and beta 2F119Q subunits with little, if any. labeling in alpha 4, beta 2, or beta 2S113R subunits. Finally. functional studies established that the beta 2F119/beta 4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation (c) 2009 Elsevier B V All rights reserved