title: Photoaffinity labeling the agonist binding domain of alpha4beta4 and alpha4beta2 neuronal nicotinic acetylcholine receptors with [(125)I]epibatidine and 5[(125)I]A-85380.
abstract: 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 alpha4beta2 and rat alpha4beta4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [(125)I]epibatidine and 5[(125)I]A-85380. Our results show that both agonists photoincorporated into the beta4 subunit with little or no labeling of the beta2 and alpha4 subunits respectively. [(125)I]epibatidine labeling in the beta4 subunit was mapped to two overlapping proteolytic fragments that begin at beta4V102 and contain Loop E (beta4I109-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 beta4Q117 in Loop E is the principal site of [(125)I]epibatidine labeling. This was accomplished by substituting residues in the beta2 subunit with the beta4 homologs and finding [(125)I]epibatidine labeling in beta4 and beta2F119Q subunits with little, if any, labeling in alpha4, beta2, or beta2S113R subunits. Finally, functional studies established that the beta2F119/beta4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.
authors: Department of Pharmacology and Neuroscience and the Center for Membrane Protein Research, School of Medicine, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430, USA.
authors: Ayman K
authors: AK
authors: Hamouda
authors: Xiaochun
authors: X
authors: Jin
authors: Mitesh
authors: M
authors: Sanghvi
authors: Shouryadeep
authors: S
authors: Srivastava
authors: Akash
authors: A
authors: Pandhare
authors: Phaneendra K
authors: PK
authors: Duddempudi
authors: Joe Henry
authors: JH
authors: Steinbach
authors: Michael P
authors: MP
authors: Blanton
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001384:Azetidines / Q000378:metabolism*; D001665:Binding Sites; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D018722:Nicotinic Agonists / Q000378:metabolism* / Q000494:pharmacology; D009865:Oocytes / Q000502:physiology; D019920:Photoaffinity Labels; D011725:Pyridines / Q000378:metabolism*; D051381:Rats; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D016415:Sequence Alignment; D014981:Xenopus
chemical_list: C104288:A 85380; D001384:Azetidines; D019086:Bridged Bicyclo Compounds, Heterocyclic; D007457:Iodine Radioisotopes; D018722:Nicotinic Agonists; D019920:Photoaffinity Labels; D011725:Pyridines; D011978:Receptors, Nicotinic; C427403:alpha(4)beta(4) nicotinic receptor; C403531:nicotinic receptor alpha4beta2; C082748:epibatidine