Identifying the binding site(s) for antidepressants on the Torpedo nicotinic acetylcholine receptor: [3H]2-azidoimipramine photolabeling and molecular dynamics studies

Mitesh Sanghvi, Ayman K. Hamouda, Krzysztof Jozwiak, Michael P. Blanton, James R. Trudell, Hugo R. Arias

Research output: Contribution to journalArticle

19 Scopus citations

Abstract

Radioligand binding, photoaffinity labeling, and docking and molecular dynamics were used to characterize the tricyclic antidepressant (TCA) binding sites in the nicotinic acetylcholine receptor (nAChR). Competition experiments indicate that the noncompetitive antagonist phencyclidine (PCP) inhibits [3H]imipramine binding to resting (closed) and desensitized nAChRs. [3H]2-azidoimipramine photoincorporates into each subunit from the desensitized nAChR with ∼ 25% of the labeling specifically inhibited by TCP (a PCP analog), whereas no TCP-inhibitable labeling was observed in the resting (closed) state. For the desensitized nAChR and within the α subunit, the majority of specific [3H]2-azidoimipramine labeling mapped to a ∼ 20 kDa Staphylococcus aureus V8 protease fragment (αV8-20; Ser173-Glu338). To further map the labeling site, the αV8-20 fragment was further digested with endoproteinase Lys-C and resolved by Tricine SDS-PAGE. The principal labeled fragment (11 kDa) was further purified by rpHPLC and subjected to N-terminal sequencing. Based on the amino terminus (αMet243) and apparent molecular weight, the 11 kDa fragment contains the channel lining M2 segment. Finally, docking and molecular dynamics results indicate that imipramine and PCP interact preferably with the M2 transmembrane segments in the middle of the ion channel. Collectively, these results are consistent with a model where PCP and TCA bind to overlapping sites within the lumen of the Torpedo nAChR ion channel.

Original languageEnglish
Pages (from-to)2690-2699
Number of pages10
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1778
Issue number12
DOIs
StatePublished - 1 Dec 2008
Externally publishedYes

Keywords

  • Molecular modeling
  • Noncompetitive inhibitors
  • Photolabeling
  • Torpedo nAChR
  • Tricyclic antidepressants

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