Fluorescent agonists for the Torpedo nicotinic acetylcholine receptor.

We have synthesized a series of fluorescent acylcholine derivatives carrying different linkers that vary in length and structure and connect the acylcholine unit to the environment-sensitive fluorophores 7-(diethylamino)coumarin-3-carbonyl (DEAC) or N-(7-nitrobenz-2-oxa-1,3-diazol-yl) (NBD). The pharmacological properties of the fluorescent analogues were investigated on heterologously expressed nicotinic acetylcholine receptor (nAChR) from Torpedo californica and on oocytes transplanted with nAChR-rich Torpedo marmorata membranes. Agonist action strongly depends on the length and the structure of the linker.

Engineered site-directed labeling of nicotinic acetylcholine receptors using reactive epibatidine derivatives: appraisal of epibatidine-docking models in neuronal and muscular receptors.

We developed an engineered site-directed labeling method (Foucaud et al., 2001) to investigate ligand receptor interactions on the acetylcholine (ACh)- binding site of nicotinic acetylcholine receptors (nAChRs). The method uses cysteine receptor mutants, together with cysteine-reactive ligand analogs, to generate a site-directed covalent reaction within the binding site.

Reorganization of the nicotinic acetylcholine receptor during desensitization probed with a photoactivatable agonist.

The nicotinic acetylcholine receptor (nAChR) from fish electric organs and vertebrate neuromuscular junctions is a well-characterized transmembrane allosteric protein, composed of four polypeptide chains assembled into a heterologous pentamer alpha2betagammadelta, which carries ACh-binding sites and contains cation-selective channel-forming elements. Topographical mapping of residues contributing to the ligand-binding domain (LBD) of Torpedo nAChR was achieved with different site-directed antagonist or agonist probes. Over two decades of biochemical investigation led to the identification of three discontinuous domains on alpha subunits, with additional residues on gamma and delta subunits (Kotzyba- Hibert et al.

Photo-induced covalent attachment of agonists as a tool to study allosteric mechanisms of nicotinic acetylcholine receptors.

Muscular and neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels and contain either two or five binding sites for acetylcholine (ACh). Binding of ACh molecules on the nAChR will trigger the fast opening of the channel and subsequent slow desensitization process. Neuronal alpha7 nicotinic receptors are made up of five identical subunits and possess five binding sites for ACh; this raises the question of how many sites must be occupied before channel opening.

Dynamic structural investigations on the torpedo nicotinic acetylcholine receptor by time-resolved photoaffinity labeling.

An increasing number of high-resolution structures of membrane-embedded ion channels (or soluble homologues) have emerged during the last couple of years. The most pressing need now is to understand the complex mechanism underlying ion-channel function. Time-resolved photoaffinity labeling is a suitable tool for investigating the molecular function of membrane proteins, especially when high-resolution structures of related proteins are available.

Probing the reorganization of the nicotinic acetylcholine receptor during desensitization by time-resolved covalent labeling using [3H]AC5, a photoactivatable agonist.

The structural reorganizations occurring on the nicotinic acetylcholine receptor (nAChR) during activation and subsequent desensitization have been investigated through time-resolved photoaffinity labeling using a photoactivatable nicotinic agonist. [(3)H]AC5 is a photosensitive nicotinic probe with high affinity for the desensitized state of the Torpedo marmorata receptor (K(D) = 5 nM) that displays full agonist activity on the Torpedo californica receptor expressed in oocytes (EC(50) = 1.2 microM).

Syntheses and biological properties of cysteine-reactive epibatidine derivatives.

The synthesis of epibatidine derivatives modified at the 2-position of the pyridine or pyrimidine rings by reactive functions are described for potential irreversible site-directed coupling reactions on cysteine mutants of neuronal nicotinic acetylcholine receptors. An improved synthesis of the 7-azabicyclo[2,2,1]hepta-2,5-diene key intermediate has been developed to allow reproducible syntheses of the epibatidine derivatives. Binding tests and electrophysiological experiments allowed to select the 2-substituted alpha-chloroacetamido 13 and the chloropyrimidine derivative 11 as potential site-directed probes for the epibatidine binding site.

Structural reorganization of the acetylcholine binding site of the torpedo nicotinic receptor as revealed by dynamic photoaffinity labeling.

We explored the structural changes that occur at the acetylcholine binding site of the Torpedo marmorata nicotinic receptor during activation by the tritiated photoactivatable agonist (diazocyclohexadienoylpropyl)trimethylammonium ([(3)H]DCTA). We quantified the incorporation of radioactivity into the receptor subunits as a function of the mixing time of [(3)H]DCTA with the receptor by using a rapid-mixing device adapted with a photochemical quenching system. A saturable increase of the specific photolabeling on the alpha and gamma subunits was observed with a half-time of about 2 minutes.