title: Engineered site-directed labeling of nicotinic acetylcholine receptors using reactive epibatidine derivatives: appraisal of epibatidine-docking models in neuronal and muscular receptors.
abstract: 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. We selected epibatidine (EPB) as a prototypical ligand, acting at all types of nAChRs with sufficient affinity to allow this study. Accordingly, we synthesized three cysteine-reactive derivatives, all modified at the C-3 of the pyridine ring of the alkaloid with NCS; -NHCOCH2Cl, and -CH2Cl groups, respectively (Fig. 1). The binding properties have been established on rat brain, alpha7-5HT3 chimera, and Torpedo membranes, respectively, whereas the functional properties were tested on alpha4beta2 and alpha7 receptor expressed in oocytes and Cys-less muscular receptor expressed in HEK cells (Sakr et al., 2005).
authors.affiliation: Université Louis Pasteur Strasbourg, Strasbourg, France.
authors.forename: Elias
authors.initials: E
authors.lastname: Sakr
authors.forename: Florence
authors.initials: F
authors.lastname: Kotzyba-Hibert
authors.forename: Joerg
authors.initials: J
authors.lastname: Grande
authors.forename: Rund
authors.initials: R
authors.lastname: Hovius
authors.forename: Horst
authors.initials: H
authors.lastname: Vogel
authors.forename: Sonia
authors.initials: S
authors.lastname: Bertrand
authors.forename: Daniel
authors.initials: D
authors.lastname: Bertrand
authors.forename: Maurice
authors.initials: M
authors.lastname: Goeldner
mesh_terms: D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000737:chemistry / Q000493:pharmacokinetics*; D002460:Cell Line; D006801:Humans; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D018722:Nicotinic Agonists / Q000737:chemistry / Q000493:pharmacokinetics*; D011725:Pyridines / Q000737:chemistry / Q000493:pharmacokinetics*; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000502:physiology*; D014101:Torpedo
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D050505:Mutant Proteins; D018722:Nicotinic Agonists; D011725:Pyridines; D011978:Receptors, Nicotinic; C082748:epibatidine