Nicotinic Acetylcholine Receptor Labeled with a Tritiated, Photoactivatable Agonist:  A New Tool for Investigating the Functional, Activated State

Abstract

Upon agonist activation, the nicotinic acetylcholine receptor undergoes allosteric transitions leading to channel opening and sodium ion influx. The molecular structure of the agonist binding site has been mapped previously by photoaffinity labeling, but most photosensitive probes used for this purpose interact only with closed receptor states (resting or desensitized). We have synthesized two novel photoactivatable 4-diazocyclohexa-2,5-dienone derivatives as cholinergic agonist candidates, with the objective of identifying structural changes at the acetylcholine binding site associated with receptor activation. One of these ligands, 9b, is a functional agonist at muscle acetylcholine receptors in human TE 671 cells. In photolabeling experiments with 9b, up to 35% inactivation of agonist binding sites was observed at Torpedo acetylcholine receptors. Tritiated 9b was synthesized, and photolabeling was found to occur mainly on the alpha-subunit in a partially protectable manner. This novel radiolabeled photoprobe appears to be suitable for future investigation of the molecular dynamics of allosteric transitions occurring at the active acetylcholine receptor binding site.

Binding, Thermodynamics, and Selectivity of a Non-peptide Antagonist to the Melanocortin-4 Receptor

“…In accordance with previous computational and experimental work carried out for other receptors ( Dror et al, 2011 ; Bock et al, 2012 ; Kruse et al, 2012 ; DeVree et al, 2016 ; Milanos et al, 2016 ; Saleh et al, 2016 , 2017a , c ), our study suggests a stepwise binding mechanism for the selective non-peptide antagonist MCL0129 to hMC4R. The Nterm plays a key role in ligand binding and recognition, trapping MCL0129 into its final binding mode.…”

“…Biomolecular [molecular dynamics (MDs)] simulations have been successful in providing missing structural information for GPCRs ( Dror et al, 2011 , 2012 , 2013 ; Grossfield, 2011 ; Kruse et al, 2012 ; Vanni and Rothlisberger, 2012 ; Rose et al, 2014 ; Clark, 2017 ). Moreover, homology models based on high-quality templates in combination with microsecond-scale unbiased MD refinement ( Clark, 2017 ) have provided robust experimentally testable structural and thermodynamic information on GPCRs ( Milanos et al, 2016 ; Saleh et al, 2016 , 2017a , b , c ).…”

Structural Reorganization of the Acetylcholine Binding Site of the Torpedo Nicotinic Receptor as Revealed by Dynamic Photoaffinity Labeling

“…Materials : [ 3 H]DCTA (0.4–0.6 Ci mmol −1 ) was synthesized and purified as described previously 15. nAChR‐rich membranes were purified from T. marmorata frozen electric organs as described elsewhere 21.…”

“…Accordingly, we developed a novel family of photosensitive agonists of suitable size and reactivity, designed to be covalently incorporated amongst the surrounding ACh‐binding residues upon UV irradiation 14. One of these photoprobes, DCTA (Figure 1) is a functional agonist of nAChR that is expressed by TE 671 cells15 and was designed to explore the ester‐binding pocket of ACh 16. The four [ 3 H]DCTA‐labeled residues (Tyr‐190, Cys‐192, Cys‐193, and Tyr‐198) in the receptor were anticipated to participate directly in the binding of the ester moiety of ACh.…”

4-Diazocyclohexa-2,5-dienone Derivatives as Photoaffinity Labels for Proteins: Scope and Limitations

“…Molecule A was designed to label the membraneassociate domains of proteins, 12 compound B was synthesized as a channel blocker associate to the GABA receptor 13 compound C was described for a potential use as a peptidic probe 14 , while molecules D and E containing either a tertiary amine or a quaternary ammonium salt were synthesized to label cholinergic proteins, acetylcholinesterase 15 and the nicotinic cholinergic receptor 16 respectively.…”