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O. Hegener, R. Jordan, H. Häberlein
Benzodiazepine Binding Studies on Living Cells: Application of Small Ligands for Fluorescence Correlation Spectroscopy
We demonstrate the applicability of fluorescence correlation spectroscopy (FCS) for receptor binding studies using low molecular weight ligands on membranes of living nerve cells. The binding of benzodiazepine Ro 7 1986/602 (Ndesdiethylfluorazepam), labeled with the fluorophore Alexa 532, 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 ligandreceptor dissociation kdiss = (1.28±0.08)10- 3 s 1 show that there is a specific and high affinity interaction between the dyelabeled ligand (RoAlexa) and the receptor site. The binding was saturated at approx. 100 and displacement of 10 nM RoAlexa, with a 1000- excess of midazolam, showed a nonspecific 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 [Dbound1 = (1.32±0.26) m2/s; Dbound2 = (2.63±0.63)10- 2 m2/s] related to binding sites, which shows a monoexponential decay in a timedependent dissociation of ligandreceptor complex.
Biological Chemistry, Walter de Gruyter
Print ISSN: 1431-6730
Volume: 383, 11/2002
Pages: 1801 - 1807