Identifying the binding site(s) for antidepressants on the Torpedo nicotinic acetylcholine receptor: [3H]2-azidoimipramine photolabeling and molecular dynamics studies.
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 approximately 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 alpha subunit, the majority of specific [3H]2-azidoimipramine labeling mapped to a approximately 20 kDa Staphylococcus aureus V8 protease fragment (alphaV8-20; Ser173-Glu338). To further map the labeling site, the alphaV8-20 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 (alphaMet243) 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.
Sanghvi
Mitesh
M
Department of Pharmacology and Neuroscience, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA.
Hamouda
Ayman K
AK
Jozwiak
Krzysztof
K
Blanton
Michael P
MP
Trudell
James R
JR
Arias
Hugo R
HR
D000595:Amino Acid Sequence; D000818:Animals; D000929:Antidepressive Agents, Tricyclic / Q000378:metabolism*; D001386:Azides / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D003198:Computer Simulation; D004557:Electric Organ / Q000378:metabolism; D020128:Inhibitory Concentration 50; D007473:Ion Channels / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019920:Photoaffinity Labels / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000378:metabolism*; D014101:Torpedo; D014316:Tritium
D000929:Antidepressive Agents, Tricyclic; D001386:Azides; D007473:Ion Channels; D019920:Photoaffinity Labels; D011978:Receptors, Nicotinic; D014316:Tritium; C033466:2-azidoimipramine