[3H]Epibatidine photolabels non-equivalent amino acids in the agonist binding site of Torpedo and α4β2 nicotinic acetylcholine receptors

Shouryadeep Srivastava, Ayman K. Hamouda, Akash Pandhare, Phaneendra K. Duddempudi, Mitesh Sanghvi, Jonathan B. Cohen, Michael P. Blanton

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Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo α2βγδ and expressed human α4β2 nAChRs with [3H]epibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of αTyr198 in agonist binding site Segment C of the principal (+) face in both α subunits and of γLeu109 and γTyr117 in Segment E of the complementary (-) face, with no labeling detected in the δ subunit. For affinity-purified α4β2 nAChRs, [3H]epibatidine photolabeled α4Tyr195 (equivalent to Torpedo αTyr190) in Segment C as well as β2Val111 and β2Ser113 in Segment E (equivalent to Torpedo γLeu109 and γTyr111 respectively). Consideration of the location of the photolabeled amino acids in homology models of the nAChRs based upon the acetylcholine-binding protein structure and the results of ligand docking simulations suggests that epibatidine binds in a single preferred orientation with the α-γ transmitter binding site, whereas it binds in two distinct orientations in α4β2 nAChR.

Original languageEnglish (US)
Pages (from-to)24939-24947
Number of pages9
JournalJournal of Biological Chemistry
Issue number37
Publication statusPublished - Sep 11 2009
Externally publishedYes


ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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