title: Benzodiazepine binding studies on living cells: application of small ligands for fluorescence correlation spectroscopy.
abstract: We demonstrate the applicability of fluorescence correlation spectroscopy (FCS) for receptor binding studies using low molecular weight ligands on the membranes of living nerve cells. The binding of the benzodiazepine Ro 7-1986/602 (N-des-diethyl-fluorazepam), labeled with the fluorophore Alexa 532, to the benzodiazepine receptor was analyzed quantitatively at the membrane of single rat hippocampal neurons. The values obtained for the dissociation constant Kd = (9.9 +/- 1.9) nm and the rate constant for ligand-receptor dissociation kdisS = (1.28 +/- 0.08) x 10(-3) s(-1) show that there is a specific and high affinity interaction between the dye-labeled ligand (Ro-Alexa) and the receptor site. The binding was saturated at approx. 100 nM and displacement of 10 nM Ro-Alexa, with a 1,000-fold excess of midazolam, showed a non-specific binding of 7-10%. Additionally, two populations of the benzodiazepine receptor that differed in their lateral mobility were detected in the membrane of rat neurons. The diffusion coefficients for these two populations [D(bound1) = (1.32 +/- 0.26) microm2/s; D(bound2) = (2.63 +/- 0.63) x 10(-2) microm2/s] are related to binding sites, which shows a mono-exponential decay in a time-dependent dissociation of the ligand-receptor complex.
authors.affiliation: Department of Pharmaceutical Biology, Philipps-University of Marburg, D-35037 Marburg, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Hegener
authors.forename: Randolf
authors.initials: R
authors.lastname: Jordan
authors.forename: Hanns
authors.initials: H
authors.lastname: Häberlein
mesh_terms: D000465:Algorithms; D000818:Animals; D000918:Antibody Specificity; D001667:Binding, Competitive / Q000187:drug effects; D004058:Diffusion; D005456:Fluorescent Dyes; D005479:Flurazepam / Q000031:analogs & derivatives* / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects; D007700:Kinetics; D008024:Ligands; D018613:Microscopy, Confocal; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D011963:Receptors, GABA-A / Q000187:drug effects / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D011963:Receptors, GABA-A; C033473:didesethylflurazepam; D005479:Flurazepam