Abstract

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.